Sample records for precursor cells derived

  1. Derivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells

    PubMed Central

    Barberi, Tiziano; Willis, Lucy M; Socci, Nicholas D; Studer, Lorenz

    2005-01-01

    Background Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. Methods and Findings Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. Conclusion Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications. PMID:15971941

  2. Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

    PubMed

    Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

    2016-01-01

    As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

  3. Bioreactor Expansion of Skin-Derived Precursor Schwann Cells.

    PubMed

    Walsh, Tylor; Biernaskie, Jeff; Midha, Rajiv; Kallos, Michael S

    2016-01-01

    Scaling up the production of cells in a culture process is a critical step when trying to develop cell-based regenerative therapies. Static cultures often cannot be easily scaled up to clinically relevant cell numbers. Alternatively, bioreactors offer a highly valuable means to develop a clinical-ready process. To culture adherent cells in suspension, such as skin-derived precursor Schwann cells (SKP-SCs), microcarriers need to be used. Microcarriers are small spherical beads suspended within the vessel that allow for higher growth surface area to volume ratio. Here we describe the procedure of combining microcarriers with the controllability of bioreactors to generate higher cell densities in smaller reactor volumes leading to a more efficient and cost-effective cell production for applications in regenerative medicine.

  4. Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

    PubMed

    Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

    2016-01-01

    The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

  5. Skin-derived neural precursors competitively generate functional myelin in adult demyelinated mice

    PubMed Central

    Mozafari, Sabah; Laterza, Cecilia; Roussel, Delphine; Bachelin, Corinne; Marteyn, Antoine; Deboux, Cyrille; Martino, Gianvito; Evercooren, Anne Baron-Van

    2015-01-01

    Induced pluripotent stem cell–derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent–derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin–derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS. PMID:26301815

  6. Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

    PubMed

    Rodrigues, Gonçalo M C; Fernandes, Tiago G; Rodrigues, Carlos A V; Cabral, Joaquim M S; Diogo, Maria Margarida

    2015-01-01

    Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

  7. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boku, Shuken, E-mail: shuboku@med.hokudai.ac.jp; Nakagawa, Shin; Takamura, Naoki

    2013-05-17

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression andmore » secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.« less

  8. Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

    PubMed

    Darabi, Radbod; Perlingeiro, Rita C R

    2016-01-01

    Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

  9. Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

    PubMed Central

    Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

    2016-01-01

    Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event

  10. The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

    PubMed

    Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

    2011-12-01

    We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Brain-Derived Neurotrophic Factor Induces Cell Survival and the Migration of Murine Adult Hippocampal Precursor Cells During Differentiation In Vitro.

    PubMed

    Ortiz-López, Leonardo; Vega-Rivera, Nelly Maritza; Babu, Harish; Ramírez-Rodríguez, Gerardo Bernabé

    2017-01-01

    The generation of new neurons during adulthood involves local precursor cell migration and terminal differentiation in the dentate gyrus. These events are influenced by the hippocampal microenvironment. Brain-derived neurotrophic factor (BDNF) is relevant for hippocampal neuronal development and behavior. Interestingly, studies that have been performed in controlled in vitro systems that involve isolated precursor cells that were derived from the dentate gyrus (AHPCs) have shown that BDNF induces the activation of the TrkB receptor and, consequentially, might activate signaling pathways that favor survival and neuronal differentiation. Based on the fact that the cellular events of AHPCs that are induced by single factors can be studied in this controlled in vitro system, we investigated the ability of BDNF and the involvement of protein kinase C (PKC), as one of the TrkB-downstream activated signaling proteins, in the regulation of migration, here reflected by motility, of AHPCs. Precursor cells were cultured following a concentration-response curve (1-640 ng/ml) for 24 or 96 h. We found that BDNF favored cell survival without altering the viability under culture proliferative conditions of the AHPCs. Concomitantly, glial- and neuronal-differentiated precursor cells increased as a consequence of survival promoted by BDNF. Additionally, pharmacological approaches showed that BDNF (40 ng/ml)-induced migration of AHPCs was blocked with the compounds K252a and GF109203x, which prevent the activation of TrkB and PKC, respectively. The results indicate that in the in vitro migration of differentiated AHPCs it is involved the BDNF and TrkB cascade. Our results provide additional information about the mechanism by which BDNF impacts adult neurogenesis in the hippocampus.

  12. Overexpression of Polysialylated Neural Cell Adhesion Molecule Improves the Migration Capacity of Induced Pluripotent Stem Cell-Derived Oligodendrocyte Precursors

    PubMed Central

    Czepiel, Marcin; Leicher, Lasse; Becker, Katja; Boddeke, Erik

    2014-01-01

    Cell replacement therapy aiming at the compensation of lost oligodendrocytes and restoration of myelination in acquired or congenital demyelination disorders has gained considerable interest since the discovery of induced pluripotent stem cells (iPSCs). Patient-derived iPSCs provide an inexhaustible source for transplantable autologous oligodendrocyte precursors (OPCs). The first transplantation studies in animal models for demyelination with iPSC-derived OPCs demonstrated their survival and remyelinating capacity, but also revealed their limited migration capacity. In the present study, we induced overexpression of the polysialylating enzyme sialyltransferase X (STX) in iPSC-derived OPCs to stimulate the production of polysialic acid-neuronal cell adhesion molecules (PSA-NCAMs), known to promote and facilitate the migration of OPCs. The STX-overexpressing iPSC-derived OPCs showed a normal differentiation and maturation pattern and were able to downregulate PSA-NCAMs when they became myelin-forming oligodendrocytes. After implantation in the demyelinated corpus callosum of cuprizone-fed mice, STX-expressing iPSC-derived OPCs demonstrated a significant increase in migration along the axons. Our findings suggest that the reach and efficacy of iPSC-derived OPC transplantation can be improved by stimulating the OPC migration potential via specific gene modulation. PMID:25069776

  13. Artificial Extracellular Matrices with Oversulfated Glycosaminoglycan Derivatives Promote the Differentiation of Osteoblast-Precursor Cells and Premature Osteoblasts

    PubMed Central

    Hempel, Ute; Preissler, Carolin; Möller, Stephanie; Becher, Jana; Rauner, Martina; Hofbauer, Lorenz C.; Dieter, Peter

    2014-01-01

    Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts. PMID:24864267

  14. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

    PubMed Central

    Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

    2016-01-01

    Introduction Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. Materials and methods We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Results Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Conclusion Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders. PMID:27920532

  15. Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy

    PubMed Central

    2017-01-01

    Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE), a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80%) and yield (>70%). Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies. PMID:28122047

  16. Ground-state transcriptional requirements for skin-derived precursors.

    PubMed

    Suflita, Michael T; Pfaltzgraff, Elise R; Mundell, Nathan A; Pevny, Larysa H; Labosky, Patricia A

    2013-06-15

    Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell.

  17. Ground-State Transcriptional Requirements for Skin-Derived Precursors

    PubMed Central

    Suflita, Michael T.; Pfaltzgraff, Elise R.; Mundell, Nathan A.; Pevny, Larysa H.

    2013-01-01

    Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell. PMID:23316968

  18. Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma

    PubMed Central

    Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

    2017-01-01

    As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion. PMID:28212546

  19. Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma.

    PubMed

    Xiong, Ye; Liu, Liyun; Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

    2017-04-11

    As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion.

  20. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages.

    PubMed

    Hoeffel, Guillaume; Wang, Yilin; Greter, Melanie; See, Peter; Teo, Pearline; Malleret, Benoit; Leboeuf, Marylène; Low, Donovan; Oller, Guillaume; Almeida, Francisca; Choy, Sharon H Y; Grisotto, Marcos; Renia, Laurent; Conway, Simon J; Stanley, E Richard; Chan, Jerry K Y; Ng, Lai Guan; Samokhvalov, Igor M; Merad, Miriam; Ginhoux, Florent

    2012-06-04

    Langerhans cells (LCs) are the dendritic cells (DCs) of the epidermis, forming one of the first hematopoietic lines of defense against skin pathogens. In contrast to other DCs, LCs arise from hematopoietic precursors that seed the skin before birth. However, the origin of these embryonic precursors remains unclear. Using in vivo lineage tracing, we identify a first wave of yolk sac (YS)-derived primitive myeloid progenitors that seed the skin before the onset of fetal liver hematopoiesis. YS progenitors migrate to the embryo proper, including the prospective skin, where they give rise to LC precursors, and the brain rudiment, where they give rise to microglial cells. However, in contrast to microglia, which remain of YS origin throughout life, YS-derived LC precursors are largely replaced by fetal liver monocytes during late embryogenesis. Consequently, adult LCs derive predominantly from fetal liver monocyte-derived cells with a minor contribution of YS-derived cells. Altogether, we establish that adult LCs have a dual origin, bridging early embryonic and late fetal myeloid development.

  1. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac–derived macrophages

    PubMed Central

    Hoeffel, Guillaume; Wang, Yilin; Greter, Melanie; See, Peter; Teo, Pearline; Malleret, Benoit; Leboeuf, Marylène; Low, Donovan; Oller, Guillaume; Almeida, Francisca; Choy, Sharon H.Y.; Grisotto, Marcos; Renia, Laurent; Conway, Simon J.; Stanley, E. Richard; Chan, Jerry K.Y.; Ng, Lai Guan; Samokhvalov, Igor M.

    2012-01-01

    Langerhans cells (LCs) are the dendritic cells (DCs) of the epidermis, forming one of the first hematopoietic lines of defense against skin pathogens. In contrast to other DCs, LCs arise from hematopoietic precursors that seed the skin before birth. However, the origin of these embryonic precursors remains unclear. Using in vivo lineage tracing, we identify a first wave of yolk sac (YS)–derived primitive myeloid progenitors that seed the skin before the onset of fetal liver hematopoiesis. YS progenitors migrate to the embryo proper, including the prospective skin, where they give rise to LC precursors, and the brain rudiment, where they give rise to microglial cells. However, in contrast to microglia, which remain of YS origin throughout life, YS-derived LC precursors are largely replaced by fetal liver monocytes during late embryogenesis. Consequently, adult LCs derive predominantly from fetal liver monocyte-derived cells with a minor contribution of YS-derived cells. Altogether, we establish that adult LCs have a dual origin, bridging early embryonic and late fetal myeloid development. PMID:22565823

  2. Skin derived precursor Schwann cell-generated acellular matrix modified chitosan/silk scaffolds for bridging rat sciatic nerve gap.

    PubMed

    Zhu, Changlai; Huang, Jing; Xue, Chengbin; Wang, Yaxian; Wang, Shengran; Bao, Shuangxi; Chen, Ruyue; Li, Yuan; Gu, Yun

    2017-12-27

    Extracellular/acellular matrix has been attracted much research interests for its unique biological characteristics, and ACM modified neural scaffolds shows the remarkable role of promoting peripheral nerve regeneration. In this study, skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) were used as parent cells to generate acellular(ACM) for constructing a ACM-modified neural scaffold. SKP-SCs were co-cultured with chitosan nerve guidance conduits (NGC) and silk fibroin filamentous fillers, followed by decellularization to stimulate ACM deposition. This NGC-based, SKP-SC-derived ACM-modified neural scaffold was used for bridging a 10 mm long rat sciatic nerve gap. Histological and functional evaluation after grafting demonstrated that regenerative outcomes achieved by this engineered neural scaffold were better than those achieved by a plain chitosan-silk fibroin scaffold, and suggested the benefits of SKP-SC-derived ACM for peripheral nerve repair. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

  3. Identification and characterization of B cell precursors in rat lymphoid tissues. I. Adoptive transfer assays for precursors of TI-1, TI-2, and TD antigen-reactive B cells.

    PubMed

    Whalen, B J; Goldschneider, I

    1993-10-01

    Quantitative adoptive transfer assays were developed to detect the precursors of TI-1, TI-2, and TD antigen-reactive B cells in rat lymphoid tissues. Studies on the immune responses in normal and athymic nude rats validate the use of TNP-lipopolysaccharide as a TI-1 antigen, TNP-Ficoll as a TI-2 antigen, and SRBC as a TD antigen in rats. The precursors to these immunologically competent B cells are detected, following transfer into irradiated histocompatible recipients, by their ability to generate expanded populations of antigen-reactive B cells capable of mounting antibody responses (splenic IgM plaque-forming cells) to these antigens. Maximal numbers of antigen-reactive B cells emerge in antigenically naive rats after an interval of 7-12 days following transfer of donor lymphoid cells and decline rapidly thereafter. The delayed responses in adoptive recipients reconstituted with spleen cells are proportional to the numbers of spleen cells transferred and are shown to be primarily donor derived using histocompatible Ig kappa chain alloantigen disparate rat strain combinations. The precursors of TI-1, TI-2, and TD antigen-reactive B cells are present in both donor spleen and bone marrow. However, precursor cells to TI-1 and TD antigens are largely absent from donor lymph node cells, whereas precursors to the TI-2 antigen are as prevalent in donor lymph node as in donor spleen. These results support the hypothesis that newly formed virginal B cells represent transient populations of precursor cells that undergo further proliferation and differentiation in the spleen before acquiring immunological competence. The results also suggest that the precursors of TI-2 antigen-reactive B cells differ developmentally from those of TI-1 and TD antigen-reactive B cells, and that the antigen-reactive progeny of these precursors require additional stimulation in order to join the pool of long-lived peripheral B cells.

  4. Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

    PubMed Central

    Sánchez-Mendoza, Eduardo H.; Bellver-Landete, Victor; Arce, Carmen; Doeppner, Thorsten R.; Hermann, Dirk M.

    2017-01-01

    The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs. PMID:28493916

  5. Identification of early B cell precursors (stage 1 and 2 hematogones) in the peripheral blood.

    PubMed

    Kurzer, Jason H; Weinberg, Olga K

    2018-05-25

    Differentiating malignant B-lymphoblasts from early benign B cell precursors (hematogones) is a vital component of the diagnosis of B-lymphoblastic leukaemia. It has been previously reported that only late-stage B cell precursors circulate in the peripheral blood. Consequently, flow cytometric detection of cells with immunophenotypic findings similar to earlier stage precursors in the peripheral blood justifiably raises concern for involvement by B-lymphoblastic leukaemia. We report here, however, that benign early B cell precursors can indeed be detected in the peripheral blood, thus complicating the interpretation of flow cytometric findings derived from these sample types. A retrospective search of our collective databases identified 13 cases containing circulating early stage B cell precursors. The patients ranged in age from 15 days to 85 years old. All positive cases demonstrated that the earlier B cell precursors were associated with later stage precursors, a finding that could help differentiate these cells from B-lymphoblastic leukaemia. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  6. Whole-cell fungal transformation of precursors into dyes

    PubMed Central

    2010-01-01

    Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important

  7. CD34(+) Liver Cancer Stem Cells Were Formed by Fusion of Hepatobiliary Stem/Progenitor Cells with Hematopoietic Precursor-Derived Myeloid Intermediates.

    PubMed

    Zeng, Changjun; Zhang, Yanling; Park, Su Cheol; Eun, Jong Ryeol; Nguyen, Ngoc Tue; Tschudy-Seney, Benjamin; Jung, Yong Jin; Theise, Neil D; Zern, Mark A; Duan, Yuyou

    2015-11-01

    A large number of cancer stem cells (CSCs) were identified and characterized; however, the origins and formation of CSCs remain elusive. In this study, we examined the origination of the newly identified CD34(+) liver CSC (LCSC). We found that CD34(+) LCSC coexpressed liver stem cell and myelomonocytic cell markers, showing a mixed phenotype, a combination of hepatobiliary stem/progenitor cells (HSPCs) and myelomonocytic cells. Moreover, human xenografts produced by CD34(+) LCSCs and the parental cells, which CD34(+) LCSC was isolated from, coexpressed liver cancer and myelomonocytic markers, also demonstrating mixed phenotypes. The xenografts and the parental cells secreted albumin demonstrating their hepatocyte origin and also expressed cytokines [interleukin (IL)-1b, IL-6, IL-12A, IL-18, tumor necrosis factor-alpha (TNF-α), and CSF1] and chemokines (IL-8, CCL2, and CCL5). Expression of these cytokines and chemokines responded to the stimuli [interferon-γ (INF-γ), IL-4, and lipopolysaccharide (LPS)]. Furthermore, human xenografts and the parental cells phagocytized Escherichia coli. CD34(+) LCSC coexpressed CD45, demonstrating that its origin appears to be from a hematopoietic precursor. The percentage of cells positive for OV6, CD34, and CD31, presenting the markers of HSPC, hematopoietic, and myelomonocytic cells, increased under treatment of CD34(+) LCSC with a drug. Cytogenetic analysis showed that CD34(+) LCSC contained a greater number of chromosomes. HBV DNA integrations and mutations in CD34(+) LCSC and the parental cells were identical to those in the literature or the database. Thus, these results demonstrated that CD34(+) LCSCs were formed by fusion of HSPC with CD34(+) hematopoietic precursor-derived myeloid intermediates; it appears that this is the first report that human CSCs have been formed by the fusion. Therefore, it represents a significant step toward better understanding of the formation of human CSC and the diverse origins of liver

  8. Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors.

    PubMed

    Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E; Cao, Mengjun; Wu, Yaojiong

    2016-12-01

    : Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. ©AlphaMed Press.

  9. Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors

    PubMed Central

    Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E.; Cao, Mengjun

    2016-01-01

    Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. Significance In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. PMID:27458264

  10. Identification and Characterization of a Dendritic Cell Precursor in Parenchymal Lung Tissue.

    PubMed

    von Garnier, Christophe; Blank, Fabian; Rothen-Rutishauser, Barbara; Goethert, Joachim R; Holt, Patrick G; Stumbles, Philip A; Strickland, Deborah H

    2017-03-01

    The pulmonary parenchymal and mucosal microenvironments are constantly exposed to the external environment and thus require continuous surveillance to maintain steady-state immunological homeostasis. This is achieved by a mobile network of pulmonary dendritic cells (DC) and macrophages (mø) that constantly sample and process microenvironmental antigens into signals that can initiate or dampen inflammation, either locally or after onward migration to draining lymph nodes. The constant steady-state turnover of pulmonary DC and mø requires replenishment from bone marrow precursors; however, the nature of the pulmonary precursor cell (PC) remains unclear, although recent studies suggest that subsets of pulmonary DC may derive from circulating monocytic precursors. In the current study, we describe a population of cells in steady-state mouse lung tissue that has the surface phenotypic and ultrastructural characteristics of a common DC progenitor. Irradiation and reconstitution studies confirmed the bone marrow origins of this PC and showed that it had rapid depletion and reconstitution kinetics that were similar to those of DC, with a 50% repopulation by donor-derived cells by Days 7-9 after reconstitution. This was significantly faster than the rates observed for mø, which showed 50% repopulation by donor-derived cells beyond Days 16-21 after reconstitution. Purified PC gained antigen-presenting function and a cell surface phenotype similar to that of pulmonary DC after maturation in vitro, with light and electron microscopy confirming a myeloid DC morphology. To the best of our knowledge, this is the first study to describe a PC for DC in lung tissue; the findings have implications for the restoration of pulmonary immunological homeostasis after bone marrow transplant.

  11. Enhanced Expansion and Sustained Inductive Function of Skin‐Derived Precursor Cells in Computer‐Controlled Stirred Suspension Bioreactors

    PubMed Central

    Agabalyan, Natacha A.; Borys, Breanna S.; Sparks, Holly D.; Boon, Kathryn; Raharjo, Eko W.; Abbasi, Sepideh; Kallos, Michael S.

    2016-01-01

    Abstract Endogenous dermal stem cells (DSCs) reside in the adult hair follicle mesenchyme and can be isolated and grown in vitro as self‐renewing colonies called skin‐derived precursors (SKPs). Following transplantation into skin, SKPs can generate new dermis and reconstitute the dermal papilla and connective tissue sheath, suggesting they could have important therapeutic value for the treatment of skin disease (alopecia) or injury. Controlled cell culture processes must be developed to efficiently and safely generate sufficient stem cell numbers for clinical use. Compared with static culture, stirred‐suspension bioreactors generated fivefold greater expansion of viable SKPs. SKPs from each condition were able to repopulate the dermal stem cell niche within established hair follicles. Both conditions were also capable of inducing de novo hair follicle formation and exhibited bipotency, reconstituting the dermal papilla and connective tissue sheath, although the efficiency was significantly reduced in bioreactor‐expanded SKPs compared with static conditions. We conclude that automated bioreactor processing could be used to efficiently generate large numbers of autologous DSCs while maintaining their inherent regenerative function. Stem Cells Translational Medicine 2017;6:434–443 PMID:28191777

  12. Cell size control and a cell-intrinsic maturation program in proliferating oligodendrocyte precursor cells.

    PubMed

    Gao, F B; Raff, M

    1997-09-22

    We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by "transition probability" models, may explain the random variability of cell cycle times seen within clonal cell lines in culture.

  13. Cell Size Control and a Cell-intrinsic Maturation Program in Proliferating Oligodendrocyte Precursor Cells

    PubMed Central

    Gao, Fen-Biao; Raff, Martin

    1997-01-01

    We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by “transition probability” models, may explain the random variability of cell cycle times seen within clonal cell lines in culture. PMID:9298991

  14. Characterization of glial-restricted precursors from rhesus monkey embryonic stem cells.

    PubMed

    Chen, Hongwei; Mao, Yu; Wang, Shufen; Li, Bin; Wang, Jinhuan; Li, Jian; Ma, Yuanye

    2015-01-01

    Glial-restricted precursor (GRP) cells, the earliest glial progenitors for both astrocytes and oligodendrocytes, have been derived from embryos and embryonic stem cells (ESC) in rodents. However, knowledge regarding the equivalent cell type in primates is limited due to restrictions imposed by ethics and resources. Here we report successful derivation and characterization of primate GRP cells from rhesus monkey ESC. The purified monkey GRP cells were A 2 B 5 -positive and FGF2-dependent for survival and proliferation. The differentiation assays indicated that they were tri-potential in vitro and bi-potential in vivo . These newly purified GRP cells will help to facilitate understanding of the molecular mechanism of glial development in primates as well as provide a source of therapeutic donor cells for use in neuroregenerative medicine.

  15. [The role of endothelial cells and endothelial precursor cells in angiogenesis].

    PubMed

    Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

    2006-01-01

    Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

  16. The Innate Lymphoid Cell Precursor.

    PubMed

    Ishizuka, Isabel E; Constantinides, Michael G; Gudjonson, Herman; Bendelac, Albert

    2016-05-20

    The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.

  17. The fate of wastewater-derived NDMA precursors in the aquatic environment.

    PubMed

    Pehlivanoglu-Mantas, Elif; Sedlak, David L

    2006-03-01

    To assess the stability of precursors of the chloramine disinfection byproduct N-nitrosodimethylamine (NDMA) under conditions expected in effluent-dominated surface waters, effluent samples from four municipal wastewater treatment plants were subjected to chlorination and chloramination followed by incubation in the presence of inocula derived from activated sludge. Samples subjected to free chlorine disinfection showed lower initial concentrations of NDMA precursors than those that were not chlorinated or were disinfected with pre-formed chloramines. For chloraminated and control (unchlorinated) treatments, the concentration of NDMA precursors decreased by an average of 24% over the 30-day incubation in samples from three of the four facilities. At the fourth facility, where samples were collected on three different days, NDMA precursor concentrations decreased by approximately 80% in one sample and decreased by less than 20% in the other two samples. In contrast to the low reactivity of the NDMA precursors, NDMA disappeared within 30 days under the conditions employed in these experiments. These results and measurements made in an effluent-dominated river suggest that although NDMA may be removed after wastewater effluent is discharged, wastewater-derived NDMA precursors could persist long enough to form significant concentrations of NDMA in drinking water treatment plants that use water originating from sources that are subjected to wastewater effluent discharges.

  18. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Liang; Dong, Chuanming; Department of Anatomy and Neurobiology, The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and weremore » associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.« less

  19. Bleogens: Cactus-Derived Anti-Candida Cysteine-Rich Peptides with Three Different Precursor Arrangements

    PubMed Central

    Loo, Shining; Kam, Antony; Xiao, Tianshu; Tam, James P.

    2017-01-01

    Cysteine-rich peptides (CRPs) play important host-defense roles in plants. However, information concerning CRPs in the Cactaceae (cactus) family is limited, with only a single cactus-derived CRP described to date. Here, we report the identification of 15 novel CRPs with three different precursor architectures, bleogens pB1-15 from Pereskia bleo of the Cactaceae family. By combining proteomic and transcriptomic methods, we showed that the prototype, bleogen pB1, contained 36 amino acid residues, a six-cysteine motif typical of the six-cysteine-hevein-like peptide (6C-HLP) family, and a type I two-domain precursor consisting of an endoplasmic reticulum (ER) and a mature domain. In contrast, the precursors of the other 14 bleogens contained a type II three-domain architecture with a propeptide domain inserted between the ER and the mature bleogen domain. Four of these 14 bleogens display a third type of architecture with a tandemly repeating bleogen domain. A search of the Onekp database revealed that <1% plant species possess three different precursor architectures for the biosynthesis of 6C-HLPs, including Lophophora williamsii, Pereskia aculeate, Portulaca cryptopetala, Portulaca oleracea, Portulaca suffruticosa, and Talinum sp. NMR analysis confirmed that bleogen pB1 has cystine-knot disulfide connectivity as well as a two-beta-sheet and a four-loop structural fold that is similar to other 6C-HLPs. Sequence analysis, structural studies, and in silico modeling revealed that bleogen pB1 has a cation-polar-cation motif, a signature heparin-binding motif that was confirmed by heparin affinity chromatography. Cell-based assays showed that bleogen pB1 is non-toxic to mammalian cells but functions as an anti-Candida peptide. Taken together, our findings provide insight into the occurrence, functions and precursor architectures of CRPs in the cactus family. PMID:29312404

  20. A Subpopulation of Smooth Muscle Cells, Derived from Melanocyte-Competent Precursors, Prevents Patent Ductus Arteriosus

    PubMed Central

    Puig, Isabel; Champeval, Delphine; Kumasaka, Mayuko; Belloir, Elodie; Bonaventure, Jacky; Mark, Manuel; Yamamoto, Hiroaki; Taketo, Mark M.; Choquet, Philippe; Etchevers, Heather C.; Beermann, Friedrich; Delmas, Véronique; Monassier, Laurent; Larue, Lionel

    2013-01-01

    Background Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes. PMID:23382837

  1. Prolactin Stimulates Precursor Cells in the Adult Mouse Hippocampus

    PubMed Central

    Walker, Tara L.; Vukovic, Jana; Koudijs, Margaretha M.; Blackmore, Daniel G.; Mackay, Eirinn W.; Sykes, Alex M.; Overall, Rupert W.; Hamlin, Adam S.; Bartlett, Perry F.

    2012-01-01

    In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory. PMID:22973440

  2. Circulating osteogentic precursor cells in non-hereditary heterotopic ossification.

    PubMed

    Egan, Kevin P; Duque, Gustavo; Keenan, Mary Ann; Pignolo, Robert J

    2018-04-01

    Non-hereditary heterotopic ossification (NHHO) may occur after musculoskeletal trauma, central nervous system (CNS) injury, or surgery. We previously described circulating osteogenic precursor (COP) cells as a bone marrow-derived type 1 collagen + CD45 + subpopulation of mononuclear adherent cells that are able of producing extraskeletal ossification in a murine in vivo implantation assay. In the current study, we performed a tissue analysis of COP cells in NHHO secondary to defined conditions, including traumatic brain injury, spinal cord injury, cerebrovascular accident, trauma without neurologic injury, and joint arthroplasty. All bone specimens revealed the presence of COP cells at 2-14 cells per high power field. COP cells were localized to early fibroproliferative and neovascular lesions of NHHO with evidence for their circulatory status supported by their presence near blood vessels in examined lesions. This study provides the first systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO. These data support that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites, such as those subject to soft tissue injury, and due to their migratory nature, may likely be involved in seeding sites distant to CNS injury. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Brain-Derived Neurotrophic Factor Promotes Vasculature-Associated Migration of Neuronal Precursors toward the Ischemic Striatum

    PubMed Central

    Grade, Sofia; Weng, Yuan C.; Snapyan, Marina; Kriz, Jasna; Malva, João O.; Saghatelyan, Armen

    2013-01-01

    Stroke induces the recruitment of neuronal precursors from the subventricular zone (SVZ) into the ischemic striatum. In injured areas, de-routed neuroblasts use blood vessels as a physical scaffold to their migration, in a process that resembles the constitutive migration seen in the rostral migratory stream (RMS). The molecular mechanism underlying injury-induced vasculature-mediated migration of neuroblasts in the post-stroke striatum remains, however, elusive. Using adult mice we now demonstrate that endothelial cells in the ischemic striatum produce brain-derived neurotrophic factor (BDNF), a neurotrophin that promotes the vasculature-mediated migration of neuronal precursors in the RMS, and that recruited neuroblasts maintain expression of p75NTR, a low-affinity receptor for BDNF. Reactive astrocytes, which are widespread throughout the damaged area, ensheath blood vessels and express TrkB, a high-affinity receptor for BDNF. Despite the absence of BDNF mRNA, we observed strong BDNF immunolabeling in astrocytes, suggesting that these glial cells trap extracellular BDNF. Importantly, this pattern of expression is reminiscent of the adult RMS, where TrkB-expressing astrocytes bind and sequester vasculature-derived BDNF, leading to the entry of migrating cells into the stationary phase. Real-time imaging of cell migration in acute brain slices revealed a direct role for BDNF in promoting the migration of neuroblasts to ischemic areas. We also demonstrated that cells migrating in the ischemic striatum display higher exploratory behavior and longer stationary periods than cells migrating in the RMS. Our findings suggest that the mechanisms involved in the injury-induced vasculature-mediated migration of neuroblasts recapitulate, at least partially, those observed during constitutive migration in the RMS. PMID:23383048

  4. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

    PubMed

    Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

    2017-10-10

    Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

  5. Cardiomyocyte-released factors stimulate oligodendrocyte precursor cells proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kuroda, Mariko; Muramatsu, Rieko; Precursory Research for Embryonic Science and Technology

    The heart produces multiple diffusible factors that are involved in a number of physiological processes, but the action of these factors on the central nervous system is not well understood. In this study, we found that one or more factors released by cardiomyocytes promote oligodendrocyte precursor cell (OPC) proliferation in vitro. Mouse OPCs co-cultured with mouse cardiomyocytes showed higher proliferative ability than OPCs cultured alone. In addition, cardiomyocyte-conditioned media was sufficient to promote OPC proliferation. The phosphorylation of phosphatidylinositol (PI) 3-kinase and extracellular signal-regulated kinase (ERK) in OPCs is necessary for the enhancement of OPC proliferation by cardiomyocyte-conditioned media. These datamore » indicate that heart-derived factors have the ability to directly regulate the function of central nervous system (CNS) cells.« less

  6. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

    PubMed Central

    Wiley, Luke A.; Burnight, Erin R.; DeLuca, Adam P.; Anfinson, Kristin R.; Cranston, Cathryn M.; Kaalberg, Emily E.; Penticoff, Jessica A.; Affatigato, Louisa M.; Mullins, Robert F.; Stone, Edwin M.; Tucker, Budd A.

    2016-01-01

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans. PMID:27471043

  7. Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.

    PubMed

    Kim, Han-Seop; Lee, Jungwoon; Lee, Da Yong; Kim, Young-Dae; Kim, Jae Yun; Lim, Hyung Jin; Lim, Sungmin; Cho, Yee Sook

    2017-06-06

    Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.

    PubMed

    Goto, Hiroaki; Naruto, Takuya; Tanoshima, Reo; Kato, Hiromi; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Fujii, Hisaki; Yokota, Shumpei; Komine, Hiromi

    2009-10-01

    Sensitivity to 10 anticancer drugs was evaluated in 6 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. Authenticity of newly established cell lines was confirmed by genomic fingerprinting. The line YCUB-5R established at relapse was more resistant to 4-hydroperoxy-cyclophosphamide, cytarabine, L-asparaginase, topotecan, fludarabine, and etoposide than YCUB-5 from the same patient at diagnosis. Of the drugs tested, etoposide and SN-38 (irinotecan) showed highest efficacy in the panel, with 50% growth inhibition at 0.22-1.8 microg/ml and 0.57-3.6 ng/ml, respectively. This cell line panel offers an in vitro model for the development of new therapies for childhood BCP-ALL.

  9. Pluripotent stem cell-derived natural killer cells for cancer therapy

    PubMed Central

    Knorr, David A.; Kaufman, Dan S.

    2010-01-01

    Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an accessible, genetically tractable and homogenous starting cell populations to efficiently study human blood cell development. These cell populations provide platforms to develop new cell-based therapies to treat both malignant and non-malignant hematological diseases. Our group has previously demonstrated the ability of hESC-derived hematopoietic precursors to produce functional natural killer (NK) cells as well as an explanation of the underlying mechanism responsible for inefficient development of T and B cells from hESCs. hESCs and iPSCs, which can be reliably engineered in vitro, provide an important new model system to study human lymphocyte development and produce enhanced cell-based therapies with potential to serve as a “universal” source of anti-tumor lymphocytes for novel clinical therapies. This review will focus on the application of hESC-derived NK cells with currently used and novel therapeutics for clinical trials, current barriers to translation, and future applications through genetic engineering approaches. PMID:20801411

  10. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors

    PubMed Central

    Doulatov, Sergei; Vo, Linda T.; Chou, Stephanie S.; Kim, Peter G.; Arora, Natasha; Li, Hu; Hadland, Brandon K.; Bernstein, Irwin D.; Collins, James J.; Zon, Leonard I.; Daley, George Q.

    2013-01-01

    Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells. PMID:24094326

  11. Generation of glucose-responsive, insulin-producing cells from human umbilical cord blood-derived mesenchymal stem cells.

    PubMed

    Prabakar, Kamalaveni R; Domínguez-Bendala, Juan; Molano, R Damaris; Pileggi, Antonello; Villate, Susana; Ricordi, Camillo; Inverardi, Luca

    2012-01-01

    We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge. These data suggest that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.

  12. Tissue-specific differentiation of a circulating CCR9- pDC-like common dendritic cell precursor.

    PubMed

    Schlitzer, Andreas; Heiseke, Alexander F; Einwächter, Henrik; Reindl, Wolfgang; Schiemann, Matthias; Manta, Calin-Petru; See, Peter; Niess, Jan-Hendrik; Suter, Tobias; Ginhoux, Florent; Krug, Anne B

    2012-06-21

    The ontogenic relationship between the common dendritic cell (DC) progenitor (CDP), the committed conventional DC precursor (pre-cDC), and cDC subpopulations in lymphoid and nonlymphoid tissues has been largely unraveled. In contrast, the sequential steps of plasmacytoid DC (pDC) development are less defined, and it is unknown at which developmental stage and location final commitment to the pDC lineage occurs. Here we show that CCR9(-) pDCs from murine BM which enter the circulation and peripheral tissues have a common DC precursor function in vivo in the steady state, in contrast to CCR9(+) pDCs which are terminally differentiated. On adoptive transfer, the fate of CCR9(-) pDC-like precursors is governed by the tissues they enter. In the BM and liver, most transferred CCR9(-) pDC-like precursors differentiate into CCR9(+) pDCs, whereas in peripheral lymphoid organs, lung, and intestine, they additionally give rise to cDCs. CCR9(-) pDC-like precursors which are distinct from pre-cDCs can be generated from the CDP. Thus, CCR9(-) pDC-like cells are novel CDP-derived circulating DC precursors with pDC and cDC potential. Their final differentiation into functionally distinct pDCs and cDCs depends on tissue-specific factors allowing adaptation to local requirements under homeostatic conditions.

  13. Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

    PubMed Central

    Debeb, Bisrat G.; Galat, Vasiliy; Epple-Farmer, Jessica; Iannaccone, Steve; Woodward, Wendy A.; Bader, Michael; Iannaccone, Philip; Binas, Bert

    2009-01-01

    Background The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. Methodology/Principal Findings Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. Conclusions/Significance Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation. PMID:19784378

  14. Intervertebral disc-derived stem cells: implications for regenerative medicine and neural repair.

    PubMed

    Erwin, W Mark; Islam, Diana; Eftekarpour, Eftekhar; Inman, Robert D; Karim, Muhammad Zia; Fehlings, Michael G

    2013-02-01

    An in vitro and in vivo evaluation of intervertebral disc (IVD)-derived stem/progenitor cells. To determine the chondrogenic, adipogenic, osteogenic, and neurogenic differentiation capacity of disc-derived stem/progenitor cells in vitro and neurogenic differentiation in vivo. Tissue repair strategies require a source of appropriate cells that could be used to replace dead or damaged cells and tissues such as stem cells. Here we examined the potential use of IVD-derived stem cells in regenerative medicine approaches and neural repair. Nonchondrodystrophic canine IVD nucleus pulposus (NP) cells were used to generate stem/progenitor cells (NP progenitor cells [NPPCs]) and the NPPCs were differentiated in vitro into chondrogenic, adipogenic, and neurogenic lineages and in vivo into the neurogenic lineage. NPPCs were compared with bone marrow-derived mesenchymal (stromal) stem cells in terms of the expression of stemness genes. The expression of the neural crest marker protein 0 and the Brachyury gene were evaluated in NP cells and NPPCs. NPPCs contain stem/progenitor cells and express "stemness" genes such as Sox2, Oct3/4, Nanog, CD133, Nestin, and neural cell adhesion molecule but differ from mesenchymal (stromal) stem cells in the higher expression of the Nanog gene by NPPCs. NPPCs do not express protein 0 or the Brachyury gene both of which are expressed by the totality of IVD NP cells. The percentage of NPPCs within the IVD is 1% of the total as derived by colony-forming assay. NPPCs are capable of differentiating along chondrogenic, adipogenic, and neurogenic lineages in vitro and into oligodendrocyte, neuron, and astroglial specific precursor cells in vivo within the compact myelin-deficient shiverer mouse. We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.

  15. Thymus-like activities of sulphur derivatives on T-cell differentiation

    PubMed Central

    1977-01-01

    Levamisole and sodium diethyldithiocarbamate can induce in vivo thymocyte differentiation from precursor spleen cells of nu/nu mice and evoke indirect plaque-forming cells in nude mice immunized with sheep red cells. These sulphur drugs induce in thymusless mice the production of a serum factor which transfer in vivo immune enhancement and in vitro thymocyte differentiation. In vivo treatment with sulphur derivative can substitute for an alleged thymice hormone. PMID:188971

  16. [Isolation and Characterization of Multipotent Precursor Cells from Murine Adipose Tissue using a Clinically Approved Cell Separation System].

    PubMed

    Krug, C; Beer, A; Saller, M M; Aszodi, A; Holzbach, T; Giunta, R E; Volkmer, E

    2016-04-01

    Recent studies underscored the clinical potential of adipose-derived multipotent stem-/precursor cells (ASPCs). One of the main hurdles en route to clinical application was to isolate cells without having to perform expansion cultures outside the OR. A new generation of clinically approved, commercially available cell separation systems claims to provide ASPCs ready for application without further expansion cultures. However, it is unclear if the new systems yield sufficient cells of adequate quality for the use in autologous murine models. The aim of this study was to isolate and characterize adipose-derived precursor cells taken from the inguinal fat pat of wistar rats using InGeneron's clinically approved ARC™-cell separation system. We isolated cells from the inguinal fat pad of 3 male Wistar rats according to the manufacturer's protocol. In order to reduce the influence of the atmospheric oxygen on the multipotent precursor cells, one half of the cell suspension was cultivated under hypoxia (2% O2) simulating physiological conditions for ASPCs. As a control, the other half of the cells were cultivated under normoxia (21% O2). Cell surface markers CD90, CD29, CD45 and CD11b/c were analyzed by FACS, and osteogenic and adipogenic differentiation of the ASPCs was performed. Finally, cellular growth characteristics were assessed by evaluation of the cumulative population doublings and CFU assay, and metabolic activity was evaluated by WST-1 assay. Processing time was 90 (± 12) min. 1 g of adipose tissue yielded approximately 60 000 plastic adhering cells. Both groups showed a high expression of the mesenchymal stem cell markers CD90 and CD29 while they were negative for the leucocyte markers CD45 and CD11b/c. A strong osteogenic differentiation and a sufficient adipogenic differentiation potential was proven for all ASPCs. Under hypoxia, ASPCs showed increased proliferation characteristics and CFU efficiency as well as a significantly increased metabolic

  17. Methods for conversion of lignocellulosic-derived products to transportation fuel precursors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lilga, Michael A.; Padmaperuma, Asanga B.

    2017-10-03

    Methods are disclosed for converting a biomass-derived product containing levulinic acid and/or gamma-valerolactone to a transportation fuel precursor product containing diesel like hydrocarbons. These methods are expected to produce fuel products at a reduced cost relative to conventional approaches.

  18. Aniline Is an Inducer, and Not a Precursor, for Indole Derivatives in Rubrivivax benzoatilyticus JA2

    PubMed Central

    Mohammed, Mujahid; Ch, Sasikala; Ch, Ramana V.

    2014-01-01

    Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway. PMID:24533057

  19. Aniline is an inducer, and not a precursor, for indole derivatives in Rubrivivax benzoatilyticus JA2.

    PubMed

    Mujahid, Mohammed; Sasikala, Ch; Ramana, Ch V

    2014-01-01

    Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway.

  20. Non-PGM cathode catalysts for fuel cell application derived from heat treated heteroatomic amines precursors

    DOEpatents

    Serov, Alexey; Halevi, Barr; Artyushkova, Kateryna; Atanassov, Plamen B; Martinez, Ulises A

    2017-04-25

    A method of preparing M-N--C catalysts utilizing a sacrificial support approach and inexpensive and readily available polymer precursors as the source of nitrogen and carbon is disclosed. Exemplary polymer precursors include non-porphyrin precursors with no initial catalytic activity. Examples of suitable non-catalytic non-porphyrin precursors include, but are not necessarily limited to low molecular weight precursors that form complexes with iron such as 4-aminoantipirine, phenylenediamine, hydroxysuccinimide, ethanolamine, and the like.

  1. Adult subependymal neural precursors, but not differentiated cells, undergo rapid cathodal migration in the presence of direct current electric fields.

    PubMed

    Babona-Pilipos, Robart; Droujinine, Ilia A; Popovic, Milos R; Morshead, Cindi M

    2011-01-01

    The existence of neural stem and progenitor cells (together termed neural precursor cells) in the adult mammalian brain has sparked great interest in utilizing these cells for regenerative medicine strategies. Endogenous neural precursors within the adult forebrain subependyma can be activated following injury, resulting in their proliferation and migration toward lesion sites where they differentiate into neural cells. The administration of growth factors and immunomodulatory agents following injury augments this activation and has been shown to result in behavioural functional recovery following stroke. With the goal of enhancing neural precursor migration to facilitate the repair process we report that externally applied direct current electric fields induce rapid and directed cathodal migration of pure populations of undifferentiated adult subependyma-derived neural precursors. Using time-lapse imaging microscopy in vitro we performed an extensive single-cell kinematic analysis demonstrating that this galvanotactic phenomenon is a feature of undifferentiated precursors, and not differentiated phenotypes. Moreover, we have shown that the migratory response of the neural precursors is a direct effect of the electric field and not due to chemotactic gradients. We also identified that epidermal growth factor receptor (EGFR) signaling plays a role in the galvanotactic response as blocking EGFR significantly attenuates the migratory behaviour. These findings suggest direct current electric fields may be implemented in endogenous repair paradigms to promote migration and tissue repair following neurotrauma.

  2. Engineering Robust and Functional Vascular Networks in Vivo with Human Adult and Cord Blood-Derived Progenitor Cells

    DTIC Science & Technology

    2008-12-01

    for other sources of ECs such as those derived from embryonic and adult progenitor cells ( Rafii ; Lyden 2003). For example, human ES-derived...functional endothelial precursors. Blood, 95, 952-958. Rafii , S., and D. Lyden, 2003: Therapeutic stem and progenitor cell transplantation for

  3. Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion.

    PubMed

    Konig, Niclas; Trolle, Carl; Kapuralin, Katarina; Adameyko, Igor; Mitrecic, Dinko; Aldskogius, Hakan; Shortland, Peter J; Kozlova, Elena N

    2017-01-01

    Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second-order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)-derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3-L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  4. Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

    PubMed Central

    Fagoonee, Sharmila; Famulari, Elvira Smeralda; Silengo, Lorenzo; Tolosano, Emanuela; Altruda, Fiorella

    2015-01-01

    One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine. PMID:26323094

  5. GH Mediates Exercise-Dependent Activation of SVZ Neural Precursor Cells in Aged Mice

    PubMed Central

    Blackmore, Daniel G.; Vukovic, Jana; Waters, Michael J.; Bartlett, Perry F.

    2012-01-01

    Here we demonstrate, both in vivo and in vitro, that growth hormone (GH) mediates precursor cell activation in the subventricular zone (SVZ) of the aged (12-month-old) brain following exercise, and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast, no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury, we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely, infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation. PMID:23209615

  6. Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

    PubMed

    Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

    2016-10-18

    Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

  7. The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis.

    PubMed

    Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L; Wang, Yanlin

    2014-08-01

    Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, and they proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. As chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly, the kidney of CXCR6 knockout mice accumulated fewer bone marrow-derived fibroblasts in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type (WT) mice. CXCR6 deficiency inhibited total collagen deposition and suppressed the expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, WT mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with WT mice engrafted with CXCR6(+/+) bone marrow cells. Transplant of WT bone marrow into CXCR6(-/-) recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may have important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

  8. Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells.

    PubMed

    Soundara Rajan, Thangavelu; Giacoppo, Sabrina; Scionti, Domenico; Diomede, Francesca; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2017-06-01

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 118: 1531-1546, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells

    PubMed Central

    Trötzmüller, Martin; Hinteregger, Barbara; Leko, Petra; Wieser, Beatrix I.; Grasmann, Gabriele; Bertsch, Alexandra L.; Züllig, Thomas; Stacher, Elvira; Valli, Alessandro; Prassl, Ruth; Olschewski, Andrea; Harris, Adrian L.; Köfeler, Harald C.; Olschewski, Horst; Hrzenjak, Andelko

    2018-01-01

    Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M–dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation. PMID:29844165

  10. Stem cell-derived kidney cells and organoids: Recent breakthroughs and emerging applications.

    PubMed

    Chuah, Jacqueline Kai Chin; Zink, Daniele

    The global rise in the numbers of kidney patients and the shortage in transplantable organs have led to an increasing interest in kidney-specific regenerative therapies, renal disease modelling and bioartificial kidneys. Sources for large quantities of high-quality renal cells and tissues would be required, also for applications in in vitro platforms for compound safety and efficacy screening. Stem cell-based approaches for the generation of renal-like cells and tissues would be most attractive, but such methods were not available until recently. This situation has drastically changed since 2013, and various protocols for the generation of renal-like cells and precursors from pluripotent stem cells (PSC) have been established. The most recent breakthroughs were related to the establishment of various protocols for the generation of PSC-derived kidney organoids. In combination with recent advances in genome editing, bioprinting and the establishment of predictive renal screening platforms this results in exciting new possibilities. This review will give a comprehensive overview over current PSC-based protocols for the generation of renal-like cells, precursors and organoids, and their current and potential applications in regenerative medicine, compound screening, disease modelling and bioartificial organs. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Improved Single-Source Precursors for Solar-Cell Absorbers

    NASA Technical Reports Server (NTRS)

    Banger, Kulbinder K.; Harris, Jerry; Hepp, Aloysius

    2007-01-01

    Improved single-source precursor compounds have been invented for use in spray chemical vapor deposition (spray CVD) of chalcopyrite semiconductor absorber layers of thin-film cells. A "single-source precursor compound" is a single molecular compound that contains all the required elements, which when used under the spray CVD conditions, thermally decomposes to form CuIn(x)Ga(1-x)S(y)Se(2-y).

  12. Vinpocetine inhibits oligodendroglial precursor cell differentiation.

    PubMed

    Torres, Klintsy Julieta; Göttle, Peter; Kremer, David; Rivera, Jose Flores; Aguirre-Cruz, Lucinda; Corona, Teresa; Hartung, Hans-Peter; Küry, Patrick

    2012-01-01

    In multiple sclerosis during periods of remission a limited degree of myelin repair can be observed mediated by oligodendroglial precursor cells. Phosphodiesterase inhibitors act as anti-inflammatory agents and might hold promise for future multiple sclerosis treatment. To investigate whether phosphodiesterase inhibitors could also influence myelin repair. We stimulated primary oligodendroglial precursor cells with cilostazol, rolipram and vinpocetine and assessed their effects on repair related cellular processes. We found that vinpocetine exerted a strong negative effect on myelin expression while cilostazol and rolipram did not show such effects. In addition, vinpocetine decreased morphological complexities suggesting an overall negative impact on oligodendroglial cell maturation. We provide evidence that this is not mediated via a blockade of phosphodiesterase-1 but rather by inhibition of IĸB kinase. These findings suggest that vinpocetine via IĸB inhibition exerts a strong negative impact on oligodendroglial cell maturation and may therefore provide the rationale to restrict its application during periods of remission in multiple sclerosis patients. This is of particular interest since vinpocetine is widely used as a health supplement thought to act as a cognitive and memory enhancer for healthy people and patients with neurological or muscle diseases. Copyright © 2012 S. Karger AG, Basel.

  13. The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

    PubMed

    Leithner, Katharina; Triebl, Alexander; Trötzmüller, Martin; Hinteregger, Barbara; Leko, Petra; Wieser, Beatrix I; Grasmann, Gabriele; Bertsch, Alexandra L; Züllig, Thomas; Stacher, Elvira; Valli, Alessandro; Prassl, Ruth; Olschewski, Andrea; Harris, Adrian L; Köfeler, Harald C; Olschewski, Horst; Hrzenjak, Andelko

    2018-06-12

    Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M ( PCK2 ), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation. Copyright © 2018 the Author(s). Published by PNAS.

  14. Coordinated Regulation of Niche and Stem Cell Precursors by Hormonal Signaling

    PubMed Central

    Gancz, Dana; Lengil, Tamar; Gilboa, Lilach

    2011-01-01

    Stem cells and their niches constitute units that act cooperatively to achieve adult body homeostasis. How such units form and whether stem cell and niche precursors might be coordinated already during organogenesis are unknown. In fruit flies, primordial germ cells (PGCs), the precursors of germ line stem cells (GSCs), and somatic niche precursors develop within the larval ovary. Together they form the 16–20 GSC units of the adult ovary. We show that ecdysone receptors are required to coordinate the development of niche and GSC precursors. At early third instar, ecdysone receptors repress precocious differentiation of both niches and PGCs. Early repression is required for correct morphogenesis of the ovary and for protecting future GSCs from differentiation. At mid-third instar, ecdysone signaling is required for niche formation. Finally, and concurrent with the initiation of wandering behavior, ecdysone signaling initiates PGC differentiation by allowing the expression of the differentiation gene bag of marbles in PGCs that are not protected by the newly formed niches. All the ovarian functions of ecdysone receptors are mediated through early repression, and late activation, of the ecdysone target gene broad. These results show that, similar to mammals, a brain-gland-gonad axis controls the initiation of oogenesis in insects. They further exemplify how a physiological cue coordinates the formation of a stem cell unit within an organ: it is required for niche establishment and to ensure that precursor cells to adult stem cells remain undifferentiated until the niches can accommodate them. Similar principles might govern the formation of additional stem cell units during organogenesis. PMID:22131903

  15. The molecular characterization of porcine egg precursor cells

    PubMed Central

    Tsai, Te-Sha; Johnson, Jacqueline; White, Yvonne; John, Justin C.

    2017-01-01

    Female-factor infertility can be caused by poor oocyte quality and depleted ovarian reserves. Egg precursor cells (EPCs), isolated from the ovarian cortex, have the potential to be used to overcome female infertility. We aimed to define the origins of EPCs by analyzing their gene expression profiles and mtDNA content using a mini-pig model. We characterized FAC-sorted DDX4+-derived porcine EPCs by performing RNA-sequencing and determined that they utilize pathways important for cell cycle and proliferation, which supports the existence of adult mitotically active oogonial cells. Expression of the pluripotent markers Sox2 and Oct4, and the primitive germ cell markers Blimp1 and Stella were not detected. However, Nanog and Ddx4 were expressed, as were the primitive germ cell markers Fragilis, c-Kit and Tert. Moreover, porcine EPCs expressed self-renewal and proliferation markers including Myc, Esrrb, Id2, Klf4, Klf5, Stat3, Fgfr1, Fgfr2 and Il6st. The presence of Zp1, Zp2, Zp3 and Nobox were not detected, indicating that porcine EPCs are not indicative of mature primordial oocytes. We performed mitochondrial DNA Next Generation Sequencing and determined that one mtDNA variant harbored by EPCs was present in oocytes, preimplantation embryos and somatic tissues over three generations in our mini-pig model indicating the potential germline origin of EPCs. PMID:28969006

  16. The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis

    PubMed Central

    Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L.; Wang, Yanlin

    2014-01-01

    Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. Since chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly fewer bone marrow-derived fibroblasts accumulated in the kidney of CXCR6 knockout mice in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type mice. CXCR6 deficiency inhibited total collagen deposition and suppressed expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, wild type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Transplant of wild type bone marrow into CXCR6−/− recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may play important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis. PMID:24646857

  17. Requirement of zebrafish pcdh10a and pcdh10b in melanocyte precursor migration.

    PubMed

    Williams, Jason S; Hsu, Jessica Y; Rossi, Christy Cortez; Artinger, Kristin Bruk

    2018-03-29

    Melanocytes derive from neural crest cells, which are a highly migratory population of cells that play an important role in pigmentation of the skin and epidermal appendages. In most vertebrates, melanocyte precursor cells migrate solely along the dorsolateral pathway to populate the skin. However, zebrafish melanocyte precursors also migrate along the ventromedial pathway, in route to the yolk, where they interact with other neural crest derivative populations. Here, we demonstrate the requirement for zebrafish paralogs pcdh10a and pcdh10b in zebrafish melanocyte precursor migration. pcdh10a and pcdh10b are expressed in a subset of melanocyte precursor and somatic cells respectively, and knockdown and TALEN mediated gene disruption of pcdh10a results in aberrant migration of melanocyte precursors resulting in fully melanized melanocytes that differentiate precociously in the ventromedial pathway. Live cell imaging analysis demonstrates that loss of pchd10a results in a reduction of directed cell migration of melanocyte precursors, caused by both increased adhesion and a loss of cell-cell contact with other migratory neural crest cells. Also, we determined that the paralog pcdh10b is upregulated and can compensate for the genetic loss of pcdh10a. Disruption of pcdh10b alone by CRISPR mutagenesis results in somite defects, while the loss of both paralogs results in enhanced migratory melanocyte precursor phenotype and embryonic lethality. These results reveal a novel role for pcdh10a and pcdh10b in zebrafish melanocyte precursor migration and suggest that pcdh10 paralogs potentially interact for proper transient migration along the ventromedial pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation.

    PubMed

    Baldassarro, Vito A; Marchesini, Alessandra; Giardino, Luciana; Calzà, Laura

    2017-07-01

    Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

    PubMed

    Diaz-Cueto, Laura; Arechavaleta-Velasco, Fabian; Diaz-Arizaga, Adriana; Dominguez-Lopez, Pablo; Robles-Flores, Martha

    2012-07-01

    Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

  20. Influence of calcium precursors on the morphology and crystallinity of sol gel-derived hydroxyapatite nanoparticles

    NASA Astrophysics Data System (ADS)

    Vijayalakshmi Natarajan, U.; Rajeswari, S.

    2008-10-01

    Nanosized hydroxyapatite (HAP) particles were prepared by sol-gel method from the water-based solution of calcium and phosphorus precursor. In this study, two calcium precursors such as calcium nitrate tetrahydrate and calcium acetate were chosen as calcium precursors. The influence of aging period, pH, viscosity and sintering temperature on crystallinity and morphology of the HAP particles were investigated for the two calcium precursors with triethyl phosphate precursor. The morphology of nano-HAP towards phosphorous precursor was dependent on the type of calcium precursor used. The HAP prepared from calcium nitrate and triethyl phosphate was spherically shaped whereas the one from calcium acetate was found to be fibrous in structure. Both HAPs were stable up to 1200 °C and their crystallinity increased with respect to the sintering temperature. The obtained sample was characterized through X-ray diffraction (XRD), P 31 nuclear magnetic resonance (NMR), scanning electronic microscopy (SEM) and TEM analysis. The sol derived from the optimized aging period for the two different calcium precursors was coated on 316L stainless-steel (SS) implant and its corrosion resistivity during long-term implantation was studied by cyclic polarization in Ringer's solution. Both HAPs have their own desirable qualities and were found to be corrosion resistive.

  1. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.

    PubMed

    Dubois, Nicole C; Craft, April M; Sharma, Parveen; Elliott, David A; Stanley, Edouard G; Elefanty, Andrew G; Gramolini, Anthony; Keller, Gordon

    2011-10-23

    To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.

  2. Reduced Osteogenesis of Human Osteogenic Precursors' Cells Cultured in the Random Positioning Machine

    NASA Astrophysics Data System (ADS)

    Gershovich, J. G.; Buravkova, L. B.

    2008-06-01

    Recent studies have shown that simulated microgravity (SMG) results in altered proliferation and differentiation not only osteoblasts but also affects on osteogenic capacity of mesenchymal stem cells (MSCs) from various sources. For present study we used system that simulates effects of microgravity produced by the Random Positioning Machine (RPM). Cultured MCSs from human bone marrow and human osteoblasts (OBs) were exposed to SMG at RPM for 10-40 days. Induced osteogenesis of these progenitor cells was compared with the appropriate static (1g) and dynamic (horizontal shaker) controls. Clinorotated OBs and MSCs showed proliferation rate lower than static and dynamic control groups of cells in the early terms of SMG. Significant reduction of ALP activity was detected after 10 days of clinorotation of MSCs. There was no such dramatic difference in ALP activity of MSCs derived cells between SMG and control groups after 20 days of clinorotation but the expression of ALP was still reduced. However, virtually no matrix mineralization was found in OBs cultured under SMG conditions in the presence of differentiation stimuli. The similar effect was observed when we assayed matrix calcification of MSCs derived cultures. Thus, our results confirm low gravity mediated reduction of osteogenesis of different osteogenic precursors' cells and can clarify the mechanisms of bone loss during spaceflight.

  3. Deactivation of wastewater-derived N-nitrosodimethylamine precursors with chlorine dioxide oxidation and the effect of pH.

    PubMed

    Uzun, Habibullah; Kim, Daekyun; Karanfil, Tanju

    2018-09-01

    In this study, the effect of chlorine dioxide (ClO 2 ) oxidation on the deactivation of wastewater (WW)-derived N-nitrosodimethylamine (NDMA) precursors was investigated under various conditions (i.e., ClO 2 application pH, dose and contact time). At pH 6.0, decreases in NDMA formation potentials (FPs) or occurrences (under uniform formation conditions [UFC]) were relatively low (<25%) with ClO 2 oxidation regardless of WW-impact. A negative removal was also observed after ClO 2 oxidation in some of the non-impacted waters. However, NDMA FP removals were significant (up to ~85%) under the same oxidation conditions in WW-impacted waters at pH 7.8. This indicates that the majority of WW-derived NDMA precursors can be deactivated with ClO 2 oxidation above neutral pH. This was attributed to the better oxidative reaction of ClO 2 with amines that have lone pair electrons to be shared at higher oxidation pH conditions. In addition, relatively short oxidation periods with ClO 2 (i.e., ≤10 min) or low Ct (concentration × time, ~10 mg ∗ min/L) values were sufficient for the deactivation of WW-derived NDMA precursors. ClO 2 oxidation was effective in freshly WW-impacted waters. Natural attenuation processes (e.g., sorption, biodegradation, etc.) can change the reactivity of WW-derived NDMA precursors for oxidation with ClO 2 . The effect of ClO 2 on the removal of THM precursors was low (<25%) and independent of oxidation conditions. Given the low formation of regulated THMs and HAAs, ClO 2 oxidation presents a viable option for the simultaneous control of NDMA and regulated DBP formation during water treatment, especially for utilities treating WW-impacted water sources. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Meninges harbor cells expressing neural precursor markers during development and adulthood.

    PubMed

    Bifari, Francesco; Berton, Valeria; Pino, Annachiara; Kusalo, Marijana; Malpeli, Giorgio; Di Chio, Marzia; Bersan, Emanuela; Amato, Eliana; Scarpa, Aldo; Krampera, Mauro; Fumagalli, Guido; Decimo, Ilaria

    2015-01-01

    Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined. In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: (i) are a highly proliferating tissue; (ii) host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and (iii) are enriched in extracellular matrix components (e.g., fractones) known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood.

  5. Meninges harbor cells expressing neural precursor markers during development and adulthood

    PubMed Central

    Bifari, Francesco; Berton, Valeria; Pino, Annachiara; Kusalo, Marijana; Malpeli, Giorgio; Di Chio, Marzia; Bersan, Emanuela; Amato, Eliana; Scarpa, Aldo; Krampera, Mauro; Fumagalli, Guido; Decimo, Ilaria

    2015-01-01

    Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined. In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: (i) are a highly proliferating tissue; (ii) host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and (iii) are enriched in extracellular matrix components (e.g., fractones) known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood. PMID:26483637

  6. Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors.

    PubMed

    Surrao, Denver C; Boon, Kathryn; Borys, Breanna; Sinha, Sarthak; Kumar, Ranjan; Biernaskie, Jeff; Kallos, Michael S

    2016-12-01

    Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an

  7. Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip.

    PubMed

    Johnston, Adam P W; Yuzwa, Scott A; Carr, Matthew J; Mahmud, Neemat; Storer, Mekayla A; Krause, Matthew P; Jones, Karen; Paul, Smitha; Kaplan, David R; Miller, Freda D

    2016-10-06

    Adult mammals have lost multi-tissue regenerative capacity, except for the distal digit, which is able to regenerate via mechanisms that remain largely unknown. Here, we show that, after adult mouse distal digit removal, nerve-associated Schwann cell precursors (SCPs) dedifferentiate and secrete growth factors that promote expansion of the blastema and digit regeneration. When SCPs were dysregulated or ablated, mesenchymal precursor proliferation in the blastema was decreased and nail and bone regeneration were impaired. Transplantation of exogenous SCPs rescued these regeneration defects. We found that SCPs secrete factors that promote self-renewal of mesenchymal precursors, and we used transcriptomic and proteomic analysis to define candidate factors. Two of these, oncostatin M (OSM) and platelet-derived growth factor AA (PDGF-AA), are made by SCPs in the regenerating digit and rescued the deficits in regeneration caused by loss of SCPs. As all peripheral tissues contain nerves, these results could have broad implications for mammalian tissue repair and regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Loss of T cell precursors after spaceflight and exposure to vector-averaged gravity

    NASA Technical Reports Server (NTRS)

    Woods, Chris C.; Banks, Krista E.; Gruener, Raphael; DeLuca, Dominick

    2003-01-01

    Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. Under both conditions, the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4+, CD8+, and CD4+CD8+ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4-CD8- T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat to produce a vector-averaged microgravity-like environment. The block in T cell development appeared to occur between the pre-T cell and CD4+CD8+ T cell stage. These data indicate that gravity plays a decisive role in the development of T cells.

  9. Biliary Tree Stem Cells, Precursors to Pancreatic Committed Progenitors: Evidence for Possible Life-long Pancreatic Organogenesis

    PubMed Central

    Wang, Yunfang; Lanzoni, Giacomo; Carpino, Guido; Cui, Cai-Bin; Dominguez-Bendala, Juan; Wauthier, Eliane; Cardinale, Vincenzo; Oikawa, Tsunekazu; Pileggi, Antonello; Gerber, David; Furth, Mark E.; Alvaro, Domenico; Gaudio, Eugenio; Inverardi, Luca; Reid, Lola M.

    2013-01-01

    Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG,OCT4,SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9,SOX17,PDX1,LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3,MUC6,insulin). Radial-axis lineages start in PBGs near the ducts’ fibromuscular layers with stem cells and end at the ducts’ lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota’s Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only ∼8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas’ committed progenitors. Both could be driven by 3-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immuno-compromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis. PMID

  10. Neurotoxicity of a Fragment of the Amyloid Precursor Associated with Alzheimer's Disease

    NASA Astrophysics Data System (ADS)

    Yankner, Bruce A.; Dawes, Linda R.; Fisher, Shannon; Villa-Komaroff, Lydia; Oster-Granite, Mary Lou; Neve, Rachael L.

    1989-07-01

    Amyloid deposition in senile plaques and the cerebral vasculature is a marker of Alzheimer's disease. Whether amyloid itself contributes to the neurodegenerative process or is simply a by-product of that process is unknown. Pheochromocytoma (PC12) and fibroblast (NIH 3T3) cell lines were transfected with portions of the gene for the human amyloid precursor protein. Stable PC12 cell transfectants expressing a specific amyloid-containing fragment of the precursor protein gradually degenerated when induced to differentiate into neuronal cells with nerve growth factor. Conditioned medium from these cells was toxic to neurons in primary hippocampal cultures, and the toxic agent could be removed by immunoabsorption with an antibody directed against the amyloid polypeptide. Thus, a peptide derived from the amyloid precursor may be neurotoxic.

  11. Lower Oncogenic Potential of Human Mesenchymal Stem Cells Derived from Cord Blood Compared to Induced Pluripotent Stem Cells

    PubMed Central

    Foroutan, T.; Najmi, M.; Kazemi, N.; Hasanlou, M.; Pedram, A.

    2015-01-01

    Background: In regenerative medicine, use of each of the mesenchymal stem cells derived from bone marrow, cord blood, and adipose tissue, has several cons and pros. Mesenchymal stem cells derived from cord blood have been considered the best source for precursor transplantation. Direct reprogramming of a somatic cell into induced pluripotent stem cells by over-expression of 6 transcription factors Oct4, Sox2, Klf4, lin28, Nanog, and c-Myc has great potential for regenerative medicine, eliminating the ethical issues of embryonic stem cells and the rejection problems of using non-autologous cells. Objective: To compare reprogramming and pluripotent markers OCT4, Sox-2, c-Myc, Klf4, Nanog, and lin28 in mesenchymal stem cells derived from cord blood and induced pluripotent stem cells. Methods: We analyzed the expression level of OCT4, Sox-2, c-Myc, Klf4, Nanog and lin28 genes in human mesenchymal stem cells derived from cord blood and induced pluripotent stem cells by cell culture and RT-PCR. Results: The expression level of pluripotent genes OCT4 and Sox-2, Nanog and lin28 in mesenchymal stem cells derived from cord blood were significantly higher than those in induced pluripotent stem cells. In contrast to OCT-4A and Sox-2, Nanog and lin28, the expression level of oncogenic factors c-Myc and Klf4 were significantly higher in induced pluripotent stem cells than in mesenchymal stem cells derived from cord blood. Conclusion: It could be concluded that mesenchymal stem cells derived from human cord blood have lower oncogenic potential compared to induced pluripotent stem cells. PMID:26306155

  12. Signaling through three chemokine receptors triggers the migration of transplanted neural precursor cells in a model of multiple sclerosis.

    PubMed

    Cohen, Mikhal E; Fainstein, Nina; Lavon, Iris; Ben-Hur, Tamir

    2014-09-01

    Multiple sclerosis (MS) is a multifocal disease, and precursor cells need to migrate into the multiple lesions in order to exert their therapeutic effects. Therefore, cell migration is a crucial element in regenerative processes in MS, dictating the route of delivery, when cell transplantation is considered. We have previously shown that inflammation triggers migration of multi-potential neural precursor cells (NPCs) into the white matter of experimental autoimmune encephalomyelitis (EAE) rodents, a widely used model of MS. Here we investigated the molecular basis of this attraction. NPCs were grown from E13 embryonic mouse brains and transplanted into the lateral cerebral ventricles of EAE mice. Transplanted NPC migration was directed by three tissue-derived chemokines. Stromal cell-derived factor-1α, monocyte chemo-attractant protein-1 and hepatocyte growth factor were expressed in the EAE brain and specifically in microglia and astrocytes. Their cognate receptors, CXCR4, CCR2 or c-Met were constitutively expressed on NPCs. Selective blockage of CXCR4, CCR2 or c-Met partially inhibited NPC migration in EAE brains. Blocking all three receptors had an additive effect and resulted in profound inhibition of NPC migration, as compared to extensive migration of control NPCs. The inflammation-triggered NPC migration into white matter tracts was dependent on a motile NPC phenotype. Specifically, depriving NPCs from epidermal growth factor (EGF) prevented the induction of glial commitment and a motile phenotype (as indicated by an in vitro motility assay), hampering their response to neuroinflammation. In conclusion, signaling via three chemokine systems accounts for most of the inflammation-induced, tissue-derived attraction of transplanted NPCs into white matter tracts during EAE. Copyright © 2014. Published by Elsevier B.V.

  13. Msx genes define a population of mural cell precursors required for head blood vessel maturation.

    PubMed

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

    2011-07-01

    Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

  14. CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

    PubMed

    Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

    2012-03-01

    Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in

  15. Distinct Mechanisms Produce Functionally Complementary Actions of Neuropeptides that are Structurally Related but Derived from Different Precursors

    PubMed Central

    Vilim, F.S.; Sasaki, K.; Rybak, J.; Alexeeva, V.; Cropper, E.; Jing, J.; Orekhova, I.V.; Brezina, V.; Price, D.; Romanova, E.V.; Rubakhin, S.S.; Hatcher, N.; Sweedler, J.V.; Weiss, K.R.

    2010-01-01

    Many bioactive neuropeptides containing RFamide at their C-terminus have been described in both invertebrates and vertebrates. To obtain insight into the functional logic of RFamide signaling, we investigate it here in the feeding system of Aplysia. We focus on the expression, localization, and actions of two families of RFamide peptides, the FRFamides and FMRFamide, in the central neuronal circuitry and the peripheral musculature that generate the feeding movements. We describe the cloning of the FRFamide precursor protein and show that the FRFamides and FMRFamide are derived from different precursors. We map the expression of the FRFamide and FMRFamide precursors in the feeding circuitry using in-situ hybridization and immunostaining, and confirm proteolytic processing of the FRFamide precursor by mass spectrometry. We show that the two precursors are expressed in different populations of sensory neurons in the feeding system. In a representative feeding muscle, we demonstrate the presence of both FRFamides and FMRFamide and their release, probably from the processes of the sensory neurons in the muscle. Both centrally and in the periphery, the FRFamides and FMRFamide act in distinct ways, apparently through distinct mechanisms, that nevertheless, from an overall functional perspective, their actions are complementary. Together, the FRFamides and FMRFamide convert feeding motor programs from ingestive to egestive, and depress feeding muscle contractions. We conclude that these structurally related peptides, even though derived from different precursors, expressed in different neurons, and acting through different mechanisms, remain related to each other in the functional roles that they play in the system. PMID:20053896

  16. Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia

    PubMed Central

    Qazi, Sanjive; Cely, Ingrid; Sahin, Kazim; Shahidzadeh, Anoush; Ozercan, Ibrahim; Yin, Qian; Gaynon, Paul; Termuhlen, Amanda; Cheng, Jianjun

    2013-01-01

    We report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)–dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL. PMID:23568490

  17. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kanakura, Y.; Kuriu, A.; Waki, N.

    Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. Large mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas medium and small mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared,more » suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast.« less

  18. CLONING AND CHARACTERIZATION OF OSTEOCLAST PRECURSORS FROM THE RAW264.7 CELL LINE

    PubMed Central

    Cuetara, Bethany L. V.; Crotti, Tania N.; O'Donoghue, Anthony J.

    2006-01-01

    SUMMARY Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell culture, which are poorly suited to molecular and transgene studies due to the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP) positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP positive multinuclear cells. Clones capable of forming large TRAP positive multinuclear cells also expressed β3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation. PMID:16948499

  19. Three-dimensional hydrogel scaffolds facilitate in vitro self-renewal of human skin-derived precursors.

    PubMed

    Wang, Xinyue; Liu, Shu; Zhao, Qian; Li, Na; Zhang, Huishan; Zhang, Xudong; Lei, Xiaohua; Zhao, Huashan; Deng, Zhili; Qiao, Jingqiao; Cao, Yujing; Ning, Lina; Liu, Shuang; Duan, Enkui

    2014-07-01

    Skin-derived precursors (SKPs) are multipotent cells with dermal stem cell properties. These easily available cells possess the capacity to reconstitute the skin in vivo, as well as a broader differentiation potential in vitro, which endows them with great prospects in regenerative medicine. However, the present authors' group and others previously found that adult human SKPs (hSKPs) expanded deficiently in vitro, which largely counteracted their research and practical values. Taking the physiological micro-environment of hSKPs into consideration, the authors sought to establish a hydrogel scaffold-based three-dimensional (3-D) culture system for hSKPs in the present study. After comparing their morphology, growth characteristics, signature gene expression and differentiation potential in different hydrogels, the present authors found that a chemically defined hyaluronic acid and denatured collagen-based hydrogel system that mimicked the natural niche of hSKPs in the dermis could alleviate hSKP senescence, support hSKP proliferation as spheres, while largely retaining their properties and potential. This study suggested that recapitulating the in vivo stem cell niche by providing them with 3-D extracellular matrix environments could help them achieve better self-renewal in vitro. In addition, the animal-origin-free and biocompatible 3-D hydrogel system will certainly benefit fundamental research and clinical applications of hSKPs in the near future. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  20. Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities

    PubMed Central

    Bain, Calum C.; Hawley, Catherine A.; Garner, Hannah; Scott, Charlotte L.; Schridde, Anika; Steers, Nicholas J.; Mack, Matthias; Joshi, Anagha; Guilliams, Martin; Mowat, Allan Mc I.; Geissmann, Frederic; Jenkins, Stephen J.

    2016-01-01

    Peritoneal macrophages are one of the most studied macrophage populations in the body, yet the composition, developmental origin and mechanisms governing the maintenance of this compartment are controversial. Here we show resident F4/80hiGATA6+ macrophages are long-lived, undergo non-stochastic self-renewal and retain cells of embryonic origin for at least 4 months in mice. However, Ly6C+ monocytes constitutively enter the peritoneal cavity in a CCR2-dependent manner, where they mature into short-lived F4/80loMHCII+ cells that act, in part, as precursors of F4/80hiGATA6+ macrophages. Notably, monocyte-derived F4/80hi macrophages eventually displace the embryonic population with age in a process that is highly gender dependent and not due to proliferative exhaustion of the incumbent embryonic population, despite the greater proliferative activity of newly recruited cells. Furthermore, although monocyte-derived cells acquire key characteristics of the embryonic population, expression of Tim4 was impaired, leading to cumulative changes in the population with age. PMID:27292029

  1. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

    DOE PAGES

    Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin; ...

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen asmore » a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.« less

  2. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen asmore » a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.« less

  3. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

    PubMed

    Jardine, Joseph G; Kulp, Daniel W; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereño-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C; Julien, Jean-Philippe; Wilson, Ian A; Burton, Dennis R; Crotty, Shane; Schief, William R

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens. Copyright © 2016, American Association for the Advancement of Science.

  4. Neuroprotective Properties of Endocannabinoids N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine Examined in Neuronal Precursors Derived from Human Pluripotent Stem Cells.

    PubMed

    Novosadova, E V; Arsenyeva, E L; Manuilova, E S; Khaspekov, L G; Bobrov, M Yu; Bezuglov, V V; Illarioshkin, S N; Grivennikov, I A

    2017-11-01

    Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1-10 µM range. However, both agents at 10 µM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.

  5. Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

    PubMed

    Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

    2008-08-01

    Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

  6. Polyimide Precursor Solid Residuum

    NASA Technical Reports Server (NTRS)

    Weiser, Erik S. (Inventor); St.Clair, Terry L. (Inventor); Echigo, Yoshiaki (Inventor); Kaneshiro, Hisayasu (Inventor)

    2001-01-01

    A polyimide precursor solid residuum is an admixture of an aromatic dianhydride or derivative thereof and an aromatic diamine or derivative thereof plus a complexing agent, which is complexed with the admixture by hydrogen bonding. The polyimide precursor solid residuum is effectively employed in the preparation of polyimide foam and the fabrication of polyimide foam structures.

  7. Autologous adipose tissue-derived stromal cells for treatment of spinal cord injury.

    PubMed

    Kang, Soo-Kyung; Shin, Myung-Joo; Jung, Jin Sup; Kim, Yong Geun; Kim, Cheul-Hong

    2006-08-01

    Isolated rat adipose tissue-derived stromal cells (rATSCs) contain pluripotent cells that can be differentiated into a variety of cell lineages, including neural cells. Recent work has shown that ATSCs can make neurosphere-like clumps and differentiate into neuron-like cells expressing neuronal markers, but their therapeutic effect is unclear. Here we report that intravenous infusion of oligodendrocyte precursor cells (OPCs) derived from rATSC autograft cells sources improve motor function in rat models of spinal cord injury (SCI). After 4-5 weeks, transplanted rATSC-OPC cells survived and migrated into the injured region of SCI very efficiently (30-35%) and migrated cells were partially differentiated into neurons and oligodendrocyte. Also, we found some of the engrafted OPCs migrated and integrated in the kidney, brain, lung, and liver through the intravenous system. Behavioral analysis revealed the locomotor functions of OPC-autografted SCI rats were significantly restored. Efficient migration of intravenously engrafted rATSC-OPCs cells into SCI lesion suggests that SCI-induced chemotaxic factors facilitate migration of rATSC-OPCs. Here, we verified that engrafted rATSCs and SCI-induced chemotaxic factors indeed play an important role in proliferation, migration, and differentiation of endogeneous spinal cord-derived neural progenitor cells in the injured region. In transplantation paradigms, the interaction between engrafted rATSC-OPCs and endogeneous spinal cord-derived neuronal progenitor cells will be important in promoting healing through fate decisions, resulting in coordinated induction of cell migration and differentiation.

  8. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

    PubMed

    Haso, Waleed; Lee, Daniel W; Shah, Nirali N; Stetler-Stevenson, Maryalice; Yuan, Constance M; Pastan, Ira H; Dimitrov, Dimiter S; Morgan, Richard A; FitzGerald, David J; Barrett, David M; Wayne, Alan S; Mackall, Crystal L; Orentas, Rimas J

    2013-02-14

    Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

  9. Thin film solar cells by selenization sulfurization using diethyl selenium as a selenium precursor

    DOEpatents

    Dhere, Neelkanth G.; Kadam, Ankur A.

    2009-12-15

    A method of forming a CIGSS absorber layer includes the steps of providing a metal precursor, and selenizing the metal precursor using diethyl selenium to form a selenized metal precursor layer (CIGSS absorber layer). A high efficiency solar cell includes a CIGSS absorber layer formed by a process including selenizing a metal precursor using diethyl selenium to form the CIGSS absorber layer.

  10. Pluripotent stem cell-derived radial glia-like cells as stable intermediate for efficient generation of human oligodendrocytes.

    PubMed

    Gorris, Raphaela; Fischer, Julia; Erwes, Kim Lina; Kesavan, Jaideep; Peterson, Daniel A; Alexander, Michael; Nöthen, Markus M; Peitz, Michael; Quandel, Tamara; Karus, Michael; Brüstle, Oliver

    2015-12-01

    Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor-mediated expansion with pre-exposure to the differentiation-inducing agent retinoic acid and subsequent immunoisolation of CD133-positive cells. This protocol yields an adherent and self-renewing population of hindbrain/spinal cord radial glia (RG)-like neural precursor cells (RGL-NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL-NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate-determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL-NPCs efficiently convert into NG2-positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL-NPCs expedite the generation of PSC-derived oligodendrocytes with O4-, 4860-, and myelin basic protein (MBP)-positive cells that already appear within 7 weeks following growth factor withdrawal-induced differentiation. Thus, RGL-NPCs may serve as robust tool for time-efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. © 2015 Wiley Periodicals, Inc.

  11. Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

    PubMed Central

    Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

    2014-01-01

    Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

  12. Gene therapy and tissue engineering based on muscle-derived stem cells.

    PubMed

    Deasy, Bridget M; Huard, Johnny

    2002-08-01

    Skeletal muscle represents a convenient source of stem cells for cell-based tissue and genetic engineering. Muscle-derived stem cells (MDSCs) exhibit both multipotentiality and self-renewal capabilities, and are considered to be distinct from the well-studied satellite cell, another type of muscle stem cell that is capable of self-renewal and myogenic lineage differentiation. The MDSC appears to have less restricted differentiation capabilities as compared with the satellite cell, and may be a precursor of the satellite cell. This review considers the evidence for the existence of MDSCs as well as their origin. We will discuss recent investigations highlighting the potential of stem cell transplantation for the treatment of skeletal, cardiac and smooth muscle injuries and disease. We will highlight challenges in bridging the gap between understanding basic stem cell biology and clinical utilization for cell therapy.

  13. Formation and specification of a Drosophila dopaminergic precursor cell.

    PubMed

    Watson, Joseph D; Crews, Stephen T

    2012-09-01

    Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb(+)/gsb-n(+) MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons.

  14. Formation and specification of a Drosophila dopaminergic precursor cell

    PubMed Central

    Watson, Joseph D.; Crews, Stephen T.

    2012-01-01

    Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb+/gsb-n+ MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons. PMID:22874915

  15. Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function

    PubMed Central

    Charles, Julia F.; Hsu, Lih-Yun; Niemi, Erene C.; Weiss, Arthur; Aliprantis, Antonios O.; Nakamura, Mary C.

    2012-01-01

    Increased osteoclastic bone resorption leads to periarticular erosions and systemic osteoporosis in RA patients. Although a great deal is known about how osteoclasts differentiate from precursors and resorb bone, the identity of an osteoclast precursor (OCP) population in vivo and its regulatory role in RA remains elusive. Here, we report the identification of a CD11b–/loLy6Chi BM population with OCP activity in vitro and in vivo. These cells, which can be distinguished from previously characterized precursors in the myeloid lineage, display features of both M1 and M2 monocytes and expand in inflammatory arthritis models. Surprisingly, in one mouse model of RA (adoptive transfer of SKG arthritis), cotransfer of OCP with SKG CD4+ T cells diminished inflammatory arthritis. Similar to monocytic myeloid-derived suppressor cells (M-MDSCs), OCPs suppressed CD4+ and CD8+ T cell proliferation in vitro through the production of NO. This study identifies a BM myeloid precursor population with osteoclastic and T cell–suppressive activity that is expanded in inflammatory arthritis. Therapeutic strategies that prevent the development of OCPs into mature bone-resorbing cells could simultaneously prevent bone resorption and generate an antiinflammatory milieu in the RA joint. PMID:23114597

  16. Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

    PubMed Central

    Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

    2016-01-01

    Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

  17. Immature MEF2C-dysregulated T-cell leukemia patients have an early T-cell precursor acute lymphoblastic leukemia gene signature and typically have non-rearranged T-cell receptors

    PubMed Central

    Zuurbier, Linda; Gutierrez, Alejandro; Mullighan, Charles G.; Canté-Barrett, Kirsten; Gevaert, A. Olivier; de Rooi, Johan; Li, Yunlei; Smits, Willem K.; Buijs-Gladdines, Jessica G.C.A.M.; Sonneveld, Edwin; Look, A. Thomas; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

    2014-01-01

    Three distinct immature T-cell acute lymphoblastic leukemia entities have been described including cases that express an early T-cell precursor immunophenotype or expression profile, immature MEF2C-dysregulated T-cell acute lymphoblastic leukemia cluster cases based on gene expression analysis (immature cluster) and cases that retain non-rearranged TRG@ loci. Early T-cell precursor acute lymphoblastic leukemia cases exclusively overlap with immature cluster samples based on the expression of early T-cell precursor acute lymphoblastic leukemia signature genes, indicating that both are featuring a single disease entity. Patients lacking TRG@ rearrangements represent only 40% of immature cluster cases, but no further evidence was found to suggest that cases with absence of bi-allelic TRG@ deletions reflect a distinct and even more immature disease entity. Immature cluster/early T-cell precursor acute lymphoblastic leukemia cases are strongly enriched for genes expressed in hematopoietic stem cells as well as genes expressed in normal early thymocyte progenitor or double negative-2A T-cell subsets. Identification of early T-cell precursor acute lymphoblastic leukemia cases solely by defined immunophenotypic criteria strongly underestimates the number of cases that have a corresponding gene signature. However, early T-cell precursor acute lymphoblastic leukemia samples correlate best with a CD1 negative, CD4 and CD8 double negative immunophenotype with expression of CD34 and/or myeloid markers CD13 or CD33. Unlike various other studies, immature cluster/early T-cell precursor acute lymphoblastic leukemia patients treated on the COALL-97 protocol did not have an overall inferior outcome, and demonstrated equal sensitivity levels to most conventional therapeutic drugs compared to other pediatric T-cell acute lymphoblastic leukemia patients. PMID:23975177

  18. A monoclonal antibody that recognizes B cells and B cell precursors in mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Coffman, R.L.; Weissman, I.L.

    1981-02-01

    The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

  19. Pericytes and endothelial precursor cells: cellular interactions and contributions to malignancy.

    PubMed

    Bagley, Rebecca G; Weber, William; Rouleau, Cecile; Teicher, Beverly A

    2005-11-01

    Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function-associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit.

  20. Protective effect of skin-derived precursors on photoaging in nude mice.

    PubMed

    Wang, Siyu; Zhong, Jianqiao; Li, Li

    2018-06-25

    Currently, innovative methods to prevent photoaging are needed. Skin-derived precursors (SKP) have been shown to play a crucial role in resisting UVB-induced apoptosis in vitro. The objective of this study was to explore the effect of SKP on preventing skin photoaging in vivo. Skin-derived precursors from neonatal BALB/c mice were isolated, identified and intradermally transplanted with a PKH26 label to track their survival. These were then injected at different concentrations into the buttock dermis of nude mice at 2-weekly intervals before UV irradiation. Photographs, assessment of live skin surface, histology with quantitative real-time polymerase chain reaction and immunohistochemistry were used to evaluate the impact of SKP on wrinkles and other relevant indicators of skin photoaging. SKP exhibited a sphere-like structure and could survive for at least 2 weeks after intradermal transplantation. A large dose of SKP transplantation (10 5 SKP +UV) at 2-weekly intervals were able to ameliorate coarse UV-induced wrinkles. Moreover, the skin smoothness value, dermal thickness and collagen percentage were significantly increased in mice that received a large dose of SKP (10 5 SKP +UV). UV radiation induced the mRNA expression of MMP-13 and decreased the mRNA and protein expression of TβRII, but these effects were diminished by SKP transplantation. The transplantation of SKP could increase the mRNA of TIMP-1. We found that transplanted SKP exert a beneficial impact on preventing UV-induced wrinkles in vivo, suggesting that SKP transplantation is a promising candidate for preventing photoaging. © 2018 The Australasian College of Dermatologists.

  1. HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

    PubMed

    Lewis, D E; Yang, L; Luo, W; Wang, X; Rodgers, J R

    1999-06-18

    To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.

  2. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    PubMed

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  3. Anti-CD22–chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia

    PubMed Central

    Haso, Waleed; Lee, Daniel W.; Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Pastan, Ira H.; Dimitrov, Dimiter S.; Morgan, Richard A.; FitzGerald, David J.; Barrett, David M.; Wayne, Alan S.; Mackall, Crystal L.

    2013-01-01

    Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3ζ constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. PMID:23243285

  4. A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles.

    PubMed

    Hoelting, Lisa; Scheinhardt, Benjamin; Bondarenko, Olesja; Schildknecht, Stefan; Kapitza, Marion; Tanavde, Vivek; Tan, Betty; Lee, Qian Yi; Mecking, Stefan; Leist, Marcel; Kadereit, Suzanne

    2013-04-01

    Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood-brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6(+) precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.

  5. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    PubMed Central

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  6. Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer.

    PubMed

    Hope, Chelsea; Emmerich, Philip B; Papadas, Athanasios; Pagenkopf, Adam; Matkowskyj, Kristina A; Van De Hey, Dana R; Payne, Susan N; Clipson, Linda; Callander, Natalie S; Hematti, Peiman; Miyamoto, Shigeki; Johnson, Michael G; Deming, Dustin A; Asimakopoulos, Fotis

    2017-09-01

    Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8 + T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8 + T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8 + T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103 + CD11c hi MHCII hi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow-derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. Effect of cooling rate on human and murine hemopoietic precursor cell recovery

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Niskanen, E.; Pirsch, G.

    1983-08-01

    The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me2SO, a cooling rate of 7 degrees C/min from -4 to -30 degrees C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 degrees C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoieticmore » precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 degrees C/min.« less

  8. Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling

    PubMed Central

    Chang, Linda; Noseda, Michela; Higginson, Michelle; Ly, Michelle; Patenaude, Alexandre; Fuller, Megan; Kyle, Alastair H.; Minchinton, Andrew I.; Puri, Mira C.; Dumont, Daniel J.; Karsan, Aly

    2012-01-01

    Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1+CD31dimvascular endothelial (VE)-cadherin−CD45− precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1+ precursor cell, but not the VE-cadherin+ endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1+ precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1+ local precursor to VSMC in a spatiotemporal fashion across all vascular beds. PMID:22509029

  9. Neural Precursor-Derived Pleiotrophin Mediates Subventricular Zone Invasion by Glioma.

    PubMed

    Qin, Elizabeth Y; Cooper, Dominique D; Abbott, Keene L; Lennon, James; Nagaraja, Surya; Mackay, Alan; Jones, Chris; Vogel, Hannes; Jackson, Peter K; Monje, Michelle

    2017-08-24

    The lateral ventricle subventricular zone (SVZ) is a frequent and consequential site of pediatric and adult glioma spread, but the cellular and molecular mechanisms mediating this are poorly understood. We demonstrate that neural precursor cell (NPC):glioma cell communication underpins this propensity of glioma to colonize the SVZ through secretion of chemoattractant signals toward which glioma cells home. Biochemical, proteomic, and functional analyses of SVZ NPC-secreted factors revealed the neurite outgrowth-promoting factor pleiotrophin, along with required binding partners SPARC/SPARCL1 and HSP90B, as key mediators of this chemoattractant effect. Pleiotrophin expression is strongly enriched in the SVZ, and pleiotrophin knock down starkly reduced glioma invasion of the SVZ in the murine brain. Pleiotrophin, in complex with the binding partners, activated glioma Rho/ROCK signaling, and ROCK inhibition decreased invasion toward SVZ NPC-secreted factors. These findings demonstrate a pathogenic role for NPC:glioma interactions and potential therapeutic targets to limit glioma invasion. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Hedgehog signalling stimulates precursor cell accumulation and impairs epithelial maturation in the murine oesophagus.

    PubMed

    van Dop, Willemijn A; Rosekrans, Sanne L; Uhmann, Anja; Jaks, Viljar; Offerhaus, G Johan A; van den Bergh Weerman, Marius A; Kasper, Maria; Heijmans, Jarom; Hardwick, James C H; Verspaget, Hein W; Hommes, Daan W; Toftgård, Rune; Hahn, Heidi; van den Brink, Gijs R

    2013-03-01

    In the intestine Hedgehog (Hh) signalling is directed from epithelium to mesenchyme and negatively regulates epithelial precursor cell fate. The role of Hh signalling in the oesophagus has not been studied in vivo. Here the authors examined the role of Hh signalling in epithelial homeostasis of oesophagus. The authors used transgenic mice in which the Hh receptor Patched1 (Ptch1) could be conditionally inactivated in a body-wide manner and mice in which Gli1 could be induced specifically in the epithelium of the skin and oesophagus. Effects on epithelial homeostasis of the oesophagus were examined using immunohistochemistry, in situ hybridisation, transmission electron microscopy and real-time PCR. Hh signalling was examined in patients with oesophageal squamous cell carcinoma (SCC) by quantitative real-time PCR. Sonic Hh is signalled in an autocrine manner in the basal layer of the oesophagus. Activation of Hh signalling resulted in an expansion of the epithelial precursor cell compartment and failure of epithelial maturation and migration. Levels of Hh targets GLI1, HHIP and PTCH1 were increased in SCC compared with normal tissue from the same patients. Here the authors find that Hh signalling positively regulates the precursor cell compartment in the oesophageal epithelium in an autocrine manner. Since Hh signalling targets precursor cells in the oesophageal epithelium and signalling is increased in SCCs, Hh signalling may be involved in oesophageal SCC formation.

  11. Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

    PubMed Central

    Schüller, Ulrich; Heine, Vivi M.; Mao, Junhao; Kho, Alvin T.; Dillon, Allison K.; Han, Young-Goo; Huillard, Emmanuelle; Sun, Tao; Ligon, Azra H.; Qian, Ying; Ma, Qiufu; Alvarez-Buylla, Arturo; McMahon, Andrew P.; Rowitch, David H.; Ligon, Keith L.

    2008-01-01

    Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors. PMID:18691547

  12. Pure erythroid leukemia following precursor B-cell lymphoblastic leukemia.

    PubMed

    Xu, Min; Finn, Laura S; Tsuchiya, Karen D; Thomson, Blythe; Pollard, Jessica; Rutledge, Joe

    2012-01-01

    Therapy-related acute myeloid leukemia is an unfortunate sequel to current multimodal intensive chemotherapy. The patient described was diagnosed with pure erythroleukemia, AML-M6b, during therapy for precursor B-cell acute lymphoblastic leukemia. To the best of our knowledge, this is the first report of this unusual association.

  13. Sustainable Life Cycles of Natural-Precursor-Derived Nanocarbons.

    PubMed

    Bazaka, Kateryna; Jacob, Mohan V; Ostrikov, Kostya Ken

    2016-01-13

    Sustainable societal and economic development relies on novel nanotechnologies that offer maximum efficiency at minimal environmental cost. Yet, it is very challenging to apply green chemistry approaches across the entire life cycle of nanotech products, from design and nanomaterial synthesis to utilization and disposal. Recently, novel, efficient methods based on nonequilibrium reactive plasma chemistries that minimize the process steps and dramatically reduce the use of expensive and hazardous reagents have been applied to low-cost natural and waste sources to produce value-added nanomaterials with a wide range of applications. This review discusses the distinctive effects of nonequilibrium reactive chemistries and how these effects can aid and advance the integration of sustainable chemistry into each stage of nanotech product life. Examples of the use of enabling plasma-based technologies in sustainable production and degradation of nanotech products are discussed-from selection of precursors derived from natural resources and their conversion into functional building units, to methods for green synthesis of useful naturally degradable carbon-based nanomaterials, to device operation and eventual disintegration into naturally degradable yet potentially reusable byproducts.

  14. IGFBP-7 inhibits the differentiation of oligodendrocyte precursor cells via regulation of Wnt/β-Catenin signaling.

    PubMed

    Li, Nan; Han, Jinfeng; Tang, Jing; Ying, Yanqin

    2018-06-01

    Oligodendrocytes (OLs) are glial cells that form myelin sheaths in the central nervous system. Myelin sheath plays important role in nervous system and loss of it in neurodegenerative diseases can lead to impairment of movement. Understanding the signals and factors that regulate OL differentiation can help to address novel strategies for improving myelin repair in neurodegenerative diseases. The aim of this study was to investigate the role of insulin-like growth factor-binding proteins 7 (IGFBP-7) in differentiating OL precursor cells (OPCs). It was found that oligodendrocyte precursors undergoing differentiation were accompanied by selective expression of IGFBP-7. In addition, knockdown of IGFBP-7 promoted differentiation of oligodendrocytes and increased formation of myelin in cultured cells. In contrast, excessive expression of IGFBP-7 inhibited differentiation of oligodendrocytes. Furthermore, overexpression of IGFBP-7 in oligodendrocyte precursor cells increased transcription of Wnt target genes and promoted β-Catenin nuclear translocation. These findings suggest that IGFBP-7 negatively regulates differentiation of oligodendrocyte precursor cells via regulation of Wnt/β-Catenin signaling. © 2017 Wiley Periodicals, Inc.

  15. An estimation of the frequency of precursor cells which generate cytotoxic lymphocytes

    PubMed Central

    1976-01-01

    The cell-mediated immune response has been generated in vitro with a polyacrylamide culture system which allows the segregation of foci (clones?) of cytotoxic lymphocytes. Using the method of limiting dilutions, the frequency of precursor cells in CBA spleen cells able to generate a cytotoxic response against DBA mastocytoma is estimated at 1 per 1,700 cells. PMID:1083894

  16. Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells

    PubMed Central

    Tai, Ta-Wei; Chen, Ching-Yu; Su, Fong-Chin; Tu, Yuan-Kun; Tsai, Tsung-Ting; Lin, Chiou-Feng; Jou, I.-Ming

    2017-01-01

    Inhibiting osteoclasts and osteoclast precursors to reduce bone resorption is an important strategy to treat osteoclast-related diseases, such as osteoporosis, inflammatory bone loss, and malignant bone metastasis. However, the mechanism by which apoptosis is induced in the osteoclasts and their precursors are not completely understood. Here, we used nitrogen-containing bisphosphonate zoledronic acid (ZA) to induce cell apoptosis in human and murine osteoclast precursors and mature osteoclast-like cells. Caspase-3-mediated cell apoptosis occurred following the ZA (100 μM) treatment. Reactive oxygen species (ROS) were also generated in a time-dependent manner. Following knock-down of the p47phox expression, which is required for ROS activation, or co-treatment with the ROS inhibitor, N-acetyl-L-cysteine, ZA-induced apoptosis was significantly suppressed in both osteoclast precursors and mature osteoclast-like cells. The ROS-activated mitogen-activated protein kinases pathways did not trigger cell apoptosis. However, a ROS-regulated Mcl-1 decrease simultaneously with glycogen synthase kinase (GSK)-3β promoted cell apoptosis. These findings show that ZA induces apoptosis in osteoclast precursors and mature osteoclast-like cells by triggering ROS- and GSK-3β-mediated Mcl-1 down-regulation. PMID:28281643

  17. Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells.

    PubMed

    Konagaya, Shuhei; Iwata, Hiroo

    2015-01-01

    Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress. hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons. Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved. hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads. Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Photoprotection against UV-induced damage by skin-derived precursors in hairless mice.

    PubMed

    Xian, Dehai; Gao, Xiaoqing; Xiong, Xia; Xu, Jixiang; Yang, Lingyu; Pan, Lun; Zhong, Jianqiao

    2017-10-01

    Skin photodamage is associated with UV-induced overproduction of reactive oxygen species (ROS) and the inactivation of NF-E2-related factor 2 (Nrf2). Skin-derived precursor cells (SKPs), a population of dermal stem cells, are considered to be involved in wound repair and skin regeneration through the activation of Nrf2. However, no reports concentrate on the treatment of skin photodamage with SKPs. To investigate the photoprotective role of SKPs against UV-induced damage in mice. Fifty Balb/c hairless mice were divided into five groups (n=10), namely, normal (no intervention), model, prevention, treatment, and control groups. The latter four groups were dorsally exposed to UVA+UVB irradiation over a 2-week period. Mice in the prevention group received weekly SKP injections for 2weeks the day before irradiation. Mice in the treatment and Hanks groups received a two-time injection of SKPs and Hanks, respectively, after irradiation. One week after final intervention, skin appearance, pathological alterations, and oxidative indicators were evaluated by enzyme-linked immunosorbent assay, immunohistochemical analysis, and western blotting. After irradiation, lesions were observed on the dorsal skin of mice, including erythema, edema, scales, and wrinkles; however, these were significantly ameliorated by subcutaneous SKP injection. Hyperkeratosis, acanthosis, and spongiosis in the epidermis, as well as dermal papillae edema and inflammatory cell infiltration, were observed in both model and control groups; however, these conditions resolved with either pretreatment or posttreatment with SKPs. In addition, SKPs increased Nrf2, heme oxygenase-1, glutathione peroxidase, superoxide dismutase, catalase, and gluthathione expression, while decreasing levels of ROS, MDA, and H 2 O 2 . These findings suggest that SKPs have a photoprotective role against UV-induced damage in mice, which may be associated with their ability to scavenge photo-oxidative insults and activate Nrf2

  19. Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

    PubMed

    Chiva-Blanch, Gemma; Suades, Rosa; Crespo, Javier; Peña, Esther; Padró, Teresa; Jiménez-Xarrié, Elena; Martí-Fàbregas, Joan; Badimon, Lina

    2016-01-01

    Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke. Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3-7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls. Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions. Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions

  20. A human bone marrow mesodermal-derived cell population with hemogenic potential.

    PubMed

    Mokhtari, Saloomeh; Colletti, Evan; Yin, Weihong; Sanada, Chad; Lamar, Zanetta; Simmons, Paul J; Walker, Steven; Bishop, Colin; Atala, Anthony; Zanjani, Esmail D; Porada, Christopher D; Almeida-Porada, Graça

    2018-02-02

    The presence, within the human bone marrow, of cells with both endothelial and hemogenic potential has been controversial. Herein, we identify, within the human fetal bone marrow, prior to establishment of hematopoiesis, a unique APLNR+, Stro-1+ cell population, co-expressing markers of early mesodermal precursors and/or hemogenic endothelium. In adult marrow, cells expressing similar markers are also found, but at very low frequency. These adult-derived cells can be extensively culture expanded in vitro without loss of potential, they preserve a biased hemogenic transcriptional profile, and, upon in vitro induction with OCT4, assume a hematopoietic phenotype. In vivo, these cells, upon transplantation into a fetal microenvironment, contribute to the vasculature, and generate hematopoietic cells that provide multilineage repopulation upon serial transplantation. The identification of this human somatic cell population provides novel insights into human ontogenetic hematovascular potential, which could lead to a better understanding of, and new target therapies for, malignant and nonmalignant hematologic disorders.

  1. Fibronectin on extracellular vesicles from microvascular endothelial cells is involved in the vesicle uptake into oligodendrocyte precursor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Osawa, Sho; Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511; Kurachi, Masashi

    We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked themore » binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway. - Highlights: • Fibronectin exists on the surface of extracellular vesicles from endothelial cells. • Integrin signaling is not involved in effects of extracellular vesicles on OPCs. • Fibronectin on the surface of extracellular vesicles mediates their uptake into OPCs.« less

  2. Generation of novel bone forming cells (monoosteophils) from the cathelicidin-derived peptide LL-37 treated monocytes.

    PubMed

    Zhang, Zhifang; Shively, John E

    2010-11-15

    Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin), which recruits circulating monocytes during injury, may play a role in bone repair. Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM). In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC) lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC), osteonectin (ON), bone sialoprotein II (BSP II), osteopontin (OP), RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CK). Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte derived lineage and their bone forming phenotype. These

  3. Local Deformation Precursors of Large Earthquakes Derived from GNSS Observation Data

    NASA Astrophysics Data System (ADS)

    Kaftan, Vladimir; Melnikov, Andrey

    2017-12-01

    Research on deformation precursors of earthquakes was of immediate interest from the middle to the end of the previous century. The repeated conventional geodetic measurements, such as precise levelling and linear-angular networks, were used for the study. Many examples of studies referenced to strong seismic events using conventional geodetic techniques are presented in [T. Rikitake, 1976]. One of the first case studies of geodetic earthquake precursors was done by Yu.A. Meshcheryakov [1968]. Rare repetitions, insufficient densities and locations of control geodetic networks made difficult predicting future places and times of earthquakes occurrences. Intensive development of Global Navigation Satellite Systems (GNSS) during the recent decades makes research more effective. The results of GNSS observations in areas of three large earthquakes (Napa M6.1, USA, 2014; El Mayor Cucapah M7.2, USA, 2010; and Parkfield M6.0, USA, 2004) are treated and presented in the paper. The characteristics of land surface deformation before, during, and after earthquakes have been obtained. The results prove the presence of anomalous deformations near their epicentres. The temporal character of dilatation and shear strain changes show existence of spatial heterogeneity of deformation of the Earth’s surface from months to years before the main shock close to it and at some distance from it. The revealed heterogeneities can be considered as deformation precursors of strong earthquakes. According to historical data and proper research values of critical deformations which are offered to be used for seismic danger scale creation based on continuous GNSS observations are received in a reference to the mentioned large earthquakes. It is shown that the approach has restrictions owing to uncertainty of the moment in the beginning of deformation accumulation and the place of expectation of another seismic event. Verification and clarification of the derived conclusions are proposed.

  4. Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

    PubMed

    Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

    2015-11-06

    NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells*

    PubMed Central

    Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E.; Ziegler, Mathias; Nikiforov, Andrey

    2015-01-01

    NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. PMID:26385918

  6. Co-ultramicronized Palmitoylethanolamide/Luteolin Promotes the Maturation of Oligodendrocyte Precursor Cells

    PubMed Central

    Barbierato, Massimo; Facci, Laura; Marinelli, Carla; Zusso, Morena; Argentini, Carla; Skaper, Stephen D.; Giusti, Pietro

    2015-01-01

    Oligodendrocytes have limited ability to repair the damage to themselves or to other nerve cells, as seen in demyelinating diseases like multiple sclerosis. An important strategy may be to replace the lost oligodendrocytes and/or promote the maturation of undifferentiated oligodendrocyte precursor cells (OPCs). Recent studies show that a composite of co-ultramicronized N-palmitoylethanolamine (PEA) and luteolin (co-ultramicronized PEA/luteolin, 10:1 by mass) is efficacious in improving outcome in experimental models of spinal cord and traumatic brain injuries. Here, we examined the ability of co-ultramicronized PEA/luteolin to promote progression of OPCs into a more differentiated phenotype. OPCs derived from newborn rat cortex were placed in culture and treated the following day with 10 μM co-ultramicronized PEA/luteolin. Cells were collected 1, 4 and 8 days later and analyzed for expression of myelin basic protein (MBP). qPCR and Western blot analyses revealed a time-dependent increase in expression of both mRNA for MBP and MBP content, along with an increased expression of genes involved in lipid biogenesis. Ultramicronized PEA or luteolin, either singly or in simple combination, were ineffective. Further, co-ultramicronized PEA/luteolin promoted morphological development of OPCs and total protein content without affecting proliferation. Co-ultramicronized PEA/luteolin may represent a novel pharmacological strategy to promote OPC maturation. PMID:26578323

  7. Co-ultramicronized Palmitoylethanolamide/Luteolin Promotes the Maturation of Oligodendrocyte Precursor Cells.

    PubMed

    Barbierato, Massimo; Facci, Laura; Marinelli, Carla; Zusso, Morena; Argentini, Carla; Skaper, Stephen D; Giusti, Pietro

    2015-11-18

    Oligodendrocytes have limited ability to repair the damage to themselves or to other nerve cells, as seen in demyelinating diseases like multiple sclerosis. An important strategy may be to replace the lost oligodendrocytes and/or promote the maturation of undifferentiated oligodendrocyte precursor cells (OPCs). Recent studies show that a composite of co-ultramicronized N-palmitoylethanolamine (PEA) and luteolin (co-ultramicronized PEA/luteolin, 10:1 by mass) is efficacious in improving outcome in experimental models of spinal cord and traumatic brain injuries. Here, we examined the ability of co-ultramicronized PEA/luteolin to promote progression of OPCs into a more differentiated phenotype. OPCs derived from newborn rat cortex were placed in culture and treated the following day with 10 μM co-ultramicronized PEA/luteolin. Cells were collected 1, 4 and 8 days later and analyzed for expression of myelin basic protein (MBP). qPCR and Western blot analyses revealed a time-dependent increase in expression of both mRNA for MBP and MBP content, along with an increased expression of genes involved in lipid biogenesis. Ultramicronized PEA or luteolin, either singly or in simple combination, were ineffective. Further, co-ultramicronized PEA/luteolin promoted morphological development of OPCs and total protein content without affecting proliferation. Co-ultramicronized PEA/luteolin may represent a novel pharmacological strategy to promote OPC maturation.

  8. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  9. Prolonged Expansion Induces Spontaneous Neural Progenitor Differentiation from Human Gingiva-Derived Mesenchymal Stem Cells.

    PubMed

    Rajan, Thangavelu Soundara; Scionti, Domenico; Diomede, Francesca; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2017-12-01

    Neural crest-derived mesenchymal stem cells (MSCs) obtained from dental tissues received considerable interest in regenerative medicine, particularly in nerve regeneration owing to their embryonic origin and ease of harvest. Proliferation efficacy and differentiation capacity into diverse cell lineages propose dental MSCs as an in vitro tool for disease modeling. In this study, we investigated the spontaneous differentiation efficiency of dental MSCs obtained from human gingiva tissue (hGMSCs) into neural progenitor cells after extended passaging. At passage 41, the morphology of hGMSCs changed from typical fibroblast-like shape into sphere-shaped cells with extending processes. Next-generation transcriptomics sequencing showed increased expression of neural progenitor markers such as NES, MEIS2, and MEST. In addition, de novo expression of neural precursor genes, such as NRN1, PHOX2B, VANGL2, and NTRK3, was noticed in passage 41. Immunocytochemistry results showed suppression of neurogenesis repressors TP53 and p21, whereas Western blot results revealed the expression of neurotrophic factors BDNF and NT3 at passage 41. Our results showed the spontaneous efficacy of hGMSCs to differentiate into neural precursor cells over prolonged passages and that these cells may assist in producing novel in vitro disease models that are associated with neural development.

  10. Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

    PubMed Central

    Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

    2014-01-01

    Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

  11. Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

    PubMed

    Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

    2015-01-01

    The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

  12. The planarian nanos-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration.

    PubMed

    Handberg-Thorsager, Mette; Saló, Emili

    2007-05-01

    Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians.

  13. A network model for the specification of vulval precursor cells and cell fusion control in Caenorhabditis elegans

    PubMed Central

    Weinstein, Nathan; Mendoza, Luis

    2013-01-01

    The vulva of Caenorhabditis elegans has been long used as an experimental model of cell differentiation and organogenesis. While it is known that the signaling cascades of Wnt, Ras/MAPK, and NOTCH interact to form a molecular network, there is no consensus regarding its precise topology and dynamical properties. We inferred the molecular network, and developed a multivalued synchronous discrete dynamic model to study its behavior. The model reproduces the patterns of activation reported for the following types of cell: vulval precursor, first fate, second fate, second fate with reversed polarity, third fate, and fusion fate. We simulated the fusion of cells, the determination of the first, second, and third fates, as well as the transition from the second to the first fate. We also used the model to simulate all possible single loss- and gain-of-function mutants, as well as some relevant double and triple mutants. Importantly, we associated most of these simulated mutants to multivulva, vulvaless, egg-laying defective, or defective polarity phenotypes. The model shows that it is necessary for RAL-1 to activate NOTCH signaling, since the repression of LIN-45 by RAL-1 would not suffice for a proper second fate determination in an environment lacking DSL ligands. We also found that the model requires the complex formed by LAG-1, LIN-12, and SEL-8 to inhibit the transcription of eff-1 in second fate cells. Our model is the largest reconstruction to date of the molecular network controlling the specification of vulval precursor cells and cell fusion control in C. elegans. According to our model, the process of fate determination in the vulval precursor cells is reversible, at least until either the cells fuse with the ventral hypoderm or divide, and therefore the cell fates must be maintained by the presence of extracellular signals. PMID:23785384

  14. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

    PubMed Central

    Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-01

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression. PMID:26700461

  15. The union of somatic gonad precursors and primordial germ cells during C. elegans embryogenesis

    PubMed Central

    Rohrschneider, Monica R.; Nance, Jeremy

    2013-01-01

    Somatic gonadal niche cells control the survival, differentiation, and proliferation of germline stem cells. The establishment of this niche-stem cell relationship is critical, and yet the precursors to these two cell types are often born at a distance from one another. The simple C. elegans gonadal primordium, which contains two somatic gonad precursors (SGPs) and two primordial germ cells (PGCs), provides an accessible model for determining how stem cell and niche cell precursors first assemble during development. To visualize the morphogenetic events that lead to formation of the gonadal primordium, we generated transgenic strains to label the cell membranes of the SGPs and PGCs and captured time-lapse movies as the gonadal primordium formed. We identify three distinct phases of SGP behavior: posterior migration along the endoderm towards the PGCs, extension of a single long projection around the adjacent PGC, and a dramatic wrapping over the PGC surfaces. We show that the endoderm and PGCs are dispensable for SGP posterior migration and initiation of projections. However, both tissues are required for the final positioning of the SGPs and the morphology of their projections, and PGCs are absolutely required for SGP wrapping behaviors. Finally, we demonstrate that the basement membrane component laminin, which localizes adjacent to the developing gonadal primordium, is required to prevent the SGPs from over-extending past the PGCs. Our findings provide a foundation for understanding the cellular and molecular regulation of the establishment of a niche-stem cell relationship. PMID:23562590

  16. Single Source Precursors for Thin Film Solar Cells

    NASA Technical Reports Server (NTRS)

    Banger, Kulbinder K.; Hollingsworth, Jennifer A.; Harris, Jerry D.; Cowen, Jonathan; Buhro, William E.; Hepp, Aloysius F.

    2002-01-01

    The development of thin film solar cells on flexible, lightweight, space-qualified substrates provides an attractive cost solution to fabricating solar arrays with high specific power, (W/kg). The use of a polycrystalline chalcopyrite absorber layer for thin film solar cells is considered as the next generation photovoltaic devices. At NASA GRC we have focused on the development of new single source precursors (SSP) and their utility to deposit the chalcopyrite semi-conducting layer (CIS) onto flexible substrates for solar cell fabrication. The syntheses and thermal modulation of SSPs via molecular engineering is described. Thin-film fabrication studies demonstrate the SSPs can be used in a spray CVD (chemical vapor deposition) process, for depositing CIS at reduced temperatures, which display good electrical properties, suitable for PV (photovoltaic) devices.

  17. Neural Growth Factor Stimulates Proliferation of Spinal Cord Derived-Neural Precursor/Stem Cells

    PubMed Central

    Han, Youngmin

    2016-01-01

    Objective Recently, regenerative therapies have been used in clinical trials (heart, cartilage, skeletal). We don't make use of these treatments to spinal cord injury (SCI) patients yet, but regenerative therapies are rising interest in recent study about SCI. Neural precursor/stem cell (NPSC) proliferation is a significant event in functional recovery of the central nervous system (CNS). However, brain NPSCs and spinal cord NPSCs (SC-NPSCs) have many differences including gene expression and proliferation. The purpose of this study was to investigate the influence of neural growth factor (NGF) on the proliferation of SC-NPSCs. Methods NPSCs (2×104) were suspended in 100 µL of neurobasal medium containing NGF-7S (Sigma-Aldrich) and cultured in a 96-well plate for 12 days. NPSC proliferation was analyzed five times for either concentration of NGF (0.02 and 2 ng/mL). Sixteen rats after SCI were randomly allocated into two groups. In group 1 (SCI-vehicle group, n=8), animals received 1.0 mL of the saline vehicle solution. In group 2 (SCI-NGF group, n=8), the animals received single doses of NGF (Sigma-Aldrich). A dose of 0.02 ng/mL of NGF or normal saline as a vehicle control was intra-thecally injected daily at 24 hour intervals for 7 days. For Immunohistochemistry analysis, rats were sacrificed after one week and the spinal cords were obtained. Results The elevation of cell proliferation with 0.02 ng/mL NGF was significant (p<0.05) but was not significant for 2 ng/mL NGF. The optical density was increased in the NGF 0.02 ng/mL group compared to the control group and NGF 2 ng/mL groups. The density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group (p<0.05). High power microscopy revealed that the density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group. Conclusion SC-NPSC proliferation is an important pathway in the functional recovery of SCI. NGF enhances SC-NPSC proliferation in vitro and in

  18. Thyroid hormone participates in the regulation of neural stem cells and oligodendrocyte precursor cells in the central nervous system of adult rat.

    PubMed

    Fernandez, M; Pirondi, S; Manservigi, M; Giardino, L; Calzà, L

    2004-10-01

    Oligodendrocyte development and myelination are under thyroid hormone control. In this study we analysed the effects of chronic manipulation of thyroid status on the expression of a wide spectrum of oligodendrocyte precursor cells (OPCs) markers and myelin basic protein (MBP) in the subventricular zone (SVZ), olfactory bulb and optic nerve, and on neural stem cell (NSC) lineage in adult rats. Hypo- and hyperthyroidism were induced in male rats, by propyl-thio-uracil (PTU) and L-thyroxin (T4) treatment, respectively. Hypothyroidism increased and hyperthyroidism downregulated proliferation in the SVZ and olfactory bulb (Ki67 immunohistochemistry and Western blotting, bromodeoxyuridine uptake). Platelet-derived growth factor receptor alpha (PDGFalpha-R) and MBP mRNA levels decreased in the optic nerve of hypothyroid rats; the same also occurred at the level of MBP protein. Hyperthyroidism slightly upregulates selected markers such as NG2 in the olfactory bulb. The lineage of cells derived from primary cultures of NSC prepared from the forebrain of adult hypo- and hyperthyroid also differs from those derived from control animals. Although no difference of in vitro proliferation of NSCs was observed in the presence of epidermal growth factor, maturation of oligodendrocytes (defined by process number and length) was enhanced in hyperthyroidism, suggesting a more mature state than in control animals. This difference was even greater when compared with the hypothyroid group, the morphology of which suggested a delay in differentiation. These results indicate that thyroid hormone affects NSC and OPC proliferation and maturation also in adulthood.

  19. Two separate defects affecting true naive or virtual memory T cell precursors combine to reduce naive T cell responses with aging.

    PubMed

    Renkema, Kristin R; Li, Gang; Wu, Angela; Smithey, Megan J; Nikolich-Žugich, Janko

    2014-01-01

    Naive T cell responses are eroded with aging. We and others have recently shown that unimmunized old mice lose ≥ 70% of Ag-specific CD8 T cell precursors and that many of the remaining precursors acquire a virtual (central) memory (VM; CD44(hi)CD62L(hi)) phenotype. In this study, we demonstrate that unimmunized TCR transgenic (TCRTg) mice also undergo massive VM conversion with age, exhibiting rapid effector function upon both TCR and cytokine triggering. Age-related VM conversion in TCRTg mice directly depended on replacement of the original TCRTg specificity by endogenous TCRα rearrangements, indicating that TCR signals must be critical in VM conversion. Importantly, we found that VM conversion had adverse functional effects in both old wild-type and old TCRTg mice; that is, old VM, but not old true naive, T cells exhibited blunted TCR-mediated, but not IL-15-mediated, proliferation. This selective proliferative senescence correlated with increased apoptosis in old VM cells in response to peptide, but decreased apoptosis in response to homeostatic cytokines IL-7 and IL-15. Our results identify TCR as the key factor in differential maintenance and function of Ag-specific precursors in unimmunized mice with aging, and they demonstrate that two separate age-related defects--drastic reduction in true naive T cell precursors and impaired proliferative capacity of their VM cousins--combine to reduce naive T cell responses with aging.

  20. Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

    PubMed

    Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

    2017-09-02

    Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. HMC-1 human mast cells synthesize neurotensin (NT) precursor, secrete bioactive NT-like peptide(s) and express NT receptor NTS1.

    PubMed

    Cochrane, David E; Carraway, Robert E; Harrington, Kimberly; Laudano, Melissa; Rawlings, Stephen; Feldberg, Ross S

    2011-12-01

    To determine if mast cells synthesize the inflammatory peptide, neurotensin (NT), secrete immunoreactive and bioactive NT, and express the NT receptor NTS1. HMC-1 cells, pleural mast cells from Sprague-Dawley rats, LAD2 mast cells, and human cord blood mast cells were used. HMC-1 cells were stimulated with NT, C48/80, mastoparan, or PGE(2). For changes in cutaneous vascular permeability, anesthetized rats were injected intravenously with Evans Blue dye and intradermally with saline, NT, histamine, diphenhydramine, and C48/80. RT-PCR was used to identify RNA transcripts. Histamine was measured by fluorometric assay. In vivo cutaneous vascular permeability assays, radio-immunoassays for NT, Western blotting for the NT precursor protein and NTS1 protein from HMC-1 cells and tissues from rats were used. Immunohistochemistry was used to identify NT precursor-like proteins in HMC-1 mast cells. HMC-1 cells express mRNAs for NT precursor, PC5A processing enzyme and NTS1 receptor. Human cord blood mast cells and LAD2 mast cells express mRNA transcripts for NT precursor and NTS1. Western blotting showed NT precursor and NTS1 receptor in HMC1. Rat tissues with high numbers of mast cells contained NT precursor proteins. NT-like peptides from HMC-1 displayed NT-like bioactivity. HMC-1 mast cells synthesize and secrete immunoreactive and bioactive NT-like peptide(s) and express the NT receptor, suggesting that NT from mast cells might serve autocrine and paracrine roles.

  2. Evidence for heterogeneity of astrocyte de-differentiation in vitro: astrocytes transform into intermediate precursor cells following induction of ACM from scratch-insulted astrocytes.

    PubMed

    Yang, Hao; Qian, Xin-Hong; Cong, Rui; Li, Jing-wen; Yao, Qin; Jiao, Xi-Ying; Ju, Gong; You, Si-Wei

    2010-04-01

    Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2(+) and A2B5(+) cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.

  3. T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells

    PubMed Central

    Plaisted, Warren C.; Weinger, Jason G.; Walsh, Craig M.; Lane, Thomas E.

    2014-01-01

    Neural precursor cells (NPCs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of NPCs remain unclear. This study demonstrates that NPCs support replication following infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV). JHMV infection leads to increased cell death and dampens IFN-γ-induced MHC class II expression. Importantly, cytokines secreted by CD4+ T cells inhibit JHMV replication in NPCs, and CD8+ T cells specifically target viral peptide-pulsed NPCs for lysis. Furthermore, treatment with IFN-γ inhibits JHMV replication in a dose-dependent manner. Together, these findings suggest that T cells play a critical role in controlling replication of a neurotropic virus in NPCs, a finding which has important implications when considering immune modulation for NPC-based therapies for treatment of human neurologic diseases. PMID:24418558

  4. Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke

    PubMed Central

    Chiva-Blanch, Gemma; Suades, Rosa; Crespo, Javier; Peña, Esther; Padró, Teresa; Jiménez-Xarrié, Elena; Martí-Fàbregas, Joan; Badimon, Lina

    2016-01-01

    Purpose Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke. Methods Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls. Results Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions. Conclusions Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process

  5. Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

    PubMed

    Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

    2018-01-01

    It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

  6. * Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

    PubMed

    Zhang, Deying; Zhang, Yong; Zhang, Yuanyuan; Yi, Hualin; Wang, Zhan; Wu, Rongpei; He, Dawei; Wei, Guanghui; Wei, Shicheng; Hu, Yun; Deng, Junhong; Criswell, Tracy; Yoo, James; Zhou, Yu; Atala, Anthony

    2017-08-01

    Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle

  7. Measles Virus Nucleocapsid (MVNP) Gene Expression and RANK Receptor Signaling in Osteoclast Precursors, Osteoclast Inhibitors Peptide Therapy for Pagets Disease

    DTIC Science & Technology

    2007-10-01

    OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c . THIS PAGE U UU 27 19b. TELEPHONE NUMBER...and c -Jun kinase activity in osteoclast precursor cells (4). Our hypothesis is that MVNP expression in osteoclast precursors modulates the status...transcription factors such as c - Fos, NFATc1 critical for OCL differentiation were significantly decreased in OIP-1 transgenic mice derived preosteoclast cells

  8. The chemokine CXCL16 induces migration and invasion of glial precursor cells via its receptor CXCR6.

    PubMed

    Hattermann, Kirsten; Ludwig, Andreas; Gieselmann, Volkmar; Held-Feindt, Janka; Mentlein, Rolf

    2008-09-01

    Chemokines are implicated in developmental and inflammatory processes in the brain. The transmembrane chemokine CXCL16 is produced in brain endothelial and reactive astroglial cells and released by shedding. Its receptor CXCR6 is detected during brain development highest at postnatal day 6, found in glial precursor cells differentiated from neural stem cells and in an A2B5-positive glial precursor cell line. Their stimulation by soluble CXCL16 induces the PI3-kinase/Akt and Erk pathways resulting in the activation of the transcription factor AP-1. As biological responses, soluble CXCL16 upregulates its own receptor, increases cell proliferation, stimulates cell migration in wound-healing and in spheroid confrontation assays. Invasion of CXCR6-positive glial cells into CXCL16-expressing spheroids can be blocked by sheddase inhibitors and CXCL16-antibody. Since CXCL16 is induced by cytokines at sites of inflammation, neurodegeneration, ischemia and malignant transformation, it should attract CXCR6-positive glial precursor cells, enhance their invasion and proliferation and thus favor astrogliosis.

  9. Early T-cell precursor acute lymphoblastic leukaemia in children treated in AIEOP centres with AIEOP-BFM protocols: a retrospective analysis.

    PubMed

    Conter, Valentino; Valsecchi, Maria Grazia; Buldini, Barbara; Parasole, Rosanna; Locatelli, Franco; Colombini, Antonella; Rizzari, Carmelo; Putti, Maria Caterina; Barisone, Elena; Lo Nigro, Luca; Santoro, Nicola; Ziino, Ottavio; Pession, Andrea; Testi, Anna Maria; Micalizzi, Concetta; Casale, Fiorina; Pierani, Paolo; Cesaro, Simone; Cellini, Monica; Silvestri, Daniela; Cazzaniga, Giovanni; Biondi, Andrea; Basso, Giuseppe

    2016-02-01

    Early T-cell precursor acute lymphoblastic leukaemia was recently recognised as a distinct leukaemia and reported as associated with poor outcomes. We aimed to assess the outcome of early T-cell precursor acute lymphoblastic leukaemia in patients from the Italian Association of Pediatric Hematology Oncology (AIEOP) centres treated with AIEOP-Berlin-Frankfurt-Münster (AIEOP-BFM) protocols. In this retrospective analysis, we included all children aged from 1 to less than 18 years with early T-cell precursor acute lymphoblastic leukaemia immunophenotype diagnosed between Jan 1, 2008, and Oct 31, 2014, from AIEOP centres. Early T-cell precursors were defined as being CD1a and CD8 negative, CD5 weak positive or negative, and positive for at least one of the following antigens: CD34, CD117, HLADR, CD13, CD33, CD11b, or CD65. Treatment was based on AIEOP-BFM acute lymphoblastic leukaemia 2000 (NCT00613457) or AIEOP-BFM acute lymphoblastic leukaemia 2009 protocols (European Clinical Trials Database 2007-004270-43). The main differences in treatment and stratification of T-cell acute lymphoblastic leukaemia between the two protocols were that in the 2009 protocol only, pegylated L-asparaginase was substituted for Escherichia coli L-asparaginase, patients with prednisone poor response received an additional dose of cyclophosphamide at day 10 of phase IA, and high minimal residual disease at day 15 assessed by flow cytometry was used as a high-risk criterion. Outcomes were assessed in terms of event-free survival, disease-free survival, and overall survival. Early T-cell precursor acute lymphoblastic leukaemia was diagnosed in 49 patients. Compared with overall T-cell acute lymphoblastic leukaemia, it was associated with absence of molecular markers for PCR detection of minimal residual disease in 25 (56%) of 45 patients; prednisone poor response in 27 (55%) of 49 patients; high minimal residual disease at day 15 after starting therapy in 25 (64%) of 39 patients (bone marrow

  10. Enhancement of viability of muscle precursor cells on 3D scaffold in a perfusion bioreactor.

    PubMed

    Cimetta, E; Flaibani, M; Mella, M; Serena, E; Boldrin, L; De Coppi, P; Elvassore, N

    2007-05-01

    The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.

  11. New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV-exposure: ultrastructural differentiation of precursor melanocytes from Langerhans cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jimbow, K.; Uesugi, T.

    1982-02-01

    Photobiological processes involving new melanogenesis after exposure to ultraviolet (UV) light were experimentally studied in C57 black adult mice by histochemistry, cytochemistry, and autoradiography. The trunk and the plantar region of the foot, where no functioning melanocytes were present before exposure, were exposed to UV-A for 14 consecutive days. Both regions revealed a basically similar pattern for new melanogenesis which involved an activation of precursor melanocytes. Essentially all of ''indeterminate'' cells appeared to be precursor melanocytes, the fine structure of which could be differentiated even from poorly developed Langerhans cells. New melanogenesis was manifested by 4 stages of cellular andmore » subcellular reactions of these cells as indicated by histochemistry of dihydroxyphenylalanine (dopa) and autoradiography of thymidine incorporation: (a) an initial lag in the activation of precursor melanocytes with development of Golgi cisternae and rough endoplasmic reticulum followed by formation of unmelanized melanosomes (day 0 to 2); (b) synthesis of active tyrosinase accumulated in Golgi cisternae and vesicles with subsequent formation of melanized melanosomes in these cells (day 3 to 5); (c) mitotic proliferation of many of these activated cells, followed by an exponential increase of new melanocytes (day 6 to 7); and (d) melanosome transfer with differentiation of 10 nm filaments and arborization of dendrites, but without any significant change in the melanocyte population (day 8 to 14). The melanosome transfer was, however, not obvious until after 7 days of exposure. The size of newly synthesized melanosomes was similar to that of tail skin where native melanocytes were present before exposure.« less

  12. Innate lymphoid cells, precursors and plasticity.

    PubMed

    Gronke, Konrad; Kofoed-Nielsen, Michael; Diefenbach, Andreas

    2016-11-01

    Innate lymphoid cells (ILC) have only recently been recognized as a separate entity of the lymphoid lineage. Their subpopulations share common characteristics in terms of early development and major transcriptional circuitry with their related cousins of the T cell world. It is currently hypothesized that ILCs constitute an evolutionary older version of the lymphoid immune system. They are found at all primary entry points for pathogens such as mucosal surfaces of the lung and gastrointestinal system, the skin and the liver, which is the central contact point for pathogens that breach the intestinal barrier and enter the circulation. There, ILC contribute to the first line defense as well as to organ homeostasis. However, ILC are not only involved in classical defense tasks, but also contribute to the organogenesis of lymphoid organs as well as tissue remodeling and even stem cell regeneration. ILC may, therefore, implement different functions according to their emergence in ontogeny, their development and their final tissue location. We will review here their early development from precursors of the fetal liver and the adult bone marrow as well as their late plasticity in adaptation to their environment. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  13. Epigenetics in myeloid derived suppressor cells: a sheathed sword towards cancer

    PubMed Central

    Zhang, Chao; Wang, Shuo; Liu, Yufeng; Yang, Cheng

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells composed of progenitors and precursors to myeloid cells, are deemed to participate in the development of tumor-favoring immunosuppressive microenvironment. Thus, the regulatory strategies targeting MDSCs' expansion, differentiation, accumulation and function could possibly be effective “weapons” in anti-tumor immunotherapies. Epigenetic mechanisms, which involve DNA modification, covalent histone modification and RNA interference, result in the heritable down-regulation or silencing of gene expression without a change in DNA sequences. Epigenetic modification of MDSC's functional plasticity leads to the remodeling of its characteristics, therefore reframing the microenvironment towards countering tumor growth and metastasis. This review summarized the pertinent findings on the DNA methylation, covalent histone modification, microRNAs and small interfering RNAs targeting MDSC in cancer genesis, progression and metastasis. The potentials as well as possible obstacles in translating into anti-cancer therapeutics were also discussed. PMID:27458169

  14. Diethanolamine alters proliferation and choline metabolism in mouse neural precursor cells.

    PubMed

    Niculescu, Mihai D; Wu, Renan; Guo, Zhong; da Costa, Kerry Ann; Zeisel, Steven H

    2007-04-01

    Diethanolamine (DEA) is a widely used ingredient in many consumer products and in a number of industrial applications. It has been previously reported that dermal administration of DEA to mice diminished hepatic stores of choline and altered brain development in the fetus. The aim of this study was to use mouse neural precursor cells in vitro to assess the mechanism underlying the effects of DEA. Cells exposed to DEA treatment (3mM) proliferated less (by 5-bromo-2-deoxyuridine incorporation) at 48 h (24% of control [CT]), and had increased apoptosis at 72 h (308% of CT). Uptake of choline into cells was reduced by DEA treatment (to 52% of CT), resulting in diminished intracellular concentrations of choline and phosphocholine (55 and 12% of CT, respectively). When choline concentration in the growth medium was increased threefold (to 210 microM), the effects of DEA exposure on cell proliferation and apoptosis were prevented, however, intracellular phosphocholine concentrations remained low. In choline kinase assays, we observed that DEA can be phosphorylated to phospho-DEA at the expense of choline. Thus, the effects of DEA are likely mediated by inhibition of choline transport into neural precursor cells and by altered metabolism of choline. Our study suggests that prenatal exposure to DEA may have a detrimental effect on brain development.

  15. Diethanolamine Alters Proliferation and Choline Metabolism in Mouse Neural Precursor Cells

    PubMed Central

    Niculescu, Mihai D.; Wu, Renan; Guo, Zhong; da Costa, Kerry Ann; Zeisel, Steven H.

    2008-01-01

    Diethanolamine (DEA) is a widely used ingredient in many consumer products and in a number of industrial applications. It has been previously reported that dermal administration of DEA to mice diminished hepatic stores of choline and altered brain development in the fetus. The aim of this study was to use mouse neural precursor cells in vitro to assess the mechanism underlying the effects of DEA. Cells exposed to DEA treatment (3mM) proliferated less (by 5-bromo-2-deoxyuridine incorporation) at 48 h (24% of control [CT]), and had increased apoptosis at 72 h (308% of CT). Uptake of choline into cells was reduced by DEA treatment (to 52% of CT), resulting in diminished intracellular concentrations of choline and phosphocholine (55 and 12% of CT, respectively). When choline concentration in the growth medium was increased threefold (to 210μM), the effects of DEA exposure on cell proliferation and apoptosis were prevented, however, intracellular phosphocholine concentrations remained low. In choline kinase assays, we observed that DEA can be phosphorylated to phospho-DEA at the expense of choline. Thus, the effects of DEA are likely mediated by inhibition of choline transport into neural precursor cells and by altered metabolism of choline. Our study suggests that prenatal exposure to DEA may have a detrimental effect on brain development. PMID:17204582

  16. Blood Cell-Derived Induced Pluripotent Stem Cells Free of Reprogramming Factors Generated by Sendai Viral Vectors

    PubMed Central

    Muench, Marcus O.; Fusaki, Noemi; Beyer, Ashley I.; Wang, Jiaming; Qi, Zhongxia; Yu, Jingwei

    2013-01-01

    The discovery of induced pluripotent stem cells (iPSCs) holds great promise for regenerative medicine since it is possible to produce patient-specific pluripotent stem cells from affected individuals for potential autologous treatment. Using nonintegrating cytoplasmic Sendai viral vectors, we generated iPSCs efficiently from adult mobilized CD34+ and peripheral blood mononuclear cells. After 5–8 passages, the Sendai viral genome could not be detected by real-time quantitative reverse transcription-polymerase chain reaction. Using the spin embryoid body method, we showed that these blood cell-derived iPSCs could efficiently be differentiated into hematopoietic stem and progenitor cells without the need of coculture with either mouse or human stromal cells. We obtained up to 40% CD34+ of which ∼25% were CD34+/CD43+ hematopoietic precursors that could readily be differentiated into mature blood cells. Our study demonstrated a reproducible protocol for reprogramming blood cells into transgene-free iPSCs by the Sendai viral vector method. Maintenance of the genomic integrity of iPSCs without integration of exogenous DNA should allow the development of therapeutic-grade stem cells for regenerative medicine. PMID:23847002

  17. Mitotic position and morphology of committed precursor cells in the zebrafish retina adapt to architectural changes upon tissue maturation.

    PubMed

    Weber, Isabell P; Ramos, Ana P; Strzyz, Paulina J; Leung, Louis C; Young, Stephen; Norden, Caren

    2014-04-24

    The development of complex neuronal tissues like the vertebrate retina requires the tight orchestration of cell proliferation and differentiation. Although the complexity of transcription factors and signaling pathways involved in retinogenesis has been studied extensively, the influence of tissue maturation itself has not yet been systematically explored. Here, we present a quantitative analysis of mitotic events during zebrafish retinogenesis that reveals three types of committed neuronal precursors in addition to the previously known apical progenitors. The identified precursor types present at distinct developmental stages and exhibit different mitotic location (apical versus nonapical), cleavage plane orientation, and morphology. Interestingly, the emergence of nonapically dividing committed bipolar cell precursors can be linked to an increase in apical crowding caused by the developing photoreceptor cell layer. Furthermore, genetic interference with neuronal subset specification induces ectopic divisions of committed precursors, underlining the finding that progressing morphogenesis can effect precursor division position. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Severe anaemia due to haematopoietic precursor cell destruction in field cases of East Coast Fever in Tanzania.

    PubMed

    Mbassa, G K; Balemba, O; Maselle, R M; Mwaga, N V

    1994-04-01

    Examinations were made on erythrocytes, thrombocytes, leukocytes, lymph nodes, thymus, haemal nodes and bone marrow in field cases of East Coast Fever (ECF) in Tanzania. Seventy-six clinically sick short-horn Zebu and Taurine-Zebu crosses, positive for Theileria parva piroplasms and schizonts and 55 apparently healthy cattle were studied. The syndrome observed was characterised by severe pancytopenia, with massive normocytic, normochromic anaemia, panleukopenia and thrombocytopenia, but no reticulocytes in peripheral blood. The erythrocyte and leukocyte counts, haematocrit and haemoglobin concentrations were greatly decreased compared with those of the healthy cattle. The means +/- SD (with values of healthy cattle in parentheses) were 2.85 +/- 1.10 (6.04 +/- 1.58) x 10(12) l-1, 2.78 +/- 1.70 (10.59 +/- 4.16) x 10(9) l-1, 0.19 +/- 0.06 (0.31 +/- 0.054)1 l-1 and 4.07 +/- 1.62 (7.29 +/- 1.39) mmol l-1 respectively. Lymphoproliferation was low, while lymphocyte destruction (lymphocytolysis) was high. There were very few small schizonts in parotid and prescapular glands. Lymphocytes were extensively destroyed in medullary cords, germinal centres of lymph nodules in cortex and paracortical regions of lymph nodes and haemal nodes. The bone marrow was hypocellular, with only a few haematopoietic precursor erythroid, granulocytic and thrombopoietic cell series. All stages of prorubriblasts and rubricytes had granulated nuclei, some with schizonts. Infection of erythrocytes by merozoites appeared to take place in precursor stages. The destruction of erythroblasts, rubricytes and other haematopoietic cells resulted in anaemia without reticulocytosis, haemoglobinuria and jaundice, accompanied by panleukopenia of extreme neutropenia, lymphopenia and eosinopenia. This indicated that this T. parva strain differs from previously described buffalo- or cattle-derived T. parva infections in causing both haemoproliferation and lymphoproliferation by extensive haematopoietic cell

  19. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    USDA-ARS?s Scientific Manuscript database

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  20. Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

    PubMed Central

    Domenis, Rossana; Cesselli, Daniela; Toffoletto, Barbara; Bourkoula, Evgenia; Caponnetto, Federica; Manini, Ivana; Beltrami, Antonio Paolo; Ius, Tamara; Skrap, Miran; Di Loreto, Carla

    2017-01-01

    A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression. PMID:28107450

  1. Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

    PubMed

    Domenis, Rossana; Cesselli, Daniela; Toffoletto, Barbara; Bourkoula, Evgenia; Caponnetto, Federica; Manini, Ivana; Beltrami, Antonio Paolo; Ius, Tamara; Skrap, Miran; Di Loreto, Carla; Gri, Giorgia

    2017-01-01

    A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

  2. Do we really need to differentiate mesenchymal stem cells into insulin-producing cells for attenuation of the autoimmune responses in type 1 diabetes: immunoprophylactic effects of precursors to insulin-producing cells.

    PubMed

    Sharma, Anshu; Rani, Rajni

    2017-07-12

    Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where pancreatic beta cells are lost before the clinical manifestations of the disease. Administration of mesenchymal stem cells (MSCs) or MSCs differentiated into insulin-producing cells (IPCs) have yielded limited success when used therapeutically. We have evaluated the immunoprophylactic potentials of precursors to insulin-producing cells (pIPCs) and IPCs in nonobese diabetic (NOD) mice to ask a basic question: do we need to differentiate MSCs into IPCs or will pIPCs suffice to attenuate autoimmune responses in T1D? Bone marrow-derived MSCs from Balb/c mice were characterized following the International Society for Cellular Therapy (ISCT) guidelines. MSCs cultured in high-glucose media for 11 to 13 passages were characterized for the expression of pancreatic lineage genes using real-time polymerase chain reaction. Expression of the PDX1 gene in pIPCs was assessed using Western blot and fluorescence-activated cell sorting (FACS). Triple-positive MSCs were differentiated into IPCs using a three-step protocol after sorting them for cell surface markers, i.e. CD29, CD44, and SCA-1. Nonobese diabetic mice were administered pIPCs, IPCs, or phosphate-buffered saline (PBS) into the tail vein at weeks 9 or 10 and followed-up for 29-30 weeks for fasting blood glucose levels. Two consecutive blood sugar levels of more than 250 mg/dl were considered diabetic. MSCs grown in high-glucose media for 11 to 13 passages expressed genes of the pancreatic lineage such as PDX1, beta2, neurogenin, PAX4, Insulin, and glucagon. Furthermore, Western blot and FACS analysis for PDX-1, a transcription factor necessary for beta cell maturation, confirmed that these cells were precursors of insulin-producing cells (pIPCs). NOD mice administered with pIPCs were better protected from developing diabetes with a protective efficacy of 78.4% (p < 0.009); however, administration of IPCs gave protective efficacy of 55% at the end of

  3. Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia

    Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145more » binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.« less

  4. OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    PubMed Central

    Kaur, Ravinder; Aiken, Christopher; Morrison, Ludivine Coudière; Rao, Radhika; Del Bigio, Marc R.; Rampalli, Shravanti; Werbowetski-Ogilvie, Tamra

    2015-01-01

    ABSTRACT Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. PMID:26398939

  5. VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.

    PubMed

    Bauer, S R; Kubagawa, H; Maclennan, I; Melchers, F

    1991-09-15

    We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.

  6. Impact of Lactic Acid on Cell Proliferation and Free Radical Induced Cell Death in Monolayer Cultures of Neural Precursor Cells

    PubMed Central

    Lampe, Kyle J.; Namba, Rachael M.; Silverman, Tyler R.; Bjugstad, Kimberly B.

    2009-01-01

    Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well-tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic-acid based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices. PMID:19408314

  7. Neural Crest-Derived Mesenchymal Cells Require Wnt Signaling for Their Development and Drive Invagination of the Telencephalic Midline

    PubMed Central

    Choe, Youngshik; Zarbalis, Konstantinos S.; Pleasure, Samuel J.

    2014-01-01

    Embryonic neural crest cells contribute to the development of the craniofacial mesenchyme, forebrain meninges and perivascular cells. In this study, we investigated the function of ß-catenin signaling in neural crest cells abutting the dorsal forebrain during development. In the absence of ß-catenin signaling, neural crest cells failed to expand in the interhemispheric region and produced ectopic smooth muscle cells instead of generating dermal and calvarial mesenchyme. In contrast, constitutive expression of stabilized ß-catenin in neural crest cells increased the number of mesenchymal lineage precursors suggesting that ß-catenin signaling is necessary for the expansion of neural crest-derived mesenchymal cells. Interestingly, the loss of neural crest-derived mesenchymal stem cells (MSCs) leads to failure of telencephalic midline invagination and causes ventricular system defects. This study shows that ß-catenin signaling is required for the switch of neural crest cells to MSCs and mediates the expansion of MSCs to drive the formation of mesenchymal structures of the head. Furthermore, loss of these structures causes striking defects in forebrain morphogenesis. PMID:24516524

  8. Opposing Effects of α2- and β-Adrenergic Receptor Stimulation on Quiescent Neural Precursor Cell Activity and Adult Hippocampal Neurogenesis

    PubMed Central

    Prosper, Boris W.; Marathe, Swanand; Husain, Basma F. A.; Kernie, Steven G.; Bartlett, Perry F.; Vaidya, Vidita A.

    2014-01-01

    Norepinephrine regulates latent neural stem cell activity and adult hippocampal neurogenesis, and has an important role in modulating hippocampal functions such as learning, memory and mood. Adult hippocampal neurogenesis is a multi-stage process, spanning from the activation and proliferation of hippocampal stem cells, to their differentiation into neurons. However, the stage-specific effects of noradrenergic receptors in regulating adult hippocampal neurogenesis remain poorly understood. In this study, we used transgenic Nestin-GFP mice and neurosphere assays to show that modulation of α2- and β-adrenergic receptor activity directly affects Nestin-GFP/GFAP-positive precursor cell population albeit in an opposing fashion. While selective stimulation of α2-adrenergic receptors decreases precursor cell activation, proliferation and immature neuron number, stimulation of β-adrenergic receptors activates the quiescent precursor pool and enhances their proliferation in the adult hippocampus. Furthermore, our data indicate no major role for α1-adrenergic receptors, as we did not observe any change in either the activation and proliferation of hippocampal precursors following selective stimulation or blockade of α1-adrenergic receptors. Taken together, our data suggest that under physiological as well as under conditions that lead to enhanced norepinephrine release, the balance between α2- and β-adrenergic receptor activity regulates precursor cell activity and hippocampal neurogenesis. PMID:24922313

  9. Exome-wide Mutation Profile in Benzo[a]pyrene-derived Post-stasis and Immortal Human Mammary Epithelial Cells

    PubMed Central

    Severson, Paul L.; Vrba, Lukas; Stampfer, Martha R.; Futscher, Bernard W.

    2014-01-01

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and towards immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes. PMID:25435355

  10. Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells.

    PubMed

    Severson, Paul L; Vrba, Lukas; Stampfer, Martha R; Futscher, Bernard W

    2014-12-01

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells

    DOE PAGES

    Severson, Paul L.; Vrba, Lukas; Stampfer, Martha R.; ...

    2014-11-04

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutationsmore » were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. In conclusion, the results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes.« less

  12. Efficient production of reactive oxygen species in neural precursor cells after exposure to 250 MeV protons.

    PubMed

    Giedzinski, Erich; Rola, Radoslaw; Fike, John R; Limoli, Charles L

    2005-10-01

    The space radiation environment is composed of highly energetic ions, dominated by protons, that pose a range of potential health risks to astronauts. Traversals of these particles through certain tissues may compromise the viability and/or function of sensitive cells, including neural precursors found within the dentate subgranular zone of the hippocampus. Irradiation has been shown to deplete these cells in vivo, and reductions of these critical cells are believed to impair neurogenesis and cognition. To more fully understand the mechanisms underlying the behavior of these precursor cells after irradiation, we have developed an in vitro neural precursor cell system and used it to assess acute (0-48 h) changes in ROS and mitochondrial end points after exposure to Bragg-peak protons of 250 MeV. Relative ROS levels were increased at nearly all doses (1-10 Gy) and postirradiation times (6-24 h) compared to unirradiated controls. The increase in ROS after proton irradiation was more rapid than that observed with X rays and showed a well-defined dose response at 6 and 24 h, increasing approximately 10% and 3% per gray, respectively. However, by 48 h postirradiation, ROS levels fell below controls and coincided with minor reductions in mitochondrial content. Use of the antioxidant alpha-lipoic acid (before or after irradiation) was shown to eliminate the radiation-induced rise in ROS levels. Our results corroborate earlier studies using X rays and provide further evidence that elevated ROS are integral to the radioresponse of neural precursor cells.

  13. Cross-link regulation of precursor N-cadherin and FGFR1 by GDNF increases U251MG cell viability.

    PubMed

    Tang, Chuan-Xi; Gu, Yan-Xia; Liu, Xin-Feng; Tong, Shu-Yan; Ayanlaja, Abiola A; Gao, Yue; Ji, Guang-Quan; Xiong, Ye; Huang, Lin-Yan; Gao, Dian-Shuai

    2018-07-01

    Glial cell line-derived neurotrophic factor (GDNF) is considered to be involved in the development of glioma. However, uncovering the underlying mechanism of the proliferation of glioma cells is a challenging work in progress. We have identified the binding of the precursor of N-cadherin (proN-cadherin) and GDNF on the cell membrane in previous studies. In the present study, we observed increased U251 Malignant glioma (U251MG) cell viability by exogenous GDNF (50 ng/ml). We also confirmed that the high expression of the proN-cadherin was stimulated by exogenous GDNF. Concurrently, we affirmed that lower expression of proN-cadherin correlated with reduced glioma cell viability. Additionally, we observed glioma cell U251MG viability as the phosphorylation level of FGFR1 at Y653 and Y654 was increased after exogenous GDNF treatment, which led to increased interaction between proN-cadherin and FGFR1 (pY653+Y654). Our experiments presented a new mechanism adopted by GDNF supporting glioma development and indicated a possible therapeutic potential via the inhibition of proN-cadherin/FGFR1 interaction.

  14. Phenotypic stability and plasticity in GMP-derived cells as determined by their underlying regulatory network.

    PubMed

    Ramírez, Carlos; Mendoza, Luis

    2018-04-01

    Blood cell formation has been recognized as a suitable system to study celular differentiation mainly because of its experimental accessibility, and because it shows characteristics such as hierarchical and gradual bifurcated patterns of commitment, which are present in several developmental processes. Although hematopoiesis has been extensively studied and there is a wealth of molecular and cellular data about it, it is not clear how the underlying molecular regulatory networks define or restrict cellular differentiation processes. Here, we infer the molecular regulatory network that controls the differentiation of a blood cell subpopulation derived from the granulocyte-monocyte precursor (GMP), comprising monocytes, neutrophils, eosinophils, basophils and mast cells. We integrate published qualitative experimental data into a model to describe temporal expression patterns observed in GMP-derived cells. The model is implemented as a Boolean network, and its dynamical behavior is studied. Steady states of the network can be clearly identified with the expression profiles of monocytes, mast cells, neutrophils, basophils, and eosinophils, under wild-type and mutant backgrounds. All scripts are publicly available at https://github.com/caramirezal/RegulatoryNetworkGMPModel. lmendoza@biomedicas.unam.mx. Supplementary data are available at Bioinformatics online.

  15. Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells

    PubMed Central

    Kondo, Toru; Raff, Martin

    2004-01-01

    We showed previously that purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. We show here that the conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, we show that the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. Our findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm. PMID:15574597

  16. Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry1[W][OA

    PubMed Central

    Alonso, Ana P.; Piasecki, Rebecca J.; Wang, Yan; LaClair, Russell W.; Shachar-Hill, Yair

    2010-01-01

    The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-13CFru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO3]−/or [H2PO4]− ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure 13C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments. PMID:20442274

  17. Uncontrolled angiogenic precursor expansion causes coronary artery anomalies in mice lacking Pofut1.

    PubMed

    Wang, Yidong; Wu, Bingruo; Lu, Pengfei; Zhang, Donghong; Wu, Brian; Varshney, Shweta; Del Monte-Nieto, Gonzalo; Zhuang, Zhenwu; Charafeddine, Rabab; Kramer, Adam H; Sibinga, Nicolas E; Frangogiannis, Nikolaos G; Kitsis, Richard N; Adams, Ralf H; Alitalo, Kari; Sharp, David J; Harvey, Richard P; Stanley, Pamela; Zhou, Bin

    2017-09-18

    Coronary artery anomalies may cause life-threatening cardiac complications; however, developmental mechanisms underpinning coronary artery formation remain ill-defined. Here we identify an angiogenic cell population for coronary artery formation in mice. Regulated by a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis, these angiogenic cells generate mature coronary arteries. The NOTCH modulator POFUT1 critically regulates this signaling axis. POFUT1 inactivation disrupts signaling events and results in excessive angiogenic cell proliferation and plexus formation, leading to anomalous coronary arteries, myocardial infarction and heart failure. Simultaneous VEGFR2 inactivation fully rescues these defects. These findings show that dysregulated angiogenic precursors link coronary anomalies to ischemic heart disease.Though coronary arteries are crucial for heart function, the mechanisms guiding their formation are largely unknown. Here, Wang et al. identify a unique, endocardially-derived angiogenic precursor cell population for coronary artery formation in mice and show that a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis is key for coronary artery development.

  18. Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

    PubMed

    Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R; Hetman, Michal

    2016-08-01

    The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10(-8) M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. © The Author(s) 2016.

  19. Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

    PubMed

    Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

    2015-05-15

    Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

  20. N-butylidenephthalide attenuates Alzheimer's disease-like cytopathy in Down syndrome induced pluripotent stem cell-derived neurons.

    PubMed

    Chang, Chia-Yu; Chen, Sheng-Mei; Lu, Huai-En; Lai, Syu-Ming; Lai, Ping-Shan; Shen, Po-Wen; Chen, Pei-Ying; Shen, Ching-I; Harn, Horng-Jyh; Lin, Shinn-Zong; Hwang, Shiaw-Min; Su, Hong-Lin

    2015-03-04

    Down syndrome (DS) patients with early-onset dementia share similar neurodegenerative features with Alzheimer's disease (AD). To recapitulate the AD cell model, DS induced pluripotent stem cells (DS-iPSCs), reprogrammed from mesenchymal stem cells in amniotic fluid, were directed toward a neuronal lineage. Neuroepithelial precursor cells with high purity and forebrain characteristics were robustly generated on day 10 (D10) of differentiation. Accumulated amyloid deposits, Tau protein hyperphosphorylation and Tau intracellular redistribution emerged rapidly in DS neurons within 45 days but not in normal embryonic stem cell-derived neurons. N-butylidenephthalide (Bdph), a major phthalide ingredient of Angelica sinensis, was emulsified by pluronic F127 to reduce its cellular toxicity and promote canonical Wnt signaling. Interestingly, we found that F127-Bdph showed significant therapeutic effects in reducing secreted Aβ40 deposits, the total Tau level and the hyperphosphorylated status of Tau in DS neurons. Taken together, DS-iPSC derived neural cells can serve as an ideal cellular model of DS and AD and have potential for high-throughput screening of candidate drugs. We also suggest that Bdph may benefit DS or AD treatment by scavenging Aβ aggregates and neurofibrillary tangles.

  1. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kawata, Shigehisa; Suzuki, Jun; Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types ofmore » cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.« less

  2. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitorsmore » in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.« less

  3. Chiral and achiral phosphine derivatives of alkylidyne tricobalt carbonyl clusters as catalyst precursors for (asymmetric) inter- and intramolecular Pauson-Khand reactions.

    PubMed

    Moberg, Viktor; Mottalib, M Abdul; Sauer, Désirée; Poplavskaya, Yulia; Craig, Donald C; Colbran, Stephen B; Deeming, Antony J; Nordlander, Ebbe

    2008-05-14

    Phosphine derivatives of alkylidyne tricobalt carbonyl clusters have been tested as catalysts/catalyst precursors in intermolecular and (asymmetric) intramolecular Pauson-Khand reactions. A number of new phosphine derivatives of the tricobalt alkylidyne clusters [Co3(micro3-CR)(CO)9] (R = H, CO2Et) were prepared and characterised. The clusters [Co3(micro3-CR)(CO)9-x(PR'3)x] (PR'3 = achiral or chiral monodentate phosphine, x = 1-3) and [Co3(micro3-CR)(CO)7)(P-P)] (P-P = chiral diphosphine; 1,1'- and 1,2-structural isomers) were assayed as catalysts for intermolecular and (asymmetric) intramolecular Pauson-Khand reactions. The phosphine-substituted tricobalt clusters proved to be viable catalysts/catalyst precursors that gave moderate to very good product yields (up to approximately 90%), but the enantiomeric excesses were too low for the clusters to be of practical use in the asymmetric reactions.

  4. MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

    PubMed Central

    Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

    2014-01-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP), and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20 ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1 hour of mechanical vibration with 20 µm displacement at a frequency of 4 Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5 days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells were determined after 1 hour mechanical vibration, while protein production of the DC-STAMP was determined after 6 hours of post incubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduce DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. PMID:23994170

  5. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    ERIC Educational Resources Information Center

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  6. Mesenchymal precursor cells in the blood of normal individuals

    PubMed Central

    Zvaifler, Nathan J; Marinova-Mutafchieva, Lilla; Adams, Gill; Edwards, Christopher J; Moss, Jill; Burger, Jan A; Maini, Ravinder N

    2000-01-01

    collagen, and BMP receptors (heterodimeric structures expressed on mesenchymal lineage cells). The cultured cells also stained strongly for the SH-2 (endoglin) antigen, a putative marker for marrow MSCs. BMPCs express the gene for SDF-1, a potent stroma-derived CXCα chemokine. Discussion: In the circulation of normal individuals is a small population of CD34- mononuclear cells that proliferate rapidly in culture as an adherent population with a variable morphology, display cytoskeletal, cytoplasmic, and surface markers of mesenchymal precursors, and differentiate into several lineages (fibroblasts, osteoblasts, and adipocytes). These are all features found in bone-marrow-derived MSCs. Therefore, autologous blood could provide cells useful for tissue engineering and gene therapy. In addition, the demonstration of similar cells in the inflammatory joint fluids and synovium of patients with rheumatoid arthritis (RA) suggests that these cells may play a role in the pathogenesis of RA. PMID:11056678

  7. Mesenchymal stem cells derived from peripheral blood protects against ischemia.

    PubMed

    Ukai, Ryo; Honmou, Osamu; Harada, Kuniaki; Houkin, Kiyohiro; Hamada, Hirofumi; Kocsis, Jeffery D

    2007-03-01

    Intravenous delivery of mesenchymal stem cells (MSCs) prepared from bone marrow (BMSCs) reduces infarction volume and ameliorates functional deficits in a rat cerebral ischemia model. MSC-like multipotent precursor cells (PMSCs) have also been suggested to exist in peripheral blood. To test the hypothesis that treatment with PMSCs may have a therapeutic benefit in stroke, we compared the efficacy of systemic delivery of BMSCs and PMSCs. A permanent middle cerebral artery occlusion (MCAO) in rat was induced by intraluminal vascular occlusion with a microfilament. Rat BMSCs and PMSCs were prepared in culture and intravenously injected into the rats 6 h after MCAO. Lesion size was assessed at 6 h, and 1, 3, and 7 days using MR imaging and histology. The hemodynamic change of cerebral blood perfusion on stroke was assessed the same times using perfusion-weighted image (PWI). Functional outcome was assessed using the treadmill stress test. Both BMSCs and PMSCs treated groups had reduced lesion volume, improved regional cerebral blood flow, and functional improvement compared to the control group. The therapeutic benefits of both MSC-treated groups were similar. These data suggest that PMSCs derived from peripheral blood could be an important cell source of cell therapy for stroke.

  8. Case report of precursor B-cell lymphoblastic lymphoma presenting as syncope and cardiac mass in a nonimmunocompromised child.

    PubMed

    Hahn, Barry; Rao, Sudha; Shah, Binita

    2007-08-01

    We report the case of a previously healthy, 10-year-old boy who presented to the emergency department with a syncopal episode. In the emergency department, the patient was diagnosed with a right atrial mass, later identified as a precursor B-cell lymphoblastic lymphoma (LL). Most causes of syncope in children are not life threatening. In most cases, it indicates a predisposition to vasovagal episodes. Lymphomas account for approximately 7% of malignancies among children younger than 20 years, are more common in white males and immunocompromised patients, and are predominantly tumors of T-cell origin. Children with non-Hodgkin lymphoma usually present with extranodal disease, most frequently involving the abdomen (31%), mediastinum (26%), or head and neck (29%). Our patient was unique in that he was a nonimmunocompromised, black boy, presenting with syncope in the setting of a large atrial mass identified as a precursor B-cell LL. To our knowledge, there are no reported cases of precursor B-cell LL presenting as syncope and a cardiac mass.

  9. In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. III. Spontaneous and natural cytotoxic effector cells.

    PubMed Central

    Herbert, A G; Le Gros, G S; Bidawid, S; Watson, J D

    1984-01-01

    Cytotoxic effector cell populations in murine spleen can be characterized by the phenotype of the cytotoxic cells or the nature of target cells. Lytic events can be antigen-specific, MHC-restricted and clonal, or target cell-specific but apparently non-MHC-restricted. Two cytotoxic effectors of this latter category are spontaneous and natural killers. Normal spleen cells from (BALB/c X DBA/2J)F1 mice (CDF1) cultured without added antigen develop a population of Thy-1+ spontaneous cytotoxic lymphocytes (SCTL) that lyse the DBA/2J mastocytoma P815, as well as the BALB/c-derived plasmacytomas MOPC-11 and SP2/0. Cold target competition experiments reveal the BALB/c-derived plasmacytomas MOPC-11, SP2/0, J558 and the A strain-derived T cell lymphoma YAC-1, but not normal lymphoblasts, block the lysis of P815 target cells. Thus, while these tumour cells appear to express common antigens which are recognized by SCTL cells, plasmacytomas such as J558 are not susceptible to lysis by SCTL. The relationship of SCTL to natural killer (NK) cells was examined. In-vivo treatment of mice with monoclonal anti-Thy-1 antibody leads to a rapid loss of SCTL and precursors from the spleen, but there is a concomitant increase in NK cell activity. PMID:6607213

  10. Functional electrical stimulation-facilitated proliferation and regeneration of neural precursor cells in the brains of rats with cerebral infarction

    PubMed Central

    Xiang, Yun; Liu, Huihua; Yan, Tiebin; Zhuang, Zhiqiang; Jin, Dongmei; Peng, Yuan

    2014-01-01

    Previous studies have shown that proliferation of endogenous neural precursor cells cannot alone compensate for the damage to neurons and axons. From the perspective of neural plasticity, we observed the effects of functional electrical stimulation treatment on endogenous neural precursor cell proliferation and expression of basic fibroblast growth factor and epidermal growth factor in the rat brain on the infarct side. Functional electrical stimulation was performed in rat models of acute middle cerebral artery occlusion. Simultaneously, we set up a placebo stimulation group and a sham-operated group. Immunohistochemical staining showed that, at 7 and 14 days, compared with the placebo group, the numbers of nestin (a neural precursor cell marker)-positive cells in the subgranular zone and subventricular zone were increased in the functional electrical stimulation treatment group. Western blot assays and reverse-transcription PCR showed that total protein levels and gene expression of epidermal growth factor and basic fibroblast growth factor were also upregulated on the infarct side. Prehensile traction test results showed that, at 14 days, prehension function of rats in the functional electrical stimulation group was significantly better than in the placebo group. These results suggest that functional electrical stimulation can promote endogenous neural precursor cell proliferation in the brains of acute cerebral infarction rats, enhance expression of basic fibroblast growth factor and epidermal growth factor, and improve the motor function of rats. PMID:25206808

  11. Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

    PubMed Central

    Paris, Irmgard; Muñoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

    2011-01-01

    Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

  12. Role of precursor crystal structure on electrochemical performance of carbide-derived carbon electrodes

    NASA Astrophysics Data System (ADS)

    Palazzo, Benjamin; Norris, Zach; Taylor, Greg; Yu, Lei; Lofland, Samuel; Hettinger, Jeffrey

    2015-03-01

    Binary carbides with hexagonal and cubic crystal structures have been synthesized by reactive magnetron sputtering of vanadium and other transition metals in acetylene or methane gas mixed with argon. The binary carbides are converted to carbide-derived carbon (CDC) films using chlorine gas in a post-deposition process in an external vacuum reaction furnace. Residual chlorine has been removed using an annealing step in a hydrogen atmosphere. The CDC materials have been characterized by x-ray diffraction, x-ray fluorescence, and scanning electron microscopy. The performance of the CDC materials in electrochemical device applications has been measured with the hexagonal phase precursor demonstrating a significantly higher specific capacitance in comparison to that of the cubic phase. We report these results and pore-size distributions of these and similar materials.

  13. Effect of secondary organic aerosol from isoprene-derived hydroxyhydroperoxides on the expression of oxidative stress response genes in human bronchial epithelial cells.

    PubMed

    Arashiro, Maiko; Lin, Ying-Hsuan; Zhang, Zhenfa; Sexton, Kenneth G; Gold, Avram; Jaspers, Ilona; Fry, Rebecca C; Surratt, Jason D

    2018-02-21

    Isoprene-derived secondary organic aerosol (SOA), which comprise a large portion of atmospheric fine particulate matter (PM 2.5 ), can be formed through various gaseous precursors, including isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene hydroxyhydroperoxides (ISOPOOH). The composition of the isoprene-derived SOA affects its reactive oxygen species (ROS) generation potential and its ability to alter oxidative stress-related gene expression. In this study we assess effects of isoprene SOA derived solely from ISOPOOH oxidation on human bronchial epithelial cells by measuring the gene expression changes in 84 oxidative stress-related genes. In addition, the thiol reactivity of ISOPOOH-derived SOA was measured through the dithiothreitol (DTT) assay. Our findings show that ISOPOOH-derived SOA alter more oxidative-stress related genes than IEPOX-derived SOA but not as many as MAE-derived SOA on a mass basis exposure. More importantly, we found that the different types of SOA derived from the various gaseous precursors (MAE, IEPOX, and ISOPOOH) have unique contributions to changes in oxidative stress-related genes that do not total all gene expression changes seen in exposures to atmospherically relevant compositions of total isoprene-derived SOA mixtures. This study suggests that amongst the different types of known isoprene-derived SOA, MAE-derived SOA are the most potent inducer of oxidative stress-related gene changes but highlights the importance of considering isoprene-derived SOA as a total mixture for pollution controls and exposure studies.

  14. Brain-derived neurotrophic factor (BDNF) and its precursor (proBDNF) in genetically defined fear-induced aggression.

    PubMed

    Ilchibaeva, Tatiana V; Kondaurova, Elena M; Tsybko, Anton S; Kozhemyakina, Rimma V; Popova, Nina K; Naumenko, Vladimir S

    2015-09-01

    The brain-derived neurotrophic factor (BDNF), its precursor (proBDNF) and BDNF mRNA levels were studied in the brain of wild rats selectively bred for more than 70 generations for either high level or for the lack of affective aggressiveness towards man. Significant increase of BDNF mRNA level in the frontal cortex and increase of BDNF level in the hippocampus of aggressive rats was revealed. In the midbrain and hippocampus of aggressive rats proBDNF level was increased, whereas BDNF/proBDNF ratio was reduced suggesting the prevalence and increased influence of proBDNF in highly aggressive rats. In the frontal cortex, proBDNF level in aggressive rats was decreased. Thus, considerable structure-specific differences in BDNF and proBDNF levels as well as in BDNF gene expression between highly aggressive and nonaggressive rats were shown. The data suggested the implication of BDNF and its precursor proBDNF in the mechanism of aggressiveness and in the creation of either aggressive or nonaggressive phenotype. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Loss of p19Arf in a Rag1−/− B-cell precursor population initiates acute B-lymphoblastic leukemia

    PubMed Central

    Hauer, Julia; Mullighan, Charles; Morillon, Estelle; Wang, Gary; Bruneau, Julie; Brousse, Nicole; Lelorc'h, Marc; Romana, Serge; Boudil, Amine; Tiedau, Daniela; Kracker, Sven; Bushmann, Frederic D.; Borkhardt, Arndt; Fischer, Alain; Hacein-Bey-Abina, Salima

    2011-01-01

    In human B-acute lymphoblastic leukemia (B-ALL), RAG1-induced genomic alterations are important for disease progression. However, given that biallelic loss of the RAG1 locus is observed in a subset of cases, RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf−/−Rag1−/− mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model, we identified a new, Rag1-independent leukemia-initiating mechanism originating from a Sca1+CD19+ precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM, a similar CD34+CD19+ population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans. PMID:21622646

  16. Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features

    PubMed Central

    Schwab, Claire J.; Chilton, Lucy; Morrison, Heather; Jones, Lisa; Al-Shehhi, Halima; Erhorn, Amy; Russell, Lisa J.; Moorman, Anthony V.; Harrison, Christine J.

    2013-01-01

    In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality. PMID:23508010

  17. High CD45 surface expression determines relapse risk in children with precursor B-cell and T-cell acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol

    PubMed Central

    Cario, Gunnar; Rhein, Peter; Mitlöhner, Rita; Zimmermann, Martin; Bandapalli, Obul R.; Romey, Renja; Moericke, Anja; Ludwig, Wolf-Dieter; Ratei, Richard; Muckenthaler, Martina U.; Kulozik, Andreas E.; Schrappe, Martin; Stanulla, Martin; Karawajew, Leonid

    2014-01-01

    Further improvement of outcome in childhood acute lymphoblastic leukemia could be achieved by identifying additional high-risk patients who may benefit from intensified treatment. We earlier identified PTPRC (CD45) gene expression as a potential new stratification marker and now analyzed the prognostic relevance of CD45 protein expression. CD45 was measured by flow cytometry in 1065 patients treated according to the ALL-BFM-2000 protocol. The 75th percentile was used as cut-off to distinguish a CD45-high from a CD45-low group. As mean CD45 expression was significantly higher in T-cell acute lymphoblastic leukemia than in B-cell-precursor acute lymphoblastic leukemia (P<0.0001), the analysis was performed separately in both groups. In B-cell-precursor acute lymphoblastic leukemia we observed a significant association of a high CD45 expression with older age, high initial white blood cell count, ETV6/RUNX1 negativity, absence of high hyperdiploidy (P<0.0001), MLL/AF4 positivity (P=0.002), BCR/ABL1 positivity (P=0.007), prednisone poor response (P=0.002) and minimal residual disease (P<0.0001). In T-cell acute lymphoblastic leukemia we observed a significant association with initial white blood cell count (P=0.0003), prednisone poor response (P=0.01), and minimal residual disease (P=0.02). Compared to CD45-low patients, CD45-high patients had a lower event-free survival rate (B-cell-precursor acute lymphoblastic leukemia: 72±3% versus 86±1%, P<0.0001; T-cell acute lymphoblastic leukemia: 60±8% versus 78±4%, P=0.02), which was mainly attributable to a higher cumulative relapse incidence (B-cell-precursor acute lymphoblastic leukemia: 22±3% versus 11±1%, P<0.0001; T-cell acute lymphoblastic leukemia: 31±8% versus 11±3%, P=0.003) and kept its significance in multivariate analysis considering sex, age, initial white blood cell count, and minimal residual disease in B-cell-precursor- and T-cell acute lymphoblastic leukemia, and additionally presence of ETV6/RUNX1

  18. High CD45 surface expression determines relapse risk in children with precursor B-cell and T-cell acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol.

    PubMed

    Cario, Gunnar; Rhein, Peter; Mitlöhner, Rita; Zimmermann, Martin; Bandapalli, Obul R; Romey, Renja; Moericke, Anja; Ludwig, Wolf-Dieter; Ratei, Richard; Muckenthaler, Martina U; Kulozik, Andreas E; Schrappe, Martin; Stanulla, Martin; Karawajew, Leonid

    2014-01-01

    Further improvement of outcome in childhood acute lymphoblastic leukemia could be achieved by identifying additional high-risk patients who may benefit from intensified treatment. We earlier identified PTPRC (CD45) gene expression as a potential new stratification marker and now analyzed the prognostic relevance of CD45 protein expression. CD45 was measured by flow cytometry in 1065 patients treated according to the ALL-BFM-2000 protocol. The 75(th) percentile was used as cut-off to distinguish a CD45-high from a CD45-low group. As mean CD45 expression was significantly higher in T-cell acute lymphoblastic leukemia than in B-cell-precursor acute lymphoblastic leukemia (P<0.0001), the analysis was performed separately in both groups. In B-cell-precursor acute lymphoblastic leukemia we observed a significant association of a high CD45 expression with older age, high initial white blood cell count, ETV6/RUNX1 negativity, absence of high hyperdiploidy (P<0.0001), MLL/AF4 positivity (P=0.002), BCR/ABL1 positivity (P=0.007), prednisone poor response (P=0.002) and minimal residual disease (P<0.0001). In T-cell acute lymphoblastic leukemia we observed a significant association with initial white blood cell count (P=0.0003), prednisone poor response (P=0.01), and minimal residual disease (P=0.02). Compared to CD45-low patients, CD45-high patients had a lower event-free survival rate (B-cell-precursor acute lymphoblastic leukemia: 72 ± 3% versus 86 ± 1%, P<0.0001; T-cell acute lymphoblastic leukemia: 60 ± 8% versus 78 ± 4%, P=0.02), which was mainly attributable to a higher cumulative relapse incidence (B-cell-precursor acute lymphoblastic leukemia: 22 ± 3% versus 11 ± 1%, P<0.0001; T-cell acute lymphoblastic leukemia: 31 ± 8% versus 11 ± 3%, P=0.003) and kept its significance in multivariate analysis considering sex, age, initial white blood cell count, and minimal residual disease in B-cell-precursor- and T-cell acute lymphoblastic leukemia, and additionally

  19. Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

    PubMed

    Rajaei, Bahareh; Shamsara, Mehdi; Amirabad, Leila Mohammadi; Massumi, Mohammad; Sanati, Mohammad Hossein

    2017-10-01

    Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic β-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional β-cells, including expression of critical β-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells

    PubMed Central

    Honda, Shozo; Kawamura, Takuya; Loher, Phillipe; Morichika, Keisuke; Rigoutsos, Isidore

    2017-01-01

    Abstract Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5′-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5′-tRNA halves as well, suggesting a previously uncharacterized link between 5′-tRNA halves and td-piRNAs. An increase in levels of the 5′-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5′-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to −1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs. PMID:28645172

  1. Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.

    PubMed

    Øbro, Nina F; Ryder, Lars P; Madsen, Hans O; Andersen, Mette K; Lausen, Birgitte; Hasle, Henrik; Schmiegelow, Kjeld; Marquart, Hanne V

    2012-01-01

    Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

  2. Immunosuppressive myeloid-derived suppressor cells can be converted into immunogenic APCs with the help of activated NKT cells: an alternative cell-based antitumor vaccine.

    PubMed

    Ko, Hyun-Jeong; Lee, Jung-Mi; Kim, Yeon-Jeong; Kim, Yun-Sun; Lee, Kyoo-A; Kang, Chang-Yuil

    2009-02-15

    Myeloid-derived suppressor cells (MDSCs), which are known to be accumulated in the blood, spleen, and bone marrow of tumor-bearing mice and cancer patients, were tested as APCs for a cellular vaccine because they have phenotypical similarity with inflammatory monocytes and may be differentiated from the same precursors as monocytes. Although MDSCs have immunosuppressive properties, in vivo transferred MDSCs, which present tumor Ag and NKT cell ligand (alpha-galactosylceramide), significantly prolonged survival time in metastatic tumor-bearing mice in a CD8(+) cell-, NK cell-, and NKT cell-dependent manner vs a CD4(+) T cell- and host dendritic cell-independent manner. Major concerns about using MDSCs as APCs in a vaccine are their suppression of CTLs and their induction of Foxp3(+) regulatory T cells. However, alpha-galactosylceramide-loaded MDSCs did not suppress CD4(+) and CD8(+) T cells and allowed for the generation of Ag-specific CTL immunity without increasing the generation of regulatory T cells. Furthermore, stimulation with activated NKT cells induced changes on MDSCs in phenotypical or maturation markers, including CD11b, CD11c, and CD86. Taken together, these findings suggest that NKT cells facilitate the conversion of immunosuppressive MDSCs into immunogenic APCs, eliciting successful antitumor immunity and providing the basis for alternative cell-based vaccines.

  3. A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation.

    PubMed

    Patenaude, Alexandre; Woerher, Stefan; Umlandt, Patricia; Wong, Fred; Ibrahim, Rawa; Kyle, Alastair; Unger, Sandy; Fuller, Megan; Parker, Jeremy; Minchinton, Andrew; Eaves, Connie J; Karsan, Aly

    2015-09-01

    Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1(+) precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146(+)/CD45(-)/CD31(-/lo) pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1(hi)/CD146(-)/CD45(-)/CD31(-). The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1(-/lo)/CD146(-)/CD45(-)/CD31(-) cells. Our results suggest that a local Sca1(hi)/CD146(-)/CD45(-)/CD31(-) pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies

    PubMed Central

    Gonsalves, Wilson I.; Hitosugi, Taro; Ghosh, Toshi; Jevremovic, Dragan; Petterson, Xuan-Mai; Wellik, Linda; Kumar, Shaji K.; Nair, K. Sreekumaran

    2018-01-01

    The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention. PMID:29321378

  5. Ewing's sarcoma precursors are highly enriched in embryonic osteochondrogenic progenitors.

    PubMed

    Tanaka, Miwa; Yamazaki, Yukari; Kanno, Yohei; Igarashi, Katsuhide; Aisaki, Ken-ichi; Kanno, Jun; Nakamura, Takuro

    2014-07-01

    Ewing's sarcoma is a highly malignant bone tumor found in children and adolescents, and the origin of this malignancy is not well understood. Here, we introduced a Ewing's sarcoma-associated genetic fusion of the genes encoding the RNA-binding protein EWS and the transcription factor ETS (EWS-ETS) into a fraction of cells enriched for osteochondrogenic progenitors derived from the embryonic superficial zone (eSZ) of long bones collected from late gestational murine embryos. EWS-ETS fusions efficiently induced Ewing's sarcoma-like small round cell sarcoma formation by these cells. Analysis of the eSZ revealed a fraction of a precursor cells that express growth/differentiation factor 5 (Gdf5), the transcription factor Erg, and parathyroid hormone-like hormone (Pthlh), and selection of the Pthlh-positive fraction alone further enhanced EWS-ETS-dependent tumor induction. Genes downstream of the EWS-ETS fusion protein were quite transcriptionally active in eSZ cells, especially in regions in which the chromatin structure of the ETS-responsive locus was open. Inhibition of β-catenin, poly (ADP-ribose) polymerase 1 (PARP1), or enhancer of zeste homolog 2 (EZH2) suppressed cell growth in a murine model of Ewing's sarcoma, suggesting the utility of the current system as a preclinical model. These results indicate that eSZ cells are highly enriched in precursors to Ewing's sarcoma and provide clues to the histogenesis of Ewing's sarcoma in bone.

  6. Adipose-derived cells.

    PubMed

    Meliga, Emanuele; Strem, Brian M; Duckers, H J; Serruys, Patrick W

    2007-01-01

    Heart failure is by far the most common cause of hospitalization in Western countries, with onerous economic consequences. Cell therapy holds great promise for use in tissue regeneration and is increasingly used in an effort to improve outcomes in cardiac disease. Recently it has been shown that adipose tissue, in addition to committed adipogenic, endothelial progenitor cells and pluripotent vascular progenitor cells, also contains multipotent cell types (adipose-derived stem cells, ADSCs) that, in cell culture conditions, have shown to have an impressive developmental plasticity including the ability to undergo multilineage differentiation and self-renewal. ADSCs express multiple CD marker antigens similar to those observed on MSCs and are also capable of secreting a large number of angiogenesis-related cytokines, including vascular endothelial growth factor, granulocyte/macrophage colony stimulating factor, stromal-derived factor-1alpha, and hepatocyte growth factor. Adipose tissue can be harvested in large quantities with minimal morbidity in several regions of the body and, on average, 100 ml of human adipose tissue yields about 1 x 10(6) stem cells. Studies conducted in porcine AMI models have shown a significant LV functional improvement, with no report of any potentially fatal arrhythmias. The APOLLO trial, a prospective, double blind, randomized, placebo-controlled trial currently in the recruiting phase, is a "first-in-man" study that explores the safety and feasibility of ADSC transplantation in patients with acute MI.

  7. Single-Cell Detection of Secreted Aβ and sAPPα from Human IPSC-Derived Neurons and Astrocytes.

    PubMed

    Liao, Mei-Chen; Muratore, Christina R; Gierahn, Todd M; Sullivan, Sarah E; Srikanth, Priya; De Jager, Philip L; Love, J Christopher; Young-Pearse, Tracy L

    2016-02-03

    Secreted factors play a central role in normal and pathological processes in every tissue in the body. The brain is composed of a highly complex milieu of different cell types and few methods exist that can identify which individual cells in a complex mixture are secreting specific analytes. By identifying which cells are responsible, we can better understand neural physiology and pathophysiology, more readily identify the underlying pathways responsible for analyte production, and ultimately use this information to guide the development of novel therapeutic strategies that target the cell types of relevance. We present here a method for detecting analytes secreted from single human induced pluripotent stem cell (iPSC)-derived neural cells and have applied the method to measure amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα), analytes central to Alzheimer's disease pathogenesis. Through these studies, we have uncovered the dynamic range of secretion profiles of these analytes from single iPSC-derived neuronal and glial cells and have molecularly characterized subpopulations of these cells through immunostaining and gene expression analyses. In examining Aβ and sAPPα secretion from single cells, we were able to identify previously unappreciated complexities in the biology of APP cleavage that could not otherwise have been found by studying averaged responses over pools of cells. This technique can be readily adapted to the detection of other analytes secreted by neural cells, which would have the potential to open new perspectives into human CNS development and dysfunction. We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single-cell analytical platform. First, we

  8. Dual-Responsive Metabolic Precursor and Light-Up AIEgen for Cancer Cell Bio-orthogonal Labeling and Precise Ablation.

    PubMed

    Hu, Fang; Yuan, Youyong; Wu, Wenbo; Mao, Duo; Liu, Bin

    2018-06-05

    Metabolic glycoengineering of unnatural glycans with bio-orthogonal chemical groups and a subsequent click reaction with fluorescent probes have been widely used in monitoring various bioprocesses. Herein, we developed a dual-responsive metabolic precursor that could specifically generate unnatural glycans with azide groups on the membrane of targeted cancer cells with high selectivity. Moreover, a water-soluble fluorescent light-up probe with aggregation-induced emission (AIE) was synthesized, which turned its fluorescence on upon a click reaction with azide groups on the cancer cell surface, enabling special cancer cell imaging with low background signal. Furthermore, the probe can generate 1 O 2 upon light irradiation, fulfilling its dual role as an imaging and therapeutic agent for cancer cells. Therefore, the concepts of the cancer-cell-specific metabolic precursor cRGD-S-Ac 3 ManNAz and the AIE light-up probe are promising in bio-orthogonal labeling and cancer-specific imaging and therapy.

  9. Synthesis of a Possible Precursor of α-Amylase in Wheat Aleurone Cells 1

    PubMed Central

    Okita, Thomas W.; Decaleya, Roberto; Rappaport, Lawrence

    1979-01-01

    α-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific λ-globulin for α-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic α-amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of α-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of α-amylase in aleurone cells. Images PMID:16660677

  10. IMMUNOLOGIC MEMORY CELLS OF BONE MARROW ORIGIN

    PubMed Central

    Miller, Harold C.; Cudkowicz, Gustavo

    1972-01-01

    Individual immunocompetent precursor cells of (C57BL/10 x C3H)F1 mouse marrow generate, on transplantation, three to five times more antibody-forming cells localized in recipient spleens during secondary than during primary immune responses. The increased burst size is immunologically specific since antigens of horse and chicken erythrocytes and of Salmonella typhimurium do not cause this effect in marrow cells responsive to sheep red blood cells. Both sensitized and nonsensitized precursors require the helper function of thymus-derived cells and antigen for the final steps of differentiation and maturation. The burst size of primed precursor cells is the same after cooperative interactions with virgin or educated helper cells of thymic origin. The greater potential of these marrow precursors may be attributable to self-replication and migration before differentiation into antibody-forming descendants. In fact, the progeny cells of primed precursor units are distributed among a multiplicity of foci, whereas those of nonimmune precursors are clustered into one focus. The described properties of specifically primed marrow precursors are those underlying immunologic memory. It remains to be established whether memory cells are induced or selected by antigens and whether the thymus plays a role in this process. PMID:4553850

  11. Polymer/Nanocrystal Hybrid Solar Cells: Influence of Molecular Precursor Design on Film Nanomorphology, Charge Generation and Device Performance

    PubMed Central

    MacLachlan, Andrew J; Rath, Thomas; Cappel, Ute B; Dowland, Simon A; Amenitsch, Heinz; Knall, Astrid-Caroline; Buchmaier, Christine; Trimmel, Gregor; Nelson, Jenny; Haque, Saif A

    2015-01-01

    In this work, molecular tuning of metal xanthate precursors is shown to have a marked effect on the heterojunction morphology of hybrid poly(3-hexylthiophene-2,5-diyl) (P3HT)/CdS blends and, as a result, the photochemical processes and overall performance of in situ fabricated hybrid solar cells. A series of cadmium xanthate complexes is synthesized for use as in situ precursors to cadmium sulfide nanoparticles in hybrid P3HT/CdS solar cells. The formation of CdS domains is studied by simultaneous GIWAXS (grazing incidence wide-angle X-ray scattering) and GISAXS (grazing incidence small-angle X-ray scattering), revealing knowledge about crystal growth and the formation of different morphologies observed using TEM (transmission electron microscopy). These measurements show that there is a strong relationship between precursor structure and heterojunction nanomorphology. A combination of TAS (transient absorption spectroscopy) and photovoltaic device performance measurements is used to show the intricate balance required between charge photogeneration and percolated domains in order to effectively extract charges to maximize device power conversion efficiencies. This study presents a strong case for xanthate complexes as a useful route to designing optimal heterojunction morphologies for use in the emerging field of hybrid organic/inorganic solar cells, due to the fact that the nanomorphology can be tuned via careful design of these precursor materials. PMID:25866496

  12. Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

    PubMed

    Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

    2016-07-01

    First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835. © 2016 AlphaMed Press.

  13. Intratumoral conversion of adrenal androgen precursors drives androgen receptor-activated cell growth in prostate cancer more potently than de novo steroidogenesis.

    PubMed

    Kumagai, Jinpei; Hofland, Johannes; Erkens-Schulze, Sigrun; Dits, Natasja F J; Steenbergen, Jacobie; Jenster, Guido; Homma, Yukio; de Jong, Frank H; van Weerden, Wytske M

    2013-11-01

    Despite an initial response to hormonal therapy, patients with advanced prostate cancer (PC) almost always progress to castration-resistant disease (CRPC). Although serum testosterone (T) is reduced by androgen deprivation therapy, intratumoral T levels in CRPC are comparable to those in prostate tissue of eugonadal men. These levels could originate from intratumoral conversion of adrenal androgens and/or from de novo steroid synthesis. However, the relative contribution of de novo steroidogenesis to AR-driven cell growth is unknown. The relative contribution of androgen biosynthetic pathways to activate androgen receptor (AR)-regulated cell growth and expression of PSA, FKBP5, and TMPRSS2 was studied at physiologically relevant levels of adrenal androgen precursors and intermediates of de novo androgen biosynthesis in human prostate cancer cell lines, PC346C, VCaP, and LNCaP. In PC346C and VCaP, responses to pregnenolone and progesterone were absent or minimal, while large effects of adrenal androgen precursors were found. VCaP CRPC clones overexpressing CYP17A1 did not acquire an increased ability to use pregnenolone or progesterone to activate AR. In contrast, all precursors stimulated growth and gene expression in LNCaP cells, presumably resulting from the mutated AR in these cells. Our data indicate that at physiological levels of T precursors PC cells can generally convert adrenal androgens, while de novo steroidogenesis is not generally possible in PC cells and is not able to support AR transactivation and PC growth. © 2013 Wiley Periodicals, Inc.

  14. Economical and green synthesis of bagasse-derived fluorescent carbon dots for biomedical applications.

    PubMed

    Du, Fengyi; Zhang, Miaomiao; Li, Xiaofeng; Li, Jianan; Jiang, Xinyi; Li, Zhang; Hua, Ye; Shao, Genbao; Jin, Jie; Shao, Qixiang; Zhou, Ming; Gong, Aihua

    2014-08-08

    Carbon quantum dots (CDs) are promising nanomaterials in biomedical, photocatalytical and photoelectronic applications. However, determining how to explore an ideal precursor for a renewable carbon resource is still an interesting challenge. Here, for the first time, we report that renewable wastes of bagasse as a new precursor were prepared for fluorescent CDs by a hydrothermal carbonization (HTC) process. The characterization results show that such bagasse-derived CDs are monodispersed, contain quasi spherical particles with a diameter of about 1.8 nm and exhibit favorable photoluminescence properties, super-high photostability and good dispersibility in water. Most importantly, bagasse-derived CDs have good biocompatibility and can be easily and quickly internalized by living cancer cells; they can also be used for multicolour biolabeling and bioimaging in cancer cells. It is suggested that bagasse-derived CDs might have potential applications in biomedical and photoelectronic fields.

  15. Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

    PubMed Central

    1995-01-01

    Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

  16. [Application of precursor ion scanning method in rapid screening of illegally added phosphodiesterase-5 inhibitors and their unknown derivatives in Chinese traditional patent medicines and health foods].

    PubMed

    Sun, Jing; Cao, Ling; Feng, Youlong; Tan, Li

    2014-11-01

    The compounds with similar structure often have similar pharmacological activities. So it is a trend for illegal addition that new derivatives of effective drugs are synthesized to avoid the statutory test. This bring challenges to crack down on illegal addition behavior, however, modified derivatives usually have similar product ions, which allow for precursor ion scanning. In this work, precursor ion scanning mode of a triple quadrupole mass spectrometer was first applied to screen illegally added drugs in complex matrix such as Chinese traditional patent medicines and healthy foods. Phosphodiesterase-5 inhibitors were used as experimental examples. Through the analysis of the structure and mass spectrum characteristics of the compounds, phosphodiesterase-5 inhibitors were classified, and their common product ions were screened by full scan of product ions of typical compounds. Then high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method with precursor ion scanning mode was established based on the optimization of MS parameters. The effect of mass parameters and the choice of fragment ions were also studied. The method was applied to determine actual samples and further refined. The results demonstrated that this method can meet the need of rapid screening of unknown derivatives of phosphodiesterase-5 inhibitors in complex matrix, and prevent unknown derivatives undetected. This method shows advantages in sensitivity, specificity and efficiency, and is worth to be further investigated.

  17. Lack of effect of a granulocyte proliferation inhibitor or their committed precursor cells.

    PubMed

    Lord, B I; Testa, N G; Wright, E G; Banerjee, R K

    1977-05-01

    Using the agar culture technique, we have measured the effect of granulocyte extracts GCE (and of erythrocyte-RCE and lymph node extracts-LNE) on the growth and proliferation of the committed granulocytic precursor cells, CFU-C. In addition we have determined their effects on the proliferation of the developing colony cells and on the ultimate cell production in the colonies. The results show that GCE has no effect on the growth or proliferative activity on the CFU-C. It does, however, reduce both the autoradiographic labelling indices of the developing colony cells and the net colony cellularities, acting as a cell cycle modulator. These are effects specific to the GCE since at the dose levels used, neither RCE nor LNE affected these measurements.

  18. Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

    PubMed

    Takata, Kazuyuki; Kozaki, Tatsuya; Lee, Christopher Zhe Wei; Thion, Morgane Sonia; Otsuka, Masayuki; Lim, Shawn; Utami, Kagistia Hana; Fidan, Kerem; Park, Dong Shin; Malleret, Benoit; Chakarov, Svetoslav; See, Peter; Low, Donovan; Low, Gillian; Garcia-Miralles, Marta; Zeng, Ruizhu; Zhang, Jinqiu; Goh, Chi Ching; Gul, Ahmet; Hubert, Sandra; Lee, Bernett; Chen, Jinmiao; Low, Ivy; Shadan, Nurhidaya Binte; Lum, Josephine; Wei, Tay Seok; Mok, Esther; Kawanishi, Shohei; Kitamura, Yoshihisa; Larbi, Anis; Poidinger, Michael; Renia, Laurent; Ng, Lai Guan; Wolf, Yochai; Jung, Steffen; Önder, Tamer; Newell, Evan; Huber, Tara; Ashihara, Eishi; Garel, Sonia; Pouladi, Mahmoud A; Ginhoux, Florent

    2017-07-18

    Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Comprehensive assessment of precursors, diagenesis, and reactivity to water treatment of dissolved and colloidal organic matter

    USGS Publications Warehouse

    Leenheer, J.A.

    2004-01-01

    A comprehensive isolation, fractionation, and characterization research approach was developed for dissolved and colloidal organic matter (DOM) in water, and it was applied to various surface- and groundwaters to assess DOM precursors, DOM diagenesis, and DOM reactivity to water treatment processes. Major precursors for natural DOM are amino sugars, condensed tannins, and terpenoids. Amino sugar colloids derived from bacterial cell walls are incompletely removed by drinking water treatment and foul reverse osmosis membranes, but are nearly quantitatively removed by soil/aquifer treatment. When chlorinated, amino sugars produce low yields of regulated disinfection by-products (DBFs) but they produce significant chlorine demand that is likely caused by chlorination of free amino groups. Condensed tannins are major precursors for "blackwater" DOM such as that found in the Suwannee River. This DOM produces high yields of DBPs upon chorination, and is efficiently removed by coagulation/flocculation treatment. Terpenoid-derived DOM appears to be biologically refractory, infiltrates readily into groundwater with little removal by soil/aquifer treatment, gives low DBF-yields upon chlorination and is poorly removed by coagulation/flocculation treatments. Peptides derived from proteins are major components of the base DOM fraction (10% or less of the mass of DOM), and this fraction produces large yields of haloacetonitriles upon chorination.

  20. Molecular precursor derived silicon boron carbonitride/carbon nanotube and silicon oxycarbide/carbon nanotube composite nanowires for energy based applications

    NASA Astrophysics Data System (ADS)

    Bhandavat, Romil

    Molecular precursor derived ceramics (also known as polymer-derived ceramics or PDCs) are high temperature glasses that have been studied for applications involving operation at elevated temperatures. Prepared from controlled thermal degradation of liquid-phase organosilicon precursors, these ceramics offer remarkable engineering properties such as resistance to crystallization up to 1400 °C, semiconductor behavior at high temperatures and intense photoluminescence. These properties are a direct result of their covalent bonded amorphous network and free (-sp2) carbon along with mixed Si/B/C/N/O bonds, which otherwise can not be obtained through conventional ceramic processing techniques. This thesis demonstrates synthesis of a unique core/shell type nanowire structure involving either siliconboroncarbonitride (SiBCN) or siliconoxycarbide (SiOC) as the shell with carbon nanotube (CNT) acting as the core. This was made possible by liquid phase functionalization of CNT surfaces with respective polymeric precursor (e.g., home-made boron-modified polyureamethylvinylsilazane for SiBCN/CNT and commercially obtained polysiloxane for SiOC/CNT), followed by controlled pyrolysis in inert conditions. This unique architecture has several benefits such as high temperature oxidation resistance (provided by the ceramic shell), improved electrical conductivity and mechanical toughness (attributed to the CNT core) that allowed us to explore its use in energy conversion and storage devices. The first application involved use of SiBCN/CNT composite as a high temperature radiation absorbant material for laser thermal calorimeter. SiBCN/CNT spray coatings on copper substrate were exposed to high energy laser beams (continuous wave at 10.6 mum 2.5 kW CO2 laser, 10 seconds) and resulting change in its microstructure was studied ex-situ. With the aid of multiple techniques we ascertained the thermal damage resistance to be 15 kW/cm -2 with optical absorbance exceeding 97%. This represents

  1. Synthesis of indolizidinone analogues of cytotoxic alkaloids: monocyclic precursors are also active.

    PubMed

    Boto, Alicia; Miguélez, Javier; Marín, Raquel; Díaz, Mario

    2012-05-15

    Readily available proline derivatives can be transformed in just two steps into analogues of cytotoxic phenanthroindolizidine alkaloids. The key step uses a sequential radical scission-oxidation-alkylation process, which yields 2-substituted pyrrolidine amides. A second process effects the cyclization to give the desired alkaloid analogues, which possess an indolizidine core. The major and minor isomers (dr 3:2 to 3:1) can be easily separated, allowing their use to study structure-activity relationships (SAR). The process is versatile and allows the introduction of aryl and heteroaryl groups (including biphenyl, halogenated phenyl, and pyrrole rings). Some of these alkaloid analogues displayed a selective cytotoxic activity against tumorogenic human neuronal and mammary cancer cells, and one derivative caused around 80% cell death in both tumor lines at micromolar doses. The cytotoxicity of some monocyclic precursors was also studied, being comparable or superior to the bicyclic derivatives. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Understanding the reconstitution of the B-cell compartment in bone marrow and blood after treatment for B-cell precursor acute lymphoblastic leukaemia.

    PubMed

    Theunissen, Prisca M J; van den Branden, Anouk; Van Der Sluijs-Gelling, Alita; De Haas, Valerie; Beishuizen, Auke; van Dongen, Jacques J M; Van Der Velden, Vincent H J

    2017-07-01

    A better understanding of the reconstitution of the B-cell compartment during and after treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) will help to assess the immunological status and needs of post-treatment BCP-ALL patients. Using 8-colour flow cytometry and proliferation-assays, we studied the composition and proliferation of both the B-cell precursor (BCP) population in the bone marrow (BM) and mature B-cell population in peripheral blood (PB) during and after BCP-ALL therapy. We found a normal BCP differentiation pattern and a delayed formation of classical CD38 dim -naive mature B-cells, natural effector B-cells and memory B-cells in patients after chemotherapy. This B-cell differentiation/maturation pattern was strikingly similar to that during initial B-cell development in healthy infants. Tissue-resident plasma cells appeared to be partly protected from chemotherapy. Also, we found that the fast recovery of naive mature B-cell numbers after chemotherapy was the result of increased de novo BCP generation, rather than enhanced B-cell proliferation in BM or PB. These results indicate that post-treatment BCP-ALL patients will eventually re-establish a B-cell compartment with a composition and B-cell receptor repertoire similar to that in healthy children. Additionally, the formation of a new memory B-cell compartment suggests that revaccination might be beneficial after BCP-ALL therapy. © 2017 John Wiley & Sons Ltd.

  3. Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles.

    PubMed

    Rezza, Amélie; Wang, Zichen; Sennett, Rachel; Qiao, Wenlian; Wang, Dongmei; Heitman, Nicholas; Mok, Ka Wai; Clavel, Carlos; Yi, Rui; Zandstra, Peter; Ma'ayan, Avi; Rendl, Michael

    2016-03-29

    The hair follicle (HF) is a complex miniorgan that serves as an ideal model system to study stem cell (SC) interactions with the niche during growth and regeneration. Dermal papilla (DP) cells are required for SC activation during the adult hair cycle, but signal exchange between niche and SC precursors/transit-amplifying cell (TAC) progenitors that regulates HF morphogenetic growth is largely unknown. Here we use six transgenic reporters to isolate 14 major skin and HF cell populations. With next-generation RNA sequencing, we characterize their transcriptomes and define unique molecular signatures. SC precursors, TACs, and the DP niche express a plethora of ligands and receptors. Signaling interaction network analysis reveals a bird's-eye view of pathways implicated in epithelial-mesenchymal interactions. Using a systematic tissue-wide approach, this work provides a comprehensive platform, linked to an interactive online database, to identify and further explore the SC/TAC/niche crosstalk regulating HF growth. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Amyloid precursor protein and Presenilin 1 interaction studied by FRET in human H4 cells.

    PubMed

    Nizzari, Mario; Venezia, Valentina; Bianchini, Paolo; Caorsi, Valentina; Diaspro, Alberto; Repetto, Emanuela; Thellung, Stefano; Corsaro, Alessandro; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

    2007-01-01

    The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

  5. Hydrogen Peroxide Activated Quinone Methide Precursors with Enhanced DNA Cross-Linking Capability and Cytotoxicity towards Cancer Cells

    PubMed Central

    Wang, Yibin; Fan, Heli; Balakrishnan, Kumudha; Lin, Zechao; Cao, Sheng; Chen, Wenbing; Fan, Yukai; Guthrie, Quibria A.; Sun, Huabing; Teske, Kelly A.; Gandhi, Varsha; Arnold, Leggy A.; Peng, Xiaohua

    2017-01-01

    Quinone methide (QM) formation induced by endogenously generated H2O2 is attractive for biological and biomedical applications. To overcome current limitations due to low biological activity of H2O2-activated QM precursors, we are introducing herein several new arylboronates with electron donating substituents at different positions of benzene ring and/or different neutral leaving groups. The reaction rate of the arylboronate esters with H2O2 and subsequent bisquinone methides formation and DNA cross-linking was accelerated with the application of Br as a leaving group instead of acetoxy groups. Additionally, a donating group placed meta to the nascent exo-methylene group of the quinone methide greatly improves H2O2-induced DNA interstrand cross-link formation as well as enhances the cellular activity. Multiple donating groups decrease the stability and DNA cross-linking capability, which lead to low cellular activity. A cell-based screen demonstrated that compounds 2a and 5a with a OMe or OH group dramatically inhibited the growth of various tissue-derived cancer cells while normal cells were less affected. Induction of H2AX phosphorylation by these compounds in CLL lymphocytes provide evidence for a correlation between cell death and DNA damage. The compounds presented herein showed potent anticancer activities and selectivity, which represent a novel scaffold for anticancer drug development. PMID:28388522

  6. Multipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System

    PubMed Central

    Hasegawa, Kouichi; Menheniott, Trevelyan; Rollo, Ben; Zhang, Dongcheng; Hough, Shelley; Alshawaf, Abdullah; Febbraro, Fabia; Ighaniyan, Samiramis; Leung, Jessie; Elliott, David A.; Newgreen, Donald F.; Pera, Martin F.

    2015-01-01

    Abstract The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named “caudal neural progenitors” (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube. Stem Cells 2015;33:1759–1770 PMID:25753817

  7. Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

    PubMed

    Ikigai, H; Ono, T; Nakae, T; Otsuru, H; Shimamura, T

    1999-01-08

    Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

  8. Ewing’s sarcoma precursors are highly enriched in embryonic osteochondrogenic progenitors

    PubMed Central

    Tanaka, Miwa; Yamazaki, Yukari; Kanno, Yohei; Igarashi, Katsuhide; Aisaki, Ken-ichi; Kanno, Jun; Nakamura, Takuro

    2014-01-01

    Ewing’s sarcoma is a highly malignant bone tumor found in children and adolescents, and the origin of this malignancy is not well understood. Here, we introduced a Ewing’s sarcoma–associated genetic fusion of the genes encoding the RNA-binding protein EWS and the transcription factor ETS (EWS-ETS) into a fraction of cells enriched for osteochondrogenic progenitors derived from the embryonic superficial zone (eSZ) of long bones collected from late gestational murine embryos. EWS-ETS fusions efficiently induced Ewing’s sarcoma–like small round cell sarcoma formation by these cells. Analysis of the eSZ revealed a fraction of a precursor cells that express growth/differentiation factor 5 (Gdf5), the transcription factor Erg, and parathyroid hormone-like hormone (Pthlh), and selection of the Pthlh-positive fraction alone further enhanced EWS-ETS–dependent tumor induction. Genes downstream of the EWS-ETS fusion protein were quite transcriptionally active in eSZ cells, especially in regions in which the chromatin structure of the ETS-responsive locus was open. Inhibition of β-catenin, poly (ADP-ribose) polymerase 1 (PARP1), or enhancer of zeste homolog 2 (EZH2) suppressed cell growth in a murine model of Ewing’s sarcoma, suggesting the utility of the current system as a preclinical model. These results indicate that eSZ cells are highly enriched in precursors to Ewing’s sarcoma and provide clues to the histogenesis of Ewing’s sarcoma in bone. PMID:24911143

  9. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator

    PubMed Central

    Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Yasuda, Hisataka; Sakamoto, Reiko; Yoshida, Nobuaki

    2016-01-01

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs. PMID:27401343

  10. Differential requirements of CD4(+) T-cell signals for effector cytotoxic T-lymphocyte (CTL) priming and functional memory CTL development at higher CD8(+) T-cell precursor frequency.

    PubMed

    Umeshappa, Channakeshava S; Nanjundappa, Roopa H; Xie, Yufeng; Freywald, Andrew; Xu, Qingyong; Xiang, Jim

    2013-04-01

    Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions. © 2012 Blackwell Publishing Ltd.

  11. Surface Expression of Precursor N-cadherin Promotes Tumor Cell Invasion12

    PubMed Central

    Maret, Deborah; Gruzglin, Eugenia; Sadr, Mohamad Seyed; Siu, Vincent; Shan, Weisong; Koch, Alexander W; Seidah, Nabil G; Del Maestro, Rolando F; Colman, David R

    2010-01-01

    The expression of N-cadherin (NCAD) has been shown to correlate with increased tumor cell motility and metastasis. However, NCAD-mediated adhesion is a robust phenomenon and therefore seems to be inconsistent with the “release” from intercellular adhesion required for invasion. We show that in the most invasive melanoma and brain tumor cells, altered posttranslational processing results in abundant nonadhesive precursor N-cadherin (proNCAD) at the cell surface, although total NCAD levels remain constant. We demonstrate that aberrantly processed proNCAD promotes cell migration and invasion in vitro. Furthermore, in human tumor specimens, we find high levels of proNCAD as well, supporting an overall conclusion that proNCAD and mature NCAD coexist on these tumor cell surfaces and that it is the ratio between these functionally antagonistic moieties that directly correlates with invasion potential. Our work provides insight into what may be a widespread mechanism for invasion and metastasis and challenges the current dogma of the functional roles played by classic cadherins in tumor progression. PMID:21170270

  12. Ca-P spots modified zirconia by liquid precursor infiltration and the effect on osteoblast-like cell responses.

    PubMed

    Li, Yongmei; Liu, Yan; Zhang, Zutai; Zhuge, Ruishen; Ding, Ning; Tian, Yueming

    2018-01-26

    Ca-P spots modified zirconia by liquid precursor infiltration and the cell responses were investigated. Pre-sintered zirconia specimens were immersed in Ca-P precursor solution. After dense sintering, scanning electron microscopy showed Ca-P spots were formed on the zirconia and anchored with zirconia substrates. The distribution density was increased with the extension of immersion time. Energy dispersive spectrometer confirmed the stoichiometric Ca/P ratio was about 1.67. After hydrothermal treatment, Ca-P spots turned into rod crystals where diffraction peaks of tricalcium phosphate and hydroxyapatite were detected by X-ray diffraction, and Ca 2+ and PO 4 3- release decreased slightly (p>0.05). There was no significant decrease on three-point bending strength (p>0.05). Osteoblast-like MC3T3-E1 cells attached and spread well and showed higher proliferation on Ca-P spots modified zirconia (p<0.05), though its initial alkaline phosphatase activity was not significant high (p>0.05). In conclusion, Ca-P liquid precursor infiltration is a potential method to modify the zirconia ceramics for improving bioactivity.

  13. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

    PubMed

    Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

    2015-10-01

    Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration. © 2015 International Federation for Cell Biology.

  14. Gene expression profiling of choline-deprived neural precursor cells isolated from mouse brain.

    PubMed

    Niculescu, Mihai D; Craciunescu, Corneliu N; Zeisel, Steven H

    2005-04-04

    Choline is an essential nutrient and an important methyl donor. Choline deficiency alters fetal development of the hippocampus in rodents and these changes are associated with decreased memory function lasting throughout life. Also, choline deficiency alters global and gene-specific DNA methylation in several models. This gene expression profiling study describes changes in cortical neural precursor cells from embryonic day 14 mice, after 48 h of exposure to a choline-deficient medium. Using Significance Analysis of Microarrays, we found the expression of 1003 genes to be significantly changed (from a total of 16,000 total genes spotted on the array), with a false discovery rate below 5%. A total of 846 genes were overexpressed while 157 were underexpressed. Classification by gene ontology revealed that 331 of these genes modulate cell proliferation, apoptosis, neuronal and glial differentiation, methyl metabolism, and calcium-binding protein classes. Twenty-seven genes that had changed expression have previously been reported to be regulated by promoter or intron methylation. These findings support our previous work suggesting that choline deficiency decreases the proliferation of neural precursors and possibly increases premature neuronal differentiation and apoptosis.

  15. Subcellular Distribution of Glutathione Precursors in Arabidopsis thaliana

    PubMed Central

    Koffler, Barbara Eva; Maier, Romana; Zechmann, Bernd

    2011-01-01

    Abstract Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) – the first enzyme of glutathione synthesis – causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells. PMID:22050910

  16. Pediatric precursor B acute lymphoblastic leukemia: are T helper cells the missing link in the infectious etiology theory?

    PubMed

    Bürgler, Simone; Nadal, David

    2017-12-01

    Precursor B acute lymphoblastic leukemia (BCP-ALL), the most common childhood malignancy, arises from an expansion of malignant B cell precursors in the bone marrow. Epidemiological studies suggest that infections or immune responses to infections may promote such an expansion and thus BCP-ALL development. Nevertheless, a specific pathogen responsible for this process has not been identified. BCP-ALL cells critically depend on interactions with the bone marrow microenvironment. The bone marrow is also home to memory T helper (Th) cells that have previously expanded during an immune response in the periphery. In secondary lymphoid organs, Th cells can interact with malignant cells of mature B cell origin, while such interactions between Th cells and malignant immature B cell in the bone marrow have not been described yet. Nevertheless, literature supports a model where Th cells-expanded during an infection in early childhood-migrate to the bone marrow and support BCP-ALL cells as they support normal B cells. Further research is required to mechanistically confirm this model and to elucidate the interaction pathways between leukemia cells and cells of the tumor microenvironment. As benefit, targeting these interactions could be included in current treatment regimens to increase therapeutic efficiency and to reduce relapses.

  17. Is there a place for human fetal-derived stem cells for cell replacement therapy in Huntington's disease?

    PubMed

    Precious, Sophie V; Zietlow, Rike; Dunnett, Stephen B; Kelly, Claire M; Rosser, Anne E

    2017-06-01

    Huntington's disease (HD) is a neurodegenerative disease that offers an excellent paradigm for cell replacement therapy because of the associated relatively focal cell loss in the striatum. The predominant cells lost in this condition are striatal medium spiny neurons (MSNs). Transplantation of developing MSNs taken from the fetal brain has provided proof of concept that donor MSNs can survive, integrate and bring about a degree of functional recovery in both pre-clinical studies and in a limited number of clinical trials. The scarcity of human fetal tissue, and the logistics of coordinating collection and dissection of tissue with neurosurgical procedures makes the use of fetal tissue for this purpose both complex and limiting. Alternative donor cell sources which are expandable in culture prior to transplantation are currently being sought. Two potential donor cell sources which have received most attention recently are embryonic stem (ES) cells and adult induced pluripotent stem (iPS) cells, both of which can be directed to MSN-like fates, although achieving a genuine MSN fate has proven to be difficult. All potential donor sources have challenges in terms of their clinical application for regenerative medicine, and thus it is important to continue exploring a wide variety of expandable cells. In this review we discuss two less well-reported potential donor cell sources; embryonic germ (EG) cells and fetal neural precursors (FNPs), both are which are fetal-derived and have some properties that could make them useful for regenerative medicine applications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Proteoglycans synthesized by smooth muscle cells derived from monkey (Macaca nemestrina) aorta.

    PubMed

    Chang, Y; Yanagishita, M; Hascall, V C; Wight, T N

    1983-05-10

    Smooth muscle cells derived from monkey aorta were cultured in medium with [35S]sulfate and [3H]glucosamine as labeling precursors. Proteoglycans in the medium and in 4 M guanidine HCl extracts of the cell layer were purified by DEAE-Sephacel and molecular sieve chromatography. Both preparations contained a predominant, large chondroitin sulfate proteoglycan (Kav = 0.30 on Sepharose CL-2B) with glycosaminoglycan chains of Mr approximately 43,000 average containing a ratio of 6-sulfate to 4-sulfate of approximately 2. Approximately 7 and 27% of the 3H label in this proteoglycan were present in N-linked and O-linked oligosaccharides, respectively. Reaggregation experiments indicated that a large proportion of these proteoglycans can form link protein-stabilized aggregates. The medium fraction also contained a smaller dermatan sulfate proteoglycan (Kav = 0.67 on Sepharose CL-2B) with glycosaminoglycan chains of Mr approximately 43,000 containing a ratio of 6-sulfate to 4-sulfate of about 0.5. This proteoglycan contained approximately the same percentage of N-linked oligosaccharides as the chondroitin sulfate proteoglycan, but few or no O-linked oligosaccharides. A smaller dermatan sulfate proteoglycan with a single chain was present only in the cell layer. Additionally, small amounts of heparan sulfate proteoglycans were synthesized by the cells.

  19. CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

    PubMed Central

    Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

    2013-01-01

    NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

  20. Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor.

    PubMed

    Fernández-Del-Río, Lucía; Nag, Anish; Gutiérrez Casado, Elena; Ariza, Julia; Awad, Agape M; Joseph, Akil I; Kwon, Ohyun; Verdin, Eric; de Cabo, Rafael; Schneider, Claus; Torres, Jorge Z; Burón, María I; Clarke, Catherine F; Villalba, José M

    2017-09-01

    Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q 10 , but due to its highly lipophilic nature, Q 10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13 C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4' of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Direct Conversion of Equine Adipose-Derived Stem Cells into Induced Neuronal Cells Is Enhanced in Three-Dimensional Culture.

    PubMed

    Petersen, Gayle F; Hilbert, Bryan J; Trope, Gareth D; Kalle, Wouter H J; Strappe, Padraig M

    2015-12-01

    The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with βIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.

  2. p57KIP2 regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex

    PubMed Central

    Mairet-Coello, Georges; Tury, Anna; Van Buskirk, Elise; Robinson, Kelsey; Genestine, Matthieu; DiCicco-Bloom, Emanuel

    2012-01-01

    During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57KIP2, an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57KIP2 regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27KIP1 controlled IPC proliferation exclusively. Furthermore, p57KIP2 deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27KIP1 increased IPC proliferation at E16.5. Consequently, loss of p57KIP2 increased primarily layer 5-6 neuron production, whereas loss of p27KIP1 increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57KIP2 and p27KIP1 control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation. PMID:22223678

  3. Sol-gel precursors and products thereof

    DOEpatents

    Warren, Scott C.; DiSalvo, Jr., Francis J.; Weisner, Ulrich B.

    2017-02-14

    The present invention provides a generalizable single-source sol-gel precursor capable of introducing a wide range of functionalities to metal oxides such as silica. The sol-gel precursor facilitates a one-molecule, one-step approach to the synthesis of metal-silica hybrids with combinations of biological, catalytic, magnetic, and optical functionalities. The single-source precursor also provides a flexible route for simultaneously incorporating functional species of many different types. The ligands employed for functionalizing the metal oxides are derived from a library of amino acids, hydroxy acids, or peptides and a silicon alkoxide, allowing many biological functionalities to be built into silica hybrids. The ligands can coordinate with a wide range of metals via a carboxylic acid, thereby allowing direct incorporation of inorganic functionalities from across the periodic table. Using the single-source precursor a wide range of functionalized nanostructures such as monolith structures, mesostructures, multiple metal gradient mesostructures and Stober-type nanoparticles can be synthesized. ##STR00001##

  4. Soluble Factors from Human Olfactory Neural Stem/Progenitor Cells Influence the Fate Decisions of Hippocampal Neural Precursor Cells.

    PubMed

    Gómez-Virgilio, Laura; Ramírez-Rodríguez, Gerardo Bernabé; Sánchez-Torres, Carmen; Ortiz-López, Leonardo; Meraz-Ríos, Marco Antonio

    2018-03-01

    Neurogenesis plays a significant role during adulthood, and the observation that neural stem cells reside in the central nervous system and the olfactory epithelium has attracted attention due to their importance in neuronal regeneration. In addition, soluble factors (SFs) release by neural stem cells may modulate the neurogenic process. Thus, in this study, we identified the SFs released by olfactory human neural stem/progenitor cells (hNS/PCs-OE). These cells express Ki67, nestin, and βIII-tubulin, indicating their neural lineage. The hNS/PCs-OE also express PSD95 and tau proteins during proliferation, but increased levels are observed after differentiation. Thus, we evaluated the effects of SFs from hNS/PCs-OE on the viability, proliferation, and differentiation potential of adult murine hippocampal neural precursor cells (AHPCs). SFs from hNS/PCs-OE maintain cells in the precursor and proliferative stages and mainly promote the astrocytic differentiation of AHPCs. These effects involved the activation, as measured by phosphorylation, of several proteins (Erk1/2; Akt/PRAS40/GSK3β and JAK/STAT) involved in key events of the neurogenic process. Moreover, according to the results from the antibody-based microarray approach, among the soluble factors, hNS/PCs-OE produce interleukin-6 (IL-6) and neurotrophin 4 (NT4). However, residual epidermal growth factor (EGF) was also detected. These proteins partially reproduced the effects of SFs from hNS/PCs-OE on AHPCs, and the mechanism underlying these effects is mediated by Src proteins, which have been implicated in EGF-induced transactivation of TrkB receptor. The results of the present study suggest the potential use of SFs from hNS/PCs-OE in controlling the differentiation potential of AHPCs. Thus, the potential clinical relevance of hNS/PCs-OE is worth pursuing.

  5. Activating Endogenous Neural Precursor Cells Using Metformin Leads to Neural Repair and Functional Recovery in a Model of Childhood Brain Injury.

    PubMed

    Dadwal, Parvati; Mahmud, Neemat; Sinai, Laleh; Azimi, Ashkan; Fatt, Michael; Wondisford, Fredric E; Miller, Freda D; Morshead, Cindi M

    2015-08-11

    The development of cell replacement strategies to repair the injured brain has gained considerable attention, with a particular interest in mobilizing endogenous neural stem and progenitor cells (known as neural precursor cells [NPCs]) to promote brain repair. Recent work demonstrated metformin, a drug used to manage type II diabetes, promotes neurogenesis. We sought to determine its role in neural repair following brain injury. We find that metformin administration activates endogenous NPCs, expanding the size of the NPC pool and promoting NPC migration and differentiation in the injured neonatal brain in a hypoxia-ischemia (H/I) injury model. Importantly, metformin treatment following H/I restores sensory-motor function. Lineage tracking reveals that metformin treatment following H/I causes an increase in the absolute number of subependyma-derived NPCs relative to untreated H/I controls in areas associated with sensory-motor function. Hence, activation of endogenous NPCs is a promising target for therapeutic intervention in childhood brain injury models. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development

    PubMed Central

    McCann, Matthew R.; Tamplin, Owen J.; Rossant, Janet; Séguin, Cheryle A.

    2012-01-01

    SUMMARY Back pain related to intervertebral disc degeneration is the most common musculoskeletal problem, with a lifetime prevalence of 82%. The lack of effective treatment for this widespread problem is directly related to our limited understanding of disc development, maintenance and degeneration. The aim of this study was to determine the developmental origins of nucleus pulposus cells within the intervertebral disc using a novel notochord-specific Cre mouse. To trace the fate of notochordal cells within the intervertebral disc, we derived a notochord-specific Cre mouse line by targeting the homeobox gene Noto. Expression of this gene is restricted to the node and the posterior notochord during gastrulation [embryonic day 7.5 (E7.5)-E12.5]. The Noto-cre mice were crossed with a conditional lacZ reporter for visualization of notochord fate in whole-mount embryos. We performed lineage-tracing experiments to examine the contribution of the notochord to spinal development from E12.5 through to skeletally mature mice (9 months). Fate mapping studies demonstrated that, following elongation and formation of the primitive axial skeleton, the notochord gives rise to the nucleus pulposus in fully formed intervertebral discs. Cellular localization of β-galactosidase (encoded by lacZ) and cytokeratin-8 demonstrated that both notochordal cells and chondrocyte-like nucleus pulposus cells are derived from the embryonic notochord. These studies establish conclusively that notochordal cells act as embryonic precursors to all cells found within the nucleus pulposus of the mature intervertebral disc. This suggests that notochordal cells might serve as tissue-specific progenitor cells within the disc and establishes the Noto-cre mouse as a unique tool to interrogate the contribution of notochordal cells to both intervertebral disc development and disc degeneration. PMID:22028328

  7. Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development.

    PubMed

    McCann, Matthew R; Tamplin, Owen J; Rossant, Janet; Séguin, Cheryle A

    2012-01-01

    Back pain related to intervertebral disc degeneration is the most common musculoskeletal problem, with a lifetime prevalence of 82%. The lack of effective treatment for this widespread problem is directly related to our limited understanding of disc development, maintenance and degeneration. The aim of this study was to determine the developmental origins of nucleus pulposus cells within the intervertebral disc using a novel notochord-specific Cre mouse. To trace the fate of notochordal cells within the intervertebral disc, we derived a notochord-specific Cre mouse line by targeting the homeobox gene Noto. Expression of this gene is restricted to the node and the posterior notochord during gastrulation [embryonic day 7.5 (E7.5)-E12.5]. The Noto-cre mice were crossed with a conditional lacZ reporter for visualization of notochord fate in whole-mount embryos. We performed lineage-tracing experiments to examine the contribution of the notochord to spinal development from E12.5 through to skeletally mature mice (9 months). Fate mapping studies demonstrated that, following elongation and formation of the primitive axial skeleton, the notochord gives rise to the nucleus pulposus in fully formed intervertebral discs. Cellular localization of β-galactosidase (encoded by lacZ) and cytokeratin-8 demonstrated that both notochordal cells and chondrocyte-like nucleus pulposus cells are derived from the embryonic notochord. These studies establish conclusively that notochordal cells act as embryonic precursors to all cells found within the nucleus pulposus of the mature intervertebral disc. This suggests that notochordal cells might serve as tissue-specific progenitor cells within the disc and establishes the Noto-cre mouse as a unique tool to interrogate the contribution of notochordal cells to both intervertebral disc development and disc degeneration.

  8. Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis[S

    PubMed Central

    Xie, Letian X.; Williams, Kevin J.; He, Cuiwen H.; Weng, Emily; Khong, San; Rose, Tristan E.; Kwon, Ohyun; Bensinger, Steven J.; Marbois, Beth N.; Clarke, Catherine F.

    2015-01-01

    Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic 13C6-ring-labeled compounds including 13C6-4HB, 13C6-pABA, 13C6-resveratrol, and 13C6-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form 13C6-Q when cultured in the presence of 13C6-pABA. However, E. coli cells treated with 13C6-pABA generated 13C6-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of 13C6-resveratrol or 13C6-coumarate were able to synthesize 13C6-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q. PMID:25681964

  9. Electrochemical Study of Hydrocarbon-Derived Electrolytes for Supercapacitors

    NASA Astrophysics Data System (ADS)

    Noorden, Zulkarnain A.; Matsumoto, Satoshi

    2013-10-01

    In this paper, we evaluate the essential electrochemical properties - capacitive and resistive behaviors - of hydrocarbon-derived electrolytes for supercapacitor application using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrolytes were systematically prepared from three hydrocarbon-derived compounds, which have different molecular structures and functional groups, by treatment with high-concentration sulfuric acid (H2SO4) at room temperature. Two-electrode cells were assembled by sandwiching an electrolyte-containing glass wool separator with two active electrodes of activated carbon sheets. The dc electrical properties of the tested cells in terms of their capacitive behavior were investigated by CV, and in order to observe the frequency characteristics of the constructed cells, EIS was carried out. Compared with the tested cell with only high-concentration H2SO4 as the electrolyte, the cell with the derived electrolytes exhibit a capacitance as high as 135 F/g with an improved overall internal resistance of 2.5 Ω. Through the use of a simple preparation method and low-cost precursors, hydrocarbon-derived electrolytes could potentially find large-scale and higher-rating supercapacitor applications.

  10. Genetically distinct leukemic stem cells in human CD34− acute myeloid leukemia are arrested at a hemopoietic precursor-like stage

    PubMed Central

    Quek, Lynn; Garnett, Catherine; Karamitros, Dimitris; Stoilova, Bilyana; Doondeea, Jessica; Kennedy, Alison; Metzner, Marlen; Ivey, Adam; Sternberg, Alexander; Hunter, Hannah; Price, Andrew; Virgo, Paul; Grimwade, David; Freeman, Sylvie; Russell, Nigel; Mead, Adam

    2016-01-01

    Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in ∼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34−, there are multiple, nonhierarchically arranged CD34+ and CD34− LSC populations. Within CD34− and CD34+ LSC–containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34− LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34− mature granulocyte–macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis. PMID:27377587

  11. Cancer cell death induced by the intracellular self-assembly of an enzyme-responsive supramolecular gelator.

    PubMed

    Tanaka, Akiko; Fukuoka, Yuki; Morimoto, Yuka; Honjo, Takafumi; Koda, Daisuke; Goto, Masahiro; Maruyama, Tatsuo

    2015-01-21

    We report cancer cell death initiated by the intracellular molecular self-assembly of a peptide lipid, which was derived from a gelator precursor. The gelator precursor was designed to form nanofibers via molecular self-assembly, after cleavage by a cancer-related enzyme (matrix metalloproteinase-7, MMP-7), leading to hydrogelation. The gelator precursor exhibited remarkable cytotoxicity to five different cancer cell lines, while the precursor exhibited low cytotoxicity to normal cells. Cancer cells secrete excessive amounts of MMP-7, which converted the precursor into a supramolecular gelator prior to its uptake by the cells. Once inside the cells, the supramolecular gelator formed a gel via molecular self-assembly, exerting vital stress on the cancer cells. The present study thus describes a new drug where molecular self-assembly acts as the mechanism of cytotoxicity.

  12. Evaluation of Accessory Lacrimal Gland in Muller's Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease.

    PubMed

    Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H; Jassim Jaboori, Assraa; Setabutr, Pete; Aakalu, Vinay K

    2017-04-01

    The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. ALG tissues obtained from Muller's muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

  13. High Tc superconducting films from metallo-organic precursors

    NASA Astrophysics Data System (ADS)

    Davison, W. W.; Shyu, S. G.; Buchanan, R. C.

    High Tc superconducting films of heavy metal soaps (derived from carboxylic acid precursors) have been prepared on Si and other substrates. The precursors were synthesized and mixed in appropriate molar ratios to form the high Tc compound YBa2Cu3O(7-x), using a high boiling point common solvent base. The precursor solution was deposited by a spin casting technique on the substrates. Film thicknesses of 0.1-1.0 micron were achieved after heat treatment at 550-850 C at not longer than 4 hours. Films were analyzed as to orientation, appropriate phase, interfacial reaction, and superconducting properties.

  14. An efficient copper phthalocyanine additive of perovskite precursor for improving the photovoltaic performance of planar perovskite solar cells

    NASA Astrophysics Data System (ADS)

    Wu, Shufang; Liu, Qingwei; Zheng, Ya; Li, Renjie; Peng, Tianyou

    2017-08-01

    Solution processable planar heterojunction perovskite solar cell has drawn much attention as a promising low-cost photovoltaic device, and much effort has been made to improve its power conversion efficiency by choosing appropriate additives for the perovskite precursor solution. Different to those additives reported, a soluble and thermal stable tert-butyl substituted copper phthalocyanine (CuPc(tBu)4) as additive is first introduced into the perovskite precursor solution of a planar perovskite solar cell that is fabricated via the one-step solution process. It is found that the pristine device without CuPc(tBu)4 additive exhibits a power conversion efficiency of 15.3%, while an extremely low concentration (4.4 × 10-3 mM) of CuPc(tBu)4 in the precursor solution leads to the corresponding device achieving an enhanced power conversion efficiency of 17.3%. CuPc(tBu)4 as an additive can improve the quality of perovskite layer with higher crystallinity and surface coverage, then resulting in enhanced light absorption and reduced charge recombination, and thus the better power conversion efficiency. The finding presented here provides a new choice for improving the quality of perovskite layer and the photovoltaic performance of the planar heterojunction perovskite solar cells.

  15. Human iPSC Derived GABA Ergic Precursor Cell Therapy for Chronic Epilepsy

    DTIC Science & Technology

    2016-10-01

    chronically epileptic rats ( CERs ) would: (1) diminish the frequency and intensity of spontaneous recurrent seizures (SRS, Specific Aim 1, SA1); and (2...epileptic rats: CERs receiving hMGE-like cell grafts and cyclosporine (an immunosuppressant to promote the survival of human cell grafts in the rat brain... CERs receiving sham-grafting surgery, CERs receiving cyclosporine only and CERs receiving no treatment. The results showed that, in comparison to

  16. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    PubMed Central

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

  17. Thrombopoietin inhibits murine mast cell differentiation

    PubMed Central

    Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

    2009-01-01

    We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

  18. Bioluminescent imaging demonstrates transplanted human embryonic stem cell-derived CD34+ cells preferentially develop into endothelial cells

    PubMed Central

    Tian, Xinghui; Hexum, Melinda K.; Penchev, Vesselin R.; Taylor, Russell J.; Shultz, Leonard D.; Kaufman, Dan S

    2010-01-01

    Human embryonic stem cells (hESCs) provide an important resource for novel regenerative medicine therapies and have been used to derive diverse cell populations, including hematopoietic and endothelial cells. However, it remains a challenge to achieve significant engraftment of hESC-derived blood cells when transplanted into animal models. To better understand mechanisms that enhance or limit the in vivo developmental potential of hESC-derived cells, we utilized hESCs that express firefly luciferase (luc) to allow non-invasive, real-time bioluminescent imaging of hESC-derived CD34+ cells transplanted into the liver of neonatal immunodeficient mice. Serial imaging demonstrated stable engraftment and expansion of the luc+ hESC-derived cells in vivo over several months. While we found that these hESC-derived CD34+ cells have bipotential ability to generate both hematopoietic and endothelial lineages in vitro, these studies demonstrate preferential differentiation into endothelial cells in vivo, with only low levels of hematopoietic cell engraftment. Therefore, these studies reveal key differences in the developmental potential of hESC-derived cells using in vitro and in vivo analyses. While transplanted hESC-derived CD34+ cells are well suited for revascularization therapies, additional measures are needed to provide higher levels of long-term hematopoietic engraftment. PMID:19711457

  19. Overexpression of Lhx2 suppresses proliferation of human T cell acute lymphoblastic leukemia-derived cells, partly by reducing LMO2 protein levels.

    PubMed

    Miyashita, Kazuya; Kitajima, Kenji; Goyama, Susumu; Kitamura, Toshio; Hara, Takahiko

    2018-01-15

    T cell acute lymphoblastic leukemia (T-ALL) is a malignant cancer with poor prognosis. The transcriptional co-factor LIM domain only 2 (LMO2) and its target gene HHEX are essential for self-renewal of T cell precursors and T-ALL etiology. LMO2 directly associates with LDB1 in a large DNA-containing nuclear complex and controls the transcription of T-ALL-related genes. Recently, we reported that overexpression of the LIM-homeodomain transcription factor, Lhx2, results in liberation of the Lmo2 protein from the Lmo2-Ldb1 complex, followed by ubiquitin proteasome mediated degradation. Here, we found that proliferation of five human T-ALL-derived cell lines, including CCRF-CEM, was significantly suppressed by retroviral overexpression of Lhx2. The majority of Lhx2-transduced CCRF-CEM cells arrested in G 0 phase and subsequently underwent apoptosis. Expression of LMO2 protein as well as HHEX, ERG, HES1 and MYC genes was repressed in CCRF-CEM cells by transduction with Lhx2. Lhx2-mediated growth inhibition was partially rescued by simultaneous overexpression of Lmo2; however, both the C-terminal LIM domain and the homeodomain of Lhx2 were required for its growth-suppressive activity. These data indicate that Lhx2 is capable of blocking proliferation of T-ALL-derived cells by both LMO2-dependent and -independent means. We propose Lhx2 as a new molecular tool for anti-T-ALL drug development. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

    PubMed Central

    Perrotta, Silverio; Cucciolla, Valeria; Ferraro, Marcella; Ronzoni, Luisa; Tramontano, Annunziata; Rossi, Francesca; Scudieri, Anna Chiara; Borriello, Adriana; Roberti, Domenico; Nobili, Bruno; Cappellini, Maria Domenica; Oliva, Adriana; Amendola, Giovanni; Migliaccio, Anna Rita; Mancuso, Patrizia; Martin-Padura, Ines; Bertolini, Francesco; Yoon, Donghoon; Prchal, Josef T.; Della Ragione, Fulvio

    2010-01-01

    Background Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. Methodology/Principal Findings We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. Conclusions/Significance Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis. PMID:20700488

  1. Overexpression of amyloid precursor protein increases copper content in HEK293 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Suazo, Miriam; Hodar, Christian; Morgan, Carlos

    2009-05-15

    Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu{sup 2+} binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu{sup 2+} reduction and {sup 64}Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu{sup 2+} reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu{sup 2+} ions. Moreover, wild-type cells exposed to bothmore » Cu{sup 2+} ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu{sup 2+} reductase activity and increased {sup 64}Cu uptake. We conclude that Cu{sup 2+} reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.« less

  2. NGFFFamide and echinotocin: structurally unrelated myoactive neuropeptides derived from neurophysin-containing precursors in sea urchins.

    PubMed

    Elphick, Maurice R; Rowe, Matthew L

    2009-04-01

    The myoactive neuropeptide NGIWYamide was originally isolated from the holothurian (sea cucumber) Apostichopus japonicus but there is evidence that NGIWYamide-like peptides also occur in other echinoderms. Here we report the discovery of a gene in the sea urchin Strongylocentrotus purpuratus that encodes two copies of an NGIWYamide-like peptide: Asn-Gly-Phe-Phe-Phe-(NH(2)) or NGFFFamide. Interestingly, the C-terminal region of the NGFFFamide precursor shares sequence similarity with neurophysins, carrier proteins hitherto uniquely associated with precursors of vasopressin/oxytocin-like neuropeptides. Thus, the NGFFFamide precursor is the first neurophysin-containing neuropeptide precursor to be discovered that does not contain a vasopressin/oxytocin-like peptide. However, it remains to be determined whether neurophysin acts as a carrier protein for NGFFFamide. The S. purpuratus genome also contains a gene encoding a precursor comprising a neurophysin polypeptide and 'echinotocin' (CFISNCPKGamide) - the first vasopressin/oxytocin-like peptide to be identified in an echinoderm. Therefore, in S. purpuratus there are two genes encoding precursors that have a neurophysin domain but which encode neuropeptides that are structurally unrelated. Furthermore, both NGFFFamide and echinotocin cause contraction of tube foot and oesophagus preparations from the sea urchin Echinus esculentus, consistent with the myoactivity of NGIWYamide in sea cucumbers and the myoactivity of vasopressin/oxytocin-like peptides in other animal phyla. Presumably the NGFFFamide precursor acquired its neurophysin domain following partial or complete duplication of a gene encoding a vasopressin/oxytocin-like peptide, but it remains to be determined when in evolutionary history this occurred.

  3. A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

    PubMed Central

    Sangawa, Takeshi; Tabata, Sanae; Suzuki, Kei; Saheki, Yasushi; Tanaka, Keiji; Takagi, Junichi

    2013-01-01

    Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. PMID:23526492

  4. Optical and mechanical behaviors of glassy silicone networks derived from linear siloxane precursors

    NASA Astrophysics Data System (ADS)

    Jang, Heejun; Seo, Wooram; Kim, Hyungsun; Lee, Yoonjoo; Kim, Younghee

    2016-01-01

    Silicon-based inorganic polymers are promising materials as matrix materials for glass fiber composites because of their good process ability, transparency, and thermal property. In this study, for utilization as a matrix precursor for a glass-fiber-reinforced composite, glassy silicone networks were prepared via hydrosilylation of linear/pendant Si-H polysiloxanes and the C=C bonds of viny-lterminated linear/cyclic polysiloxanes. 13C nuclear magnetic resonance spectroscopy was used to determine the structure of the cross-linked states, and a thermal analysis was performed. To assess the mechanical properties of the glassy silicone networks, we performed nanoindentation and 4-point bending tests. Cross-linked networks derived from siloxane polymers are thermally and optically more stable at high temperatures. Different cross-linking agents led to final networks with different properties due to differences in the molecular weights and structures. After stepped postcuring, the Young's modulus and the hardness of the glassy silicone networks increased; however, the brittleness also increased. The characteristics of the cross-linking agent played an important role in the functional glassy silicone networks.

  5. The skeletal cell-derived molecule sclerostin drives bone marrow adipogenesis.

    PubMed

    Fairfield, Heather; Falank, Carolyne; Harris, Elizabeth; Demambro, Victoria; McDonald, Michelle; Pettitt, Jessica A; Mohanty, Sindhu T; Croucher, Peter; Kramer, Ina; Kneissel, Michaela; Rosen, Clifford J; Reagan, Michaela R

    2018-02-01

    The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications. © 2017 Wiley Periodicals, Inc.

  6. Derivation of Stromal (Skeletal and Mesenchymal) Stem-Like Cells from Human Embryonic Stem Cells

    PubMed Central

    Harkness, Linda; Abdallah, Basem M.; Elsafadi, Mona; Al-Nbaheen, May S.; Aldahmash, Abdullah; Kassem, Moustapha

    2012-01-01

    Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for

  7. Comprehensive proteomic characterization of stem cell-derived extracellular matrices.

    PubMed

    Ragelle, Héloïse; Naba, Alexandra; Larson, Benjamin L; Zhou, Fangheng; Prijić, Miralem; Whittaker, Charles A; Del Rosario, Amanda; Langer, Robert; Hynes, Richard O; Anderson, Daniel G

    2017-06-01

    In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    PubMed Central

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  9. Kinetic analysis for cyclic CO2 capture using lithium orthosilicate sorbents derived from different silicon precursors.

    PubMed

    Zhao, Ming; Fan, Hanlu; Yan, Feng; Song, Yinqiang; He, Xu; Memon, Muhammad Zaki; Bhatia, Suresh K; Ji, Guozhao

    2018-06-21

    A series of Li4SiO4 was synthesized using LiNO3 and six different silicon precursors. The precipitated-silica-derived Li4SiO4 presented the highest CO2 capacity in a 10 h sorption test, and ZSM-5-derived Li4SiO4 demonstrated the most rapid CO2 sorption. The CO2 sorption kinetics predominantly followed the nucleation mode and could be accurately described by the Avrami-Erofeev model. The Avrami-Erofeev model provided an in-depth analysis of correlation between sorption performance and material properties. Both the nucleation speed and nucleation dimensionality affected the overall sorption kinetics. The kinetics and pore-size distribution suggest that the sorption kinetics was dependent on the quantity of ∼4 nm-pores which favors nucleation dimensionality. For the cyclic tests, the precipitated-silica-derived sample presented the poorest performance with the capacity decreasing from 31.33 wt% at the 1st cycle to only 11.52 wt% at the 30th cycle. However, the sample made from fumed silica displayed an opposite trend with the capacity increasing from 19.90 wt% at the 1st cycle to 34.23 wt% at the 30th cycle. The radically distinct behaviour of samples during cycles was on account of the alternation of sorption kinetics. The decrease in ∼4 nm-pores after cycles was responsible for the decrease of nucleation dimensionality for the precipitated-silica-derived sample. The rearrangement during cycles could enrich the pores of ∼4 nm for the fumed silica-derived sample, which improved the nucleation growth, thus enhancing the kinetics with cycles.

  10. Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.

    PubMed

    Sova, Matej; Žižak, Željko; Stanković, Jelena A Antic; Prijatelj, Matevž; Turk, Samo; Juranić, Zorica D; Mlinarič-Raščan, Irena; Gobec, Stanislav

    2013-08-01

    Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.

  11. Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

    PubMed Central

    Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J.; Iannaccone, Philip M.; Hendrix, Mary J.C.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

  12. Prolonged cultivation of hippocampal neural precursor cells shifts their differentiation potential and selects for aneuploid cells.

    PubMed

    Nguyen, The Duy; Widera, Darius; Greiner, Johannes; Müller, Janine; Martin, Ina; Slotta, Carsten; Hauser, Stefan; Kaltschmidt, Christian; Kaltschmidt, Barbara

    2013-12-01

    Neural precursor cells (NPCs) are lineage-restricted neural stem cells with limited self-renewal, giving rise to a broad range of neural cell types such as neurons, astrocytes, and oligodendrocytes. Despite this developmental potential, the differentiation capacity of NPCs has been controversially discussed concerning the trespassing lineage boundaries, for instance resulting in hematopoietic competence. Assessing their in vitro plasticity, we isolated nestin+/Sox2+, NPCs from the adult murine hippocampus. In vitro-expanded adult NPCs were able to form neurospheres, self-renew, and differentiate into neuronal, astrocytic, and oligodendrocytic cells. Although NPCs cultivated in early passage efficiently gave rise to neuronal cells in a directed differentiation assay, extensively cultivated NPCs revealed reduced potential for ectodermal differentiation. We further observed successful differentiation of long-term cultured NPCs into osteogenic and adipogenic cell types, suggesting that NPCs underwent a fate switch during culture. NPCs cultivated for more than 12 passages were aneuploid (abnormal chromosome numbers such as 70 chromosomes). Furthermore, they showed growth factor-independent proliferation, a hallmark of tumorigenic transformation. In conclusion, our findings substantiate the lineage restriction of NPCs from adult mammalian hippocampus. Prolonged cultivation results, however, in enhanced differentiation potential, which may be attributed to transformation events leading to aneuploid cells.

  13. Evaluation of Accessory Lacrimal Gland in Muller’s Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease

    PubMed Central

    Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H.; Jaboori, Assraa Jassim; Setabutr, Pete; Aakalu, Vinay K.

    2017-01-01

    Purpose The accessory lacrimal glands (ALG) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. Materials and Methods ALG tissues obtained from Muller’s Muscle Conjunctival Resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Results Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2 and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Conclusions Thus, we report for the first time that human ALG tissues contain precursor marker positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES and isolate candidate precursor cells from ALG tissue. PMID:27612554

  14. Laser ablation of persistent twist cells in Drosophila: muscle precursor fate is not segmentally restricted

    NASA Technical Reports Server (NTRS)

    Farrell, E. R.; Keshishian, H.

    1999-01-01

    In Drosophila the precursors of the adult musculature arise during embryogenesis. These precursor cells have been termed Persistent Twist Cells (PTCs), as they continue to express the transcription factor Twist after that gene ceases expression elsewhere in the mesoderm. In the larval abdomen, the PTCs are associated with peripheral nerves in stereotypic ventral, dorsal, and lateral clusters, which give rise, respectively, to the ventral, dorsal, and lateral muscle fiber groups of the adult. We tested the developmental potential of the PTCs by using a microbeam laser to ablate specific clusters in larvae. We found that the ablation of a single segmental PTC cluster does not usually result in the deletion of the corresponding adult fibers of that segment. Instead, normal or near normal numbers of adult fibers can form after the ablation. Examination of pupae following ablation showed that migrating PTCs from adjacent segments are able to invade the affected segment, replenishing the ablated cells. However, the ablation of homologous PTCs in multiple segments does result in the deletion of the corresponding adult muscle fibers. These data indicate that the PTCs in an abdominal segment can contribute to the formation of muscle fibers in adjacent abdominal segments, and thus are not inherently restricted to the formation of muscle fibers within their segment of origin.

  15. A Review of Single Source Precursors for the Deposition of Ternary Chalcopyrite Materials

    NASA Technical Reports Server (NTRS)

    Banger, K. K.; Cowen, J.; Harris, J.; McClarnon, R.; Hehemann, D. G.; Duraj, S. A.; Scheiman, D.; Hepp, A. F.

    2002-01-01

    The development of thin-film solar cells on flexible, lightweight, space-qualified durable substrates (i.e. Kapton) provides an attractive solution to fabricating solar arrays with high specific power, (W/kg). The syntheses and thermal modulation of ternary single source precursors, based on the [{LR}2Cu(SR')2In(SR')2] architecture in good yields are described. Thermogravimetric analyses (TGA) and Low temperature Differential Scanning Caloriometry, (DSC) demonstrate that controlled manipulation of the steric and electronic properties of either the group five-donor and/or chalcogenide moiety permits directed adjustment of the thermal stability and physical properties of the precursors. TGA-Evolved Gas Analysis, confirms that single precursors decompose by the initial extrusion of the sulphide moiety, followed by the loss of the neutral donor group, (L) to release the ternary chalcopyrite matrix. X-ray diffraction studies, EDS and SEM on the non-volatile pyrolized material demonstrate that these derivatives afford single-phase CuInS2/CuInSe2 materials at low temperature. Thin-film fabrication studies demonstrate that these single source precursors can be used in a spray chemical vapor deposition process, for depositing CuInS2 onto flexible polymer substrates at temperatures less than 400 C.

  16. GBM secretome induces transient transformation of human neural precursor cells.

    PubMed

    Venugopal, Chitra; Wang, X Simon; Manoranjan, Branavan; McFarlane, Nicole; Nolte, Sara; Li, Meredith; Murty, Naresh; Siu, K W Michael; Singh, Sheila K

    2012-09-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, with a uniformly poor prognosis. The tumor microenvironment is composed of both supportive cellular substrates and exogenous factors. We hypothesize that exogenous factors secreted by brain tumor initiating cells (BTICs) could predispose normal neural precursor cells (NPCs) to transformation. When NPCs are grown in GBM-conditioned media, and designated as "tumor-conditioned NPCs" (tcNPCs), they become highly proliferative and exhibit increased stem cell self-renewal, or the unique ability of stem cells to asymmetrically generate another stem cell and a daughter cell. tcNPCs also show an increased transcript level of stem cell markers such as CD133 and ALDH and growth factor receptors such as VEGFR1, VEGFR2, EGFR and PDGFRα. Media analysis by ELISA of GBM-conditioned media reveals an elevated secretion of growth factors such as EGF, VEGF and PDGF-AA when compared to normal neural stem cell-conditioned media. We also demonstrate that tcNPCs require prolonged or continuous exposure to the GBM secretome in vitro to retain GBM BTIC characteristics. Our in vivo studies reveal that tcNPCs are unable to form tumors, confirming that irreversible transformation events may require sustained or prolonged presence of the GBM secretome. Analysis of GBM-conditioned media by mass spectrometry reveals the presence of secreted proteins Chitinase-3-like 1 (CHI3L1) and H2A histone family member H2AX. Collectively, our data suggest that GBM-secreted factors are capable of transiently altering normal NPCs, although for retention of the transformed phenotype, sustained or prolonged secretome exposure or additional transformation events are likely necessary.

  17. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

    PubMed Central

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

    2016-01-01

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

  18. α-Secretase-derived Fragment of Cellular Prion, N1, Protects against Monomeric and Oligomeric Amyloid β (Aβ)-associated Cell Death*

    PubMed Central

    Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Ferreira, Sergio T.; Marzolo, Maria-Paz; Bauer, Charlotte; Thevenet, Aurélie; Checler, Frédéric

    2012-01-01

    In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrPc) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APPLDN) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APPLDN-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease. PMID:22184125

  19. Hydroxyapatite coatings deposited by liquid precursor plasma spraying: controlled dense and porous microstructures and osteoblastic cell responses.

    PubMed

    Huang, Yi; Song, Lei; Liu, Xiaoguang; Xiao, Yanfeng; Wu, Yao; Chen, Jiyong; Wu, Fang; Gu, Zhongwei

    2010-12-01

    Hydroxyapatite coatings were deposited on Ti-6Al-4V substrates by a novel plasma spraying process, the liquid precursor plasma spraying (LPPS) process. X-ray diffraction results showed that the coatings obtained by the LPPS process were mainly composed of hydroxyapatite. The LPPS process also showed excellent control on the coating microstructure, and both nearly fully dense and highly porous hydroxyapatite coatings were obtained by simply adjusting the solid content of the hydroxyapatite liquid precursor. Scanning electron microscope observations indicated that the porous hydroxyapatite coatings had pore size in the range of 10-200 µm and an average porosity of 48.26 ± 0.10%. The osteoblastic cell responses to the dense and porous hydroxyapatite coatings were evaluated with human osteoblastic cell MG-63, in respect of the cell morphology, proliferation and differentiation, with the hydroxyapatite coatings deposited by the atmospheric plasma spraying (APS) process as control. The cell experiment results indicated that the heat-treated LPPS coatings with a porous structure showed the best cell proliferation and differentiation among all the hydroxyapatite coatings. Our results suggest that the LPPS process is a promising plasma spraying technique for fabricating hydroxyapatite coatings with a controllable microstructure, which has great potential in bone repair and replacement applications.

  20. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon

    The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specificallymore » to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.« less

  1. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

    PubMed

    Woll, Petter S; Martin, Colin H; Miller, Jeffrey S; Kaufman, Dan S

    2005-10-15

    Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

  2. Identification of tumor-initiating cells derived from two canine rhabdomyosarcoma cell lines.

    PubMed

    Kishimoto, Takuya Evan; Yashima, Shoko; Nakahira, Rei; Onozawa, Eri; Azakami, Daigo; Ujike, Makoto; Ochiai, Kazuhiko; Ishiwata, Toshiyuki; Takahashi, Kimimasa; Michishita, Masaki

    2017-07-07

    Cancer stem cells or tumor-initiating cells (TICs) are a small subpopulation of cells that have the capacity to self-renew, differentiate and initiate tumors. These cells may function in tumor initiation, aggression and recurrence. Whether spheres derived from canine rhabdomyosarcoma cells have stem cell-like properties is unclear. We induced sphere formation in the canine rhabdomyosarcoma cell lines, CMS-C and CMS-J, and characterized the spheres in vitro and in vivo. Sphere-forming cells were more resistant to vincristine, mitoxantrone and doxorubicin than adherent cells. Xenograft transplantation demonstrated that 1 × 10 3 sphere-forming cells derived from CMS-C were sufficient for tumor formation. The sphere assay showed that the sphere-forming cells were present in these tumors. These results suggest that the spheres derived from canine rhabdomyosarcoma cells may possess characteristics of TICs. This study provides the foundation for elucidating the contribution of TICs to rhabdomyosarcoma tumorigenesis.

  3. Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma.

    PubMed

    Sun, Xi-Tai; Yuan, Xian-Wen; Zhu, Hai-Tao; Deng, Zheng-Ming; Yu, De-Cai; Zhou, Xiang; Ding, Yi-Tao

    2012-09-21

    To investigate the role of bone marrow-derived endothelial progenitor cells (EPCs) in the angiogenesis of hepatocellular carcinoma (HCC). The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein (GFP) + bone marrow cells. The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting. Serum and tissue levels of vascular endothelial growth factor (VEGF) and colony-stimulating factor (CSF) were quantified by enzyme-linked immunosorbent assay. The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction. The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry. The proportion of EPCs in vessels was then calculated. The HCC model was successful established. The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively. These values are much higher than in the sham-operation group (0.11% ± 0.13%, 0.05% ± 0.11%, n = 9) at 14 d after modeling. At 21 d, the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%, 0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%, 0.12% ± 0.11% in control. Compared to the transient increase observed in controls, the higher level of circulating EPCs were induced by HCC. In addition, the level of serum VEGF and CSF increased gradually in HCC, reaching its peak 14 d after modeling, then slowly decreased. Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels. Under fluorescence microscopy, the bone-marrow (BM)-derived cells labeled with GFP were concentrated in the same area. The relative levels of CD133 and CD34 gene expression were elevated in tumors, around 5.0 and 3.8 times that

  4. Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells.

    PubMed

    Park, Yong-Beom; Seo, Sinji; Kim, Jin-A; Heo, Jin-Chul; Lim, Young-Cheol; Ha, Chul-Won

    2015-06-24

    The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.

  5. Enabling Surgical Placement of Hydrogels through Achieving Paste-Like Rheological Behavior in Hydrogel Precursor Solutions

    PubMed Central

    Beck, Emily C.; Lohman, Brooke L.; Tabakh, Daniel B.; Kieweg, Sarah L.; Gehrke, Stevin H.; Berkland, Cory J.; Detamore, Michael S.

    2015-01-01

    Hydrogels are a promising class of materials for tissue regeneration, but they lack the ability to be molded into a defect site by a surgeon because hydrogel precursors are liquid solutions that are prone to leaking during placement. Therefore, although the main focus of hydrogel technology and developments are on hydrogels in their crosslinked form, our primary focus is on improving the fluid behavior of hydrogel precursor solutions. In this work, we introduce a method to achieve paste-like hydrogel precursor solutions by combining hyaluronic acid nanoparticles with traditional crosslinked hyaluronic acid hydrogels. Prior to crosslinking, the samples underwent rheological testing to assess yield stress and recovery using linear hyaluronic acid as a control. The experimental groups containing nanoparticles were the only solutions that exhibited a yield stress, demonstrating that the nanoparticulate rather than the linear form of hyaluronic acid was necessary to achieve paste-like behavior. The gels were also photocrosslinked and further characterized as solids, where it was demonstrated that the inclusion of nanoparticles did not adversely affect the compressive modulus and that encapsulated bone marrow-derived mesenchymal stem cells remained viable. Overall, this nanoparticle-based approach provides a platform hydrogel system that exhibits a yield stress prior to crosslinking, and can then be crosslinked into a hydrogel that is capable of encapsulating cells that remain viable. This behavior may hold significant impact for hydrogel applications where a paste-like behavior is desired in the hydrogel precursor solution. PMID:25691398

  6. Melatonin and its precursors in Y79 human retinoblastoma cells - Effect of sodium butyrate

    NASA Technical Reports Server (NTRS)

    Deng, Mei H.; Lopez G.-Coviella, Ignacio; Lynch, Harry J.; Wurtman, Richard J.

    1991-01-01

    We studied the release of melatonin and the production of its precursors, 5-hydroxytryptophan and serotonin, in cultured Y79 human retinoblastoma cells. This biosynthetic capability was found to be dependent on cell differentiation, which was initiated by culturing Y79 cells for 7 days in dishes coated with poly-D-lysine to promote cell adhesion to the surface of the culture dishes. Differentiation was further induced by exposing the cell monolayer to sodium butyrate (3 mM) for three days. This protocol dramatically increased the release of melatonin, and the syntheses of 5-hydroxytryptophan and serotonin in response to forskolin stimulation. Exposure to dopamine or L-DOPA markedly diminished the forskolin-stimulated release of melatonin, as well as the production of 5-hydroxytryptophan and serotonin. These observations indicate that Y79 cells represent a primitive cell line which, following appropriate differentiation can display biochemical characteristics similar to those of the human retina. Moreover, serotonin synthesis and melatonin release appear to be coupled in Y79 ceils.

  7. Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

    PubMed

    Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

    2015-09-18

    BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

  8. Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.

    2011-04-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry,more » EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a

  9. Skin appendage-derived stem cells: cell biology and potential for wound repair.

    PubMed

    Xie, Jiangfan; Yao, Bin; Han, Yutong; Huang, Sha; Fu, Xiaobing

    2016-01-01

    Stem cells residing in the epidermis and skin appendages are imperative for skin homeostasis and regeneration. These stem cells also participate in the repair of the epidermis after injuries, inducing restoration of tissue integrity and function of damaged tissue. Unlike epidermis-derived stem cells, comprehensive knowledge about skin appendage-derived stem cells remains limited. In this review, we summarize the current knowledge of skin appendage-derived stem cells, including their fundamental characteristics, their preferentially expressed biomarkers, and their potential contribution involved in wound repair. Finally, we will also discuss current strategies, future applications, and limitations of these stem cells, attempting to provide some perspectives on optimizing the available therapy in cutaneous repair and regeneration.

  10. iPS-cell derived dendritic cells and macrophages for cancer therapy.

    PubMed

    Senju, Satoru

    2016-08-01

    Antibody-based anti-cancer immunotherapy was recently recognized as one of the truly effective therapies for cancer patients. Antibodies against cell surface cancer antigens, such as CD20, and also those against immune-inhibitory molecules called "immune checkpoint blockers", such as CTLA4 or PD1, have emerged. Large-scale clinical trials have confirmed that, in some cases, antibody-based drugs are superior to conventional chemotherapeutic agents. These antibody-based drugs are now being manufactured employing a mass-production system by pharmaceutical companies. Anti-cancer therapy by immune cells, i.e. cell-based immunotherapy, is expected to be more effective than antibody therapy, because immune cells can recognize, infiltrate, and act in cancer tissues more directly than antibodies. In order to achieve cell-based anti-cancer immunotherapy, it is necessary to develop manufacturing systems for mass-production of immune cells. Our group has been studying immunotherapy with myeloid cells derived from ES cells or iPS cells. These pluripotent stem cells can be readily propagated under constant culture conditions, with expansion into a large quantity. We consider these stem cells to be the most suitable cellular source for mass-production of immune cells. This review introduces our studies on anti-cancer therapy with iPS cell-derived dendritic cells and iPS cell-derived macrophages.

  11. Human Myocardial Pericytes: Multipotent Mesodermal Precursors Exhibiting Cardiac Specificity

    PubMed Central

    Chen, William C.W.; Baily, James E.; Corselli, Mirko; Diaz, Mary; Sun, Bin; Xiang, Guosheng; Gray, Gillian A.; Huard, Johnny; Péault, Bruno

    2015-01-01

    Perivascular mesenchymal precursor cells (i.e. pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, PDGFRβ, PDGFRα, αSMA, and SM-MHC, but not CD117, CD133 and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express MSC markers in situ and exhibited osteo- chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte co-culture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells. PMID:25336400

  12. Identification of tumor-initiating cells derived from two canine rhabdomyosarcoma cell lines

    PubMed Central

    KISHIMOTO, Takuya Evan; YASHIMA, Shoko; NAKAHIRA, Rei; ONOZAWA, Eri; AZAKAMI, Daigo; UJIKE, Makoto; OCHIAI, Kazuhiko; ISHIWATA, Toshiyuki; TAKAHASHI, Kimimasa; MICHISHITA, Masaki

    2017-01-01

    Cancer stem cells or tumor-initiating cells (TICs) are a small subpopulation of cells that have the capacity to self-renew, differentiate and initiate tumors. These cells may function in tumor initiation, aggression and recurrence. Whether spheres derived from canine rhabdomyosarcoma cells have stem cell-like properties is unclear. We induced sphere formation in the canine rhabdomyosarcoma cell lines, CMS-C and CMS-J, and characterized the spheres in vitro and in vivo. Sphere-forming cells were more resistant to vincristine, mitoxantrone and doxorubicin than adherent cells. Xenograft transplantation demonstrated that 1 × 103 sphere-forming cells derived from CMS-C were sufficient for tumor formation. The sphere assay showed that the sphere-forming cells were present in these tumors. These results suggest that the spheres derived from canine rhabdomyosarcoma cells may possess characteristics of TICs. This study provides the foundation for elucidating the contribution of TICs to rhabdomyosarcoma tumorigenesis. PMID:28529244

  13. CD22 is required for formation of memory B cell precursors within germinal centers.

    PubMed

    Chappell, Craig P; Draves, Kevin E; Clark, Edward A

    2017-01-01

    CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens.

  14. CD22 is required for formation of memory B cell precursors within germinal centers

    PubMed Central

    Chappell, Craig P.; Draves, Kevin E.

    2017-01-01

    CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens. PMID:28346517

  15. Differentiation of osteoblast and osteoclast precursors on pure and silicon-substituted synthesized hydroxyapatites.

    PubMed

    Lehmann, Giorgia; Cacciotti, Ilaria; Palmero, Paola; Montanaro, Laura; Bianco, Alessandra; Campagnolo, Luisa; Camaioni, Antonella

    2012-10-01

    Calcium phosphate-based materials should show excellent bone-bonding and cell-mediated resorption characteristics at the same time, in order to be employed for bone replacement. In this perspective, pure (HAp) and silicon-substituted hydroxyapatite (Si-HAp, 1.4% wt) porous cylinders were prepared starting from synthesized powders and polyethylene spheres used as porogens, and investigated as supports for osteoblast and osteoclast progenitor differentiation. A systematic and detailed biological characterization is reported, in terms of cell adhesion, viability, proliferation, differentiation and bioresorption, aimed at proposing a complete and reliable picture of bone cell in vitro behavior, comprehensive of both the osteogenesis and the bone resorption processes. In order to achieve this purpose, cytocompatibility, differentiation and gene expression by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were carried out using parietal bone-derived pre-osteoblasts obtained from neonatal mice and the bioresorption capability was assessed by seeding human peripheral blood monocytes, as osteoclast precursors. It resulted that both pure and Si-substituted HAps were able to promote differentiation of precursor cells in mature osteoblasts and osteoclasts. In particular, the Si-HAps enhanced the pre-osteoblast proliferation and showed higher osteoclast-mediated bioresorption capability, as supported by the presence of larger and more numerous resorption lacunae, whereas HAps promoted a more robust cell differentiation in terms of both osteocalcin gene expression by qRT-PCR and cell morphological evaluation by SEM analysis.

  16. Tumor suppressors BTG1 and IKZF1 cooperate during mouse leukemia development and increase relapse risk in B-cell precursor acute lymphoblastic leukemia patients.

    PubMed

    Scheijen, Blanca; Boer, Judith M; Marke, René; Tijchon, Esther; van Ingen Schenau, Dorette; Waanders, Esmé; van Emst, Liesbeth; van der Meer, Laurens T; Pieters, Rob; Escherich, Gabriele; Horstmann, Martin A; Sonneveld, Edwin; Venn, Nicola; Sutton, Rosemary; Dalla-Pozza, Luciano; Kuiper, Roland P; Hoogerbrugge, Peter M; den Boer, Monique L; van Leeuwen, Frank N

    2017-03-01

    Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1 -deleted B-cell precursor acute lymphoblastic leukemia ( P =0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival ( P =0.0003) and a higher 5-year cumulative incidence of relapse ( P =0.005), when compared with IKZF1 -deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1 , did not affect the outcome of IKZF1 -deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1 -deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1 +/- mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1 +/- displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1 -deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function. Copyright© Ferrata Storti Foundation.

  17. Toxicological effects of three types of silver nanoparticles and their salt precursors acting on human U-937 and HL-60 cells.

    PubMed

    Barbasz, Anna; Oćwieja, Magdalena; Walas, Stanisław

    2017-01-01

    The growing popularity of nanomaterials requires a systematic study of their effects on the human body. Silver nanoparticles (AgNPs), due to their antiseptic properties, are used in almost every area of life. The purpose of the study was to examine whether the precursor used for the synthesis of nanoparticles affects their bio-influence and modifies their impact on cells of the human immune system. To compare the effects of precursor silver salts (AgNO 3 , CH 3 COOAg and AgClO 4 ) and corresponding nanoparticles (TAN TAA and TAC) cytotoxicity study was conducted on two cell lines U-937 and HL-60. For both cell lines, silver salts are more toxic than the corresponding nanoparticles. Cell viability after treatment with the two forms of silver (salt/particle) is dependent on silver dose and degree of cells differentiation. Addition of the silver salt of doses greater than 5 mg/L results in decreased cell viability by over 60%, whereas nanoparticles' addition reduces cell viability on average by 30%. On the basis of the determined LD 50 values it can be stated that for the tested cells the most toxic are AgClO 4 and TAC. Production of nitric oxide, which is a mediator of inflammation, is the greatest after treatment of the cells by TAC. Different interactions of studied nanoparticles with albumin has been found and it was shown that addition of albumin to the cells treated by nanoparticles reduces their toxic effects. Obtained by us highly purified, mono-disperse AgNPs exhibit diverse effects relative to the biological systems, depending on the precursor salt used.

  18. Optimizing a multifunctional microsphere scaffold to improve neural precursor cell transplantation for traumatic brain injury repair.

    PubMed

    Skop, Nolan B; Calderon, Frances; Cho, Cheul H; Gandhi, Chirag D; Levison, Steven W

    2016-10-01

    Tissue engineering using stem cells is widely used to repair damaged tissues in diverse biological systems; however, this approach has met with less success in regenerating the central nervous system (CNS). In this study we optimized and characterized the surface chemistry of chitosan-based scaffolds for CNS repair. To maintain radial glial cell (RGC) character of primitive neural precursors, fibronectin was adsorbed to chitosan. The chitosan was further modified by covalently linking heparin using genipin, which then served as a linker to immobilize fibroblast growth factor-2 (FGF-2), creating a multifunctional film. Fetal rat neural precursors plated onto this multifunctional film proliferated and remained multipotent for at least 3 days without providing soluble FGF-2. Moreover, they remained less mature and more highly proliferative than cells maintained on fibronectin-coated substrates in culture medium supplemented with soluble FGF-2. To create a vehicle for cell transplantation, a 3% chitosan solution was electrosprayed into a coagulation bath to generate microspheres (range 30-100 µm, mean 64 µm) that were subsequently modified. Radial glial cells seeded onto these multifunctional microspheres proliferated for at least 7 days in culture and the microspheres containing cells were small enough to be injected, using 23 Gauge Hamilton syringes, into the brains of adult rats that had previously sustained cortical contusion injuries. When analysed 3 days later, the transplanted RGCs were positive for the stem cell/progenitor marker Nestin. These results demonstrate that this multifunctional scaffold can be used as a cellular and growth factor delivery vehicle for the use in developing cell transplantation therapies for traumatic brain injuries. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion.

    PubMed

    Gage, P J; Roller, M L; Saunders, T L; Scarlett, L M; Camper, S A

    1996-01-01

    The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cells lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters containing different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df<-->+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.

  20. Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

    PubMed

    Body, A; Brownstein, B H

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

  1. Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

    PubMed Central

    Body, Barbara A.; Brownstein, Bernard H.

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

  2. A new pro-migratory activity on human myogenic precursor cells for a synthetic peptide within the E domain of the mechano growth factor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mills, Philippe; Lafreniere, Jean-Francois; Benabdallah, Basma Fattouma

    2007-02-01

    Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferationmore » while delaying the differentiation of C{sub 2}C{sub 12} cells. Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor. Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine.« less

  3. CCR6 Defines Memory B Cell Precursors in Mouse and Human Germinal Centers, Revealing Light-Zone Location and Predominant Low Antigen Affinity.

    PubMed

    Suan, Dan; Kräutler, Nike J; Maag, Jesper L V; Butt, Danyal; Bourne, Katherine; Hermes, Jana R; Avery, Danielle T; Young, Clara; Statham, Aaron; Elliott, Michael; Dinger, Marcel E; Basten, Antony; Tangye, Stuart G; Brink, Robert

    2017-12-19

    Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6 + GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells from Patients with Sickle Cell Disease.

    PubMed

    Uchida, Naoya; Haro-Mora, Juan J; Fujita, Atsushi; Lee, Duck-Yeon; Winkler, Thomas; Hsieh, Matthew M; Tisdale, John F

    2017-03-01

    Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent an ideal source for in vitro modeling of erythropoiesis and a potential alternative source for red blood cell transfusions. However, iPS cell-derived erythroid cells predominantly produce ε- and γ-globin without β-globin production. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production. In this study, we generated several iPS cell lines derived from bone marrow stromal cells (MSCs) and peripheral blood erythroid progenitors (EPs) from sickle cell disease patients, and evaluated hematopoietic stem/progenitor cell (HSPC) generation after iPS sac induction as well as subsequent erythroid differentiation. MSC-derived iPS sacs yielded greater amounts of immature hematopoietic progenitors (VEGFR2 + GPA-), definitive HSPCs (CD34 + CD45+), and megakaryoerythroid progenitors (GPA + CD41a+), as compared to EP-derived iPS sacs. Erythroid differentiation from MSC-derived iPS sacs resulted in greater amounts of erythroid cells (GPA+) and higher β-globin (and βS-globin) expression, comparable to ES sac-derived cells. These data demonstrate that human MSC-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, likely due to heightened emergence of immature progenitors. Our findings should be important for iPS cell-derived erythroid cell generation. Stem Cells 2017;35:586-596. © 2016 AlphaMed Press.

  5. Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal

    PubMed Central

    Chen, Hongxin; Goodus, Matthew T; de Toledo, Sonia M; Azzam, Edouard I; Levison, Steven W

    2015-01-01

    Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to 137Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure. PMID:26056396

  6. The Role of the Progressive Ankylosis Protein (ANK) in Adipogenic/Osteogenic Fate Decision of Precursor Cells

    PubMed Central

    Minashima, Takeshi; Quirno, Martin; Lee, You Jin; Kirsch, Thorsten

    2017-01-01

    The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5 weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation. PMID:28286238

  7. Generation of the heterodimeric precursor GP3 of the Chlamydomonas cell wall.

    PubMed

    Voigt, Jürgen; Kiess, Michael; Getzlaff, Rita; Wöstemeyer, Johannes; Frank, Ronald

    2010-09-01

    The cell wall of the unicellular green alga Chlamydomonas reinhardtii exclusively consists of hydroxyproline-containing glycoproteins. Protein chemical analysis of its polypeptide constituents was hindered by their cross-linking via peroxidase-catalysed intermolecular isodityrosine formation and transaminase-dependent processes. To overcome this problem, we have identified putative soluble precursors using polyclonal antibodies raised against deglycosylation products of the highly purified insoluble wall fraction and analysed their amino acid sequence. The occurrence of the corresponding polypeptide in the insoluble glycoprotein framework was finally probed by epitope mapping of the polyclonal antibodies using overlapping scan peptides which, together, cover the whole amino acid sequence of the putative precursor. As a control, peptide fragments released from the insoluble wall fraction by trypsin treatment were analysed by mass spectroscopy. By this approach, the heterodimeric, chaotrope-soluble glycoprotein GP3 proved to be a constituent of the insoluble extracellular matrix of Chlamydomonas reinhardtii. Furthermore, we have shown that the polypeptide backbones of both GP3 subunits are encoded by the same gene and differ by a C-terminal truncation in the case of GP3A. © 2010 Blackwell Publishing Ltd.

  8. JAK2 aberrations in childhood B-cell precursor acute lymphoblastic leukemia

    PubMed Central

    de Goffau-Nobel, Willemieke; Hoogkamer, Alex Q.; Boer, Judith M.; Boeree, Aurélie; van de Ven, Cesca; Koudijs, Marco J.; Besselink, Nicolle J.M.; de Groot-Kruseman, Hester A.; Zwaan, Christian Michel; Horstmann, Martin A.; Pieters, Rob; den Boer, Monique L.

    2017-01-01

    JAK2 abnormalities may serve as target for precision medicines in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In the current study we performed a screening for JAK2 mutations and translocations, analyzed the clinical outcome and studied the efficacy of two JAK inhibitors in primary BCP-ALL cells. Importantly, we identify a number of limitations of JAK inhibitor therapy. JAK2 mutations mainly occurred in the poor prognostic subtypes BCR-ABL1-like and non- BCR-ABL1-like B-other (negative for sentinel cytogenetic lesions). JAK2 translocations were restricted to BCR-ABL1-like cases. Momelotinib and ruxolitinib were cytotoxic in both JAK2 translocated and JAK2 mutated cells, although efficacy in JAK2 mutated cells highly depended on cytokine receptor activation by TSLP. However, our data also suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and microenvironment-induced resistance. Furthermore, inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon release of the inhibitors. This preclinical evidence implies that further optimization and evaluation of JAK inhibitor treatment is necessary prior to its clinical integration in pediatric BCP-ALL. PMID:29163799

  9. Langerhans cell precursors acquire RANK/CD265 in prenatal human skin

    PubMed Central

    Schöppl, Alice; Botta, Albert; Prior, Marion; Akgün, Johnnie; Schuster, Christopher; Elbe-Bürger, Adelheid

    2015-01-01

    The skin is the first barrier against foreign pathogens and the prenatal formation of a strong network of various innate and adaptive cells is required to protect the newborn from perinatal infections. While many studies about the immune system in healthy and diseased adult human skin exist, our knowledge about the cutaneous prenatal/developing immune system and especially about the phenotype and function of antigen-presenting cells such as epidermal Langerhans cells (LCs) in human skin is still scarce. It has been shown previously that LCs in healthy adult human skin express receptor activator of NF-κB (RANK), an important molecule prolonging their survival. In this study, we investigated at which developmental stage LCs acquire this important molecule. Immunofluorescence double-labeling of cryostat sections revealed that LC precursors in prenatal human skin either do not yet [10–11 weeks of estimated gestational age (EGA)] or only faintly (13–15 weeks EGA) express RANK. LCs express RANK at levels comparable to adult LCs by the end of the second trimester. Comparable with adult skin, dermal antigen-presenting cells at no gestational age express this marker. These findings indicate that epidermal leukocytes gradually acquire RANK during gestation – a phenomenon previously observed also for other markers on LCs in prenatal human skin. PMID:25722033

  10. Can Human Embryonic Stem Cell-Derived Stromal Cells Serve a Starting Material for Myoblasts?

    PubMed Central

    Ando, Yu; Saito, Marie; Machida, Masakazu; Yoshida-Noro, Chikako; Akutsu, Hidenori; Takahashi, Masataka

    2017-01-01

    A large number of myocytes are necessary to treat intractable muscular disorders such as Duchenne muscular dystrophy with cell-based therapies. However, starting materials for cellular therapy products such as myoblasts, marrow stromal cells, menstrual blood-derived cells, and placenta-derived cells have a limited lifespan and cease to proliferate in vitro. From the viewpoints of manufacturing and quality control, cells with a long lifespan are more suitable as a starting material. In this study, we generated stromal cells for future myoblast therapy from a working cell bank of human embryonic stem cells (ESCs). The ESC-derived CD105+ cells with extensive in vitro proliferation capability exhibited myogenesis and genetic stability in vitro. These results imply that ESC-derived CD105+ cells are another cell source for myoblasts in cell-based therapy for patients with genetic muscular disorders. Since ESCs are immortal, mesenchymal stromal cells generated from ESCs can be manufactured at a large scale in one lot for pharmaceutical purposes. PMID:28706537

  11. Red blood cell-derived microparticles: An overview.

    PubMed

    Westerman, Maxwell; Porter, John B

    2016-07-01

    The red blood cell (RBC) is historically the original parent cell of microparticles (MPs). In this overview, we describe the discovery and the early history of red cell-derived microparticles (RMPs) and present an overview of the evolution of RMP. We report the formation, characteristics, effects of RMP and factors which may affect RMP evaluation. The review examines RMP derived from both normal and pathologic RBC. The pathologic RBC studies include sickle cell anemia (SCA), sickle cell trait (STr), thalassemia intermedia (TI), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary stomatocytosis (HSt) and glucose-6-phosphate dehydrogenase deficiency (G6PD). Copyright © 2016 Elsevier Inc. All rights reserved.

  12. The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function.

    PubMed

    Wang, Hongtao; Hughes, Inna; Planer, William; Parsadanian, Alexander; Grider, John R; Vohra, Bhupinder P S; Keller-Peck, Cynthia; Heuckeroth, Robert O

    2010-01-27

    Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.

  13. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

    PubMed

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

    2016-04-15

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Cell Therapy From Bench to Bedside Translation in CNS Neurorestoratology Era

    PubMed Central

    Huang, Hongyun; Chen, Lin; Sanberg, Paul

    2010-01-01

    Recent advances in cell biology, neural injury and repair, and the progress towards development of neurorestorative interventions are the basis for increased optimism. Based on the complexity of the processes of demyelination and remyelination, degeneration and regeneration, damage and repair, functional loss and recovery, it would be expected that effective therapeutic approaches will require a combination of strategies encompassing neuroplasticity, immunomodulation, neuroprotection, neurorepair, neuroreplacement, and neuromodulation. Cell-based restorative treatment has become a new trend, and increasing data worldwide have strongly proven that it has a pivotal therapeutic value in CNS disease. Moreover, functional neurorestoration has been achieved to a certain extent in the CNS clinically. Up to now, the cells successfully used in preclinical experiments and/or clinical trial/treatment include fetal/embryonic brain and spinal cord tissue, stem cells (embryonic stem cells, neural stem/progenitor cells, hematopoietic stem cells, adipose-derived adult stem/precursor cells, skin-derived precursor, induced pluripotent stem cells), glial cells (Schwann cells, oligodendrocyte, olfactory ensheathing cells, astrocytes, microglia, tanycytes), neuronal cells (various phenotypic neurons and Purkinje cells), mesenchymal stromal cells originating from bone marrow, umbilical cord, and umbilical cord blood, epithelial cells derived from the layer of retina and amnion, menstrual blood-derived stem cells, Sertoli cells, and active macrophages, etc. Proof-of-concept indicates that we have now entered a new era in neurorestoratology. PMID:21359168

  15. Perfluoroalkyl Acids (PFAAs) and Selected Precursors in the Baltic Sea Environment: Do Precursors Play a Role in Food Web Accumulation of PFAAs?

    PubMed

    Gebbink, Wouter A; Bignert, Anders; Berger, Urs

    2016-06-21

    The present study examined the presence of perfluoroalkyl acids (PFAAs) and selected precursors in the Baltic Sea abiotic environment and guillemot food web, and investigated the relative importance of precursors in food web accumulation of PFAAs. Sediment, water, zooplankton, herring, sprat, and guillemot eggs were analyzed for perfluoroalkane sulfonic acids (PFSAs; C4,6,8,10) and perfluoroalkyl carboxylic acids (PFCAs; C6-15) along with six perfluoro-octane sulfonic acid (PFOS) precursors and 11 polyfluoroalkyl phosphoric acid diesters (diPAPs). FOSA, FOSAA and its methyl and ethyl derivatives (Me- and EtFOSAA), and 6:2/6:2 diPAP were detected in sediment and water. While FOSA and the three FOSAAs were detected in all biota, a total of nine diPAPs were only detected in zooplankton. Concentrations of PFOS precursors and diPAPs exceeded PFOS and PFCA concentrations, respectively, in zooplankton, but not in fish and guillemot eggs. Although PFOS precursors were present at all trophic levels, they appear to play a minor role in food web accumulation of PFOS based on PFOS precursor/PFOS ratios and PFOS and FOSA isomer patterns. The PFCA pattern in fish could not be explained by the intake pattern based on PFCAs and analyzed precursors, that is, diPAPs. Exposure to additional precursors might therefore be a dominant exposure pathway compared to direct PFCA exposure for fish.

  16. Unusual neutral oligosaccharides in mature Sindbis virus glycoproteins are synthesized from truncated precursor oligosaccharides in Chinese hamster ovary cells.

    PubMed

    Davidson, S K; Hunt, L A

    1983-03-01

    We have previously demonstrated the presence of unusual small asparaginyl-oligosaccharides [(Man)3GlcNAc2-ASN] in the mature glycoproteins of Sindbis virus released from both wild-type and lectin-resistant Chinese hamster ovary cells, but the mechanism of synthesis of these structures was not determined. Gel filtration and endo-beta-N-acetylglucosaminidase analyses of Pronase-digested glycopeptides from [3H]mannose-labelled Sindbis virus released at different times after infection of a phytohaemagglutinin-resistant line of Chinese hamster ovary cells demonstrated that these small asparaginyl-oligosaccharides were present in similar relative amounts in virus released throughout the virus infection, rather than arising primarily at late times when cytopathic effects were maximal. Similar analyses of pulse-labelled, cell-associated viral glycopeptides suggested that these small oligosaccharides on mature virus glycoprotein resulted from the normal alpha 1,2-mannosidase processing of truncated precursor oligosaccharides (containing five rather than nine mannoses), rather than from aberrant processing or degradation of the full-size precursor oligosaccharides or normal intermediates.

  17. Stabilized metal nanoparticles from organometallic precursors for low temperature fuel cells.

    PubMed

    Ramirez-Meneses, E; Dominguez-Crespo, M A; Torres-Huerta, A M

    2013-01-01

    In this work, a review of articles and patents related to the utilization of colloidal metal nanoparticles produced by the decomposition of organometallic precursors as supported electrocatalysts in different electrochemical reactions including hydrogen evolution reaction (HER), oxygen reduction reaction (ORR) and methanol oxidation reaction (MOR) is discussed. In the case of stabilized metal nanoparticles, the kind of functional group contained in the stabilizer as well as the metal/stabilizer ratio, to evaluate the effect of particle size on the electrochemical performance, were also debated. Potential applications and perspectives of these electrocatalysts in proton exchange membrane fuel cells (PEMFC) are contended with reference to the role played by the coordination compounds and costs.

  18. Myeloid-derived suppressor cells modulate B-cell responses.

    PubMed

    Lelis, Felipe J N; Jaufmann, Jennifer; Singh, Anurag; Fromm, Katja; Teschner, Annkathrin Chiara; Pöschel, Simone; Schäfer, Iris; Beer-Hammer, Sandra; Rieber, Nikolaus; Hartl, Dominik

    2017-08-01

    Myeloid-derived suppressor cells (MDSCs) are key regulators of adaptive immunity by suppressing T-cell functions. However, their potential action on or interaction with B cells remained poorly understood. Here we demonstrate that human polymorphonuclear MDSCs differentially modulate B-cell function by suppressing B-cell proliferation and antibody production. We further demonstrate that this MDSC-mediated effect is cell contact dependent and involves established mediators such as arginase-1, nitric oxide (NO), reactive oxygen species (ROS) as well as B-cell death. Collectively, our studies provide novel evidence that human MDSCs modulate B cells, which could have future implications for immunotherapy approaches. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  19. Deformation strain is the main physical driver for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation.

    PubMed

    Ramani-Mohan, Ram-Kumar; Schwedhelm, Ivo; Finne-Wistrand, Anna; Krug, Melanie; Schwarz, Thomas; Jakob, Franz; Walles, Heike; Hansmann, Jan

    2018-03-01

    Mesenchymal stem cells play a major role during bone remodelling and are thus of high interest for tissue engineering and regenerative medicine applications. Mechanical stimuli, that is, deformation strain and interstitial fluid-flow-induced shear stress, promote osteogenic lineage commitment. However, the predominant physical stimulus that drives early osteogenic cell maturation is not clearly identified. The evaluation of each stimulus is challenging, as deformation and fluid-flow-induced shear stress interdepend. In this study, we developed a bioreactor that was used to culture mesenchymal stem cells harbouring a strain-responsive AP-1 luciferase reporter construct, on porous scaffolds. In addition to the reporter, mineralization and vitality of the cells was investigated by alizarin red staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Quantification of the expression of genes associated to bone regeneration and bone remodelling was used to confirm alizarin red measurements. Controlled perfusion and deformation of the 3-dimensional scaffold facilitated the alteration of the expression of osteogenic markers, luciferase activity, and calcification. To isolate the specific impact of scaffold deformation, a computational model was developed to derive a perfusion flow profile that results in dynamic shear stress conditions present in periodically loaded scaffolds. In comparison to actually deformed scaffolds, a lower expression of all measured readout parameters indicated that deformation strain is the predominant stimulus for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation. Copyright © 2017 John Wiley & Sons, Ltd.

  20. Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

    PubMed

    Hansen-Hagge, T E; Yokota, S; Reuter, H J; Schwarz, K; Bartram, C R

    1992-11-01

    Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.

  1. Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis.

    PubMed

    Kaku, M; Kitami, M; Rosales Rocabado, J M; Ida, T; Akiba, Y; Uoshima, K

    2017-08-01

    The periodontal ligament (PDL) is a non-mineralized connective tissue that exists between the alveolar bone and root surface cementum and plays important roles in tooth function. The PDL harbors a remarkable reserve of multipotent stem cells, which maintain various types of cells. However, the sources of these stem cells, other than their developmental origin, are not well understood. To elucidate the recruitment of bone marrow (BM)-derived stem cells in the PDL, green fluorescent protein (GFP)-expressing BM-derived cells were transplanted into the femoral BM of immunodeficient rats, and the distribution and expression of stem cell markers in the PDL were analyzed in vivo. To evaluate the functional significance of BM-derived cells to the PDL, tooth replantation was performed and the expression of stromal cell-derived factor (SDF)-1, a critical chemotactic signal for mesenchymal stem cell recruitment, was analyzed. To confirm the SDF-1-dependency of BM-derived cell migration to the PDL, PDL-conditioned medium (CM) was prepared, and BM-derived cell migration was analyzed using a transwell culture system. Four weeks after cell transplantation, GFP-positive cells were detected in the PDL, and some of them were also positive for stem cell markers (i.e., CD29, SSEA4, and αSMA). Seven days after tooth replantation, the number of GFP- and SDF-1-positive cells significantly increased in PDL. Concurrently, the concentration of SDF-1 and the number of colony-forming units of fibroblasts in peripheral blood were increased. BM-derived cell migration increased in PDL-CM and was inhibited by an inhibitor of C-X-C chemokine receptor type 4 (CXCR4), an SDF-1 receptor. These results indicate that stem cells and their progeny in PDL are not only derived from their developmental origin but are also supplied from the BM via the blood as the need arises. Moreover, this BM-derived cell recruitment appears to be regulated, at least partially, by the SDF-1/CXCR4 axis. © 2017 John Wiley

  2. A dock derived compound against laminin receptor (37 LR) exhibits anti-cancer properties in a prostate cancer cell line model.

    PubMed

    Umbaugh, Charles Samuel; Diaz-Quiñones, Adriana; Neto, Manoel Figueiredo; Shearer, Joseph J; Figueiredo, Marxa L

    2018-01-19

    Laminin receptor (67 LR) is a 67 kDa protein derived from a 37 kDa precursor (37 LR). 37/67 LR is a strong clinical correlate for progression, aggression, and chemotherapeutic relapse of several cancers including breast, prostate, and colon. The ability of 37/67 LR to promote cancer cell aggressiveness is further increased by its ability to transduce physiochemical and mechanosensing signals in endothelial cells and modulate angiogenesis. Recently, it was demonstrated that 37/67 LR modulates the anti-angiogenic potential of the secreted glycoprotein pigment epithelium-derived factor (PEDF). Restoration of PEDF balance is a desirable therapeutic outcome, and we sought to identify a small molecule that could recapitulate known signaling properties of PEDF but without the additional complications of peptide formulation or gene delivery safety validation. We used an in silico drug discovery approach to target the interaction interface between PEDF and 37 LR. Following cell based counter screening and binding validation, we characterized a hit compound's anti-viability, activation of PEDF signaling-related genes, anti-wound healing, and anti-cancer signaling properties. This hit compound has potential for future development as a lead compound for treating tumor growth and inhibiting angiogenesis.

  3. Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells.

    PubMed

    Peng, L A; Schousboe, A; Hertz, L

    1991-01-01

    Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transmination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.

  4. Adipose-derived mesenchymal stem cells and regenerative medicine.

    PubMed

    Konno, Masamitsu; Hamabe, Atsushi; Hasegawa, Shinichiro; Ogawa, Hisataka; Fukusumi, Takahito; Nishikawa, Shimpei; Ohta, Katsuya; Kano, Yoshihiro; Ozaki, Miyuki; Noguchi, Yuko; Sakai, Daisuke; Kudoh, Toshihiro; Kawamoto, Koichi; Eguchi, Hidetoshi; Satoh, Taroh; Tanemura, Masahiro; Nagano, Hiroaki; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2013-04-01

    Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β-cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  5. NK Cell-derived Exosomes From NK Cells Previously Exposed to Neuroblastoma Cells Augment the Antitumor Activity of Cytokine-activated NK Cells.

    PubMed

    Shoae-Hassani, Alireza; Hamidieh, Amir Ali; Behfar, Maryam; Mohseni, Rashin; Mortazavi-Tabatabaei, Seyed A; Asgharzadeh, Shahab

    2017-09-01

    Immune cell-derived exosomes can increase immunity against tumors. In contrast, tumor-derived exosomes can reduce the immunity and can change the tumor microenvironment to further develop and provide metastasis. These effects take place by an alteration in the innate and adaptive immune cell functions. In this experiment, we studied the natural killer (NK) cells' effectiveness on tumor cells after expansion and thereafter incubated it with exosomes. The exosomes were derived from 2 populations of NK cells: (1) naive NK cells and, (2) NK cells previously exposed to neuroblastoma (NB) cells. Moreover, we have studied the NB-derived exosomes on NK cell function. The molecular load of the characterized exosomes (by means of nanoparticle-tracking analysis, flow cytometry, scanning electron microscopy, and western blot) from NK cells exposed to the NB cell revealed their expression of natural killer cell receptors in addition to CD56, NKG2D, and KIR2DL2 receptors. These exosomes were used to treat NK cells and thereafter administered to NB tumor cells both in vitro and in vivo. Our results showed some kind of NK cells' education by the exosomes. This education from NK cells previously exposed to NB cell-derived exosomes caused efficient and greater cytotoxicity against NB tumors, but NB-derived exosomes act as tumor promoters by providing a tumor supporting niche. Hence, this method of preparing the exosomes has a dramatic effect on activation of anti-NK cells against NB cells.

  6. Aqueous-Containing Precursor Solutions for Efficient Perovskite Solar Cells.

    PubMed

    Liu, Dianyi; Traverse, Christopher J; Chen, Pei; Elinski, Mark; Yang, Chenchen; Wang, Lili; Young, Margaret; Lunt, Richard R

    2018-01-01

    Perovskite semiconductors have emerged as competitive candidates for photovoltaic applications due to their exceptional optoelectronic properties. However, the impact of moisture instability on perovskite films is still a key challenge for perovskite devices. While substantial effort is focused on preventing moisture interaction during the fabrication process, it is demonstrated that low moisture sensitivity, enhanced crystallization, and high performance can actually be achieved by exposure to high water content (up to 25 vol%) during fabrication with an aqueous-containing perovskite precursor. The perovskite solar cells fabricated by this aqueous method show good reproducibility of high efficiency with average power conversion efficiency (PCE) of 18.7% and champion PCE of 20.1% under solar simulation. This study shows that water-perovskite interactions do not necessarily negatively impact the perovskite film preparation process even at the highest efficiencies and that exposure to high contents of water can actually enable humidity tolerance during fabrication in air.

  7. Synthesis and structures of metal chalcogenide precursors

    NASA Technical Reports Server (NTRS)

    Hepp, Aloysius F.; Duraj, Stan A.; Eckles, William E.; Andras, Maria T.

    1990-01-01

    The reactivity of early transition metal sandwich complexes with sulfur-rich molecules such as dithiocarboxylic acids was studied. Researchers recently initiated work on precursors to CuInSe2 and related chalcopyrite semiconductors. Th every high radiation tolerance and the high absorption coefficient of CuInSe2 makes this material extremely attractive for lightweight space solar cells. Their general approach in early transition metal chemistry, the reaction of low-valent metal complexes or metal powders with sulfur and selenium rich compounds, was extended to the synthesis of chalcopyrite precursors. Here, the researchers describe synthesis, structures, and and routes to single molecule precursors to metal chalcogenides.

  8. Neural Stem Cells Derived Directly from Adipose Tissue.

    PubMed

    Petersen, Eric D; Zenchak, Jessica R; Lossia, Olivia V; Hochgeschwender, Ute

    2018-05-01

    Neural stem cells (NSCs) are characterized as self-renewing cell populations with the ability to differentiate into the multiple tissue types of the central nervous system. These cells can differentiate into mature neurons, astrocytes, and oligodendrocytes. This category of stem cells has been shown to be a promisingly effective treatment for neurodegenerative diseases and neuronal injury. Most treatment studies with NSCs in animal models use embryonic brain-derived NSCs. This approach presents both ethical and feasibility issues for translation to human patients. Adult tissue is a more practical source of stem cells for transplantation therapies in humans. Some adult tissues such as adipose tissue and bone marrow contain a wide variety of stem cell populations, some of which have been shown to be similar to embryonic stem cells, possessing many pluripotent properties. Of these stem cell populations, some are able to respond to neuronal growth factors and can be expanded in vitro, forming neurospheres analogous to cells harvested from embryonic brain tissue. In this study, we describe a method for the collection and culture of cells from adipose tissue that directly, without going through intermediates such as mesenchymal stem cells, results in a population of NSCs that are able to be expanded in vitro and be differentiated into functional neuronal cells. These adipose-derived NSCs display a similar phenotype to those directly derived from embryonic brain. When differentiated into neurons, cells derived from adipose tissue have spontaneous spiking activity with network characteristics similar to that of neuronal cultures.

  9. Successful Vaccination Induces Multifunctional Memory T-Cell Precursors Associated with Early Control of Hepatitis C Virus

    PubMed Central

    Park, Su-Hyung; Shin, Eui-Cheol; Capone, Stefania; Caggiari, Laura; De Re, Valli; Nicosia, Alfredo; Folgori, Antonella; Rehermann, Barbara

    2012-01-01

    Background & Aims T cells are an important component for development of a vaccine against hepatitis C virus (HCV), but little is known about the features of successful vaccine-induced T cells. Methods We compared the phenotype, function, and kinetics of vaccine-induced and infection-induced T cells in chimpanzees with HCV infection using multicolor flow cytometry and real-time PCR. Results In chimpanzees successfully vaccinated with recombinant adenovirus and DNA against HCV NS3-NS5, HCV-specific T cells appeared earlier, maintained better functionality, and persisted at higher frequencies, for a longer time after HCV-challenge, than those of mock-vaccinated chimpanzees. Vaccine-induced T cells displayed higher levels of CD127, a marker of memory precursors, and lower levels of programmed death (PD)-1 than infection-induced T cells. Vaccine-induced, but not infection-induced T cells, were multifunctional; their ability to secrete interferon-γ and tumor necrosis factor-α correlated with early expression of CD127 but not PD-1. Based on a comparison of vaccine-induced and infection-induced T cells from the same chimpanzee, the CD127+ memory precursor phenotype was induced by the vaccine itself, rather than by low viremia. In contrast, PD-1 induction correlated with viremia, and levels of intrahepatic PD-1, PD-L1, and 2,5-OAS-1 mRNAs correlated with peak titers of HCV. Conclusions Compared with infection, vaccination induced HCV-specific CD127+ T cells with high functionality that persisted at higher levels for a longer time. Control of viremia prevented upregulation of PD-1 on T cells, and induction of PD-1, PD-L1, and 2,5-OAS-1 in the liver. Early development of a memory T-cell phenotype and, via control of viremia, attenuation of the inhibitory PD1–PD-L1 pathway might be necessary components of successful vaccine-induced protection against HCV. PMID:22705008

  10. Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

    PubMed Central

    Ageenko, Natalya V.; Kiselev, Konstantin V.; Dmitrenok, Pavel S.; Odintsova, Nelly A.

    2014-01-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

  11. Cdk5 phosphorylation of WAVE2 regulates oligodendrocyte precursor cell migration through nonreceptor tyrosine kinase Fyn.

    PubMed

    Miyamoto, Yuki; Yamauchi, Junji; Tanoue, Akito

    2008-08-13

    Myelin formation of the CNS is a complex and dynamic process. Before the onset of myelination, oligodendrocytes (OLs), the myelin-forming glia of the CNS, proliferate and migrate along axons. Little is known about the molecular mechanisms underlying the early myelination processes. Here, we show that platelet-derived growth factor (PDGF), the crucial physiological ligand in early OL development, controls the migration of oligodendrocyte precursor cells (OPCs) through cyclin-dependent kinase 5 (Cdk5). PDGF stimulates Cdk5 activity in a time-dependent manner, whereas suppression of Cdk5 by the specific inhibitor roscovitine or by the retrovirus encoding short-hairpin RNA for Cdk5 impairs PDGF-dependent OPC migration. The activation of Cdk5 by PDGF is mediated by the phosphorylation of the nonreceptor tyrosine kinase, Fyn, whose inhibition reduces PDGF-dependent OPC migration. Furthermore, Cdk5 regulates PDGF-dependent OPC migration through the direct phosphorylation of WASP (Wiskott-Aldrich syndrome protein)-family verprolin-homologous protein 2 (WAVE2). Cdk5 phosphorylates WAVE2 at Ser-137 in vitro. Infection of the WAVE2 construct harboring the Ser-137-to-Ala reduces PDGF-dependent migration. Together, PDGF regulates OPC migration through an as-yet-unidentified signaling cascade coupling Fyn kinase to Cdk5 phosphorylation of WAVE2. These results provide new insights into both the role of Cdk5 in glial cells and the molecular mechanisms controlling the early developmental stage of OLs.

  12. [Endometrial carcinomas and precursor lesions--new aspects].

    PubMed

    Schmidt, D

    2009-07-01

    Endometrial carcinomas can be separated into two groups which are designated as type I and type II carcinomas today. Both groups of tumors are clearly different with regard to conventional light microscopy, immunohistochemistry, molecular pathology and clinical features. Only type I carcinomas are associated with hyperestrogenism. The group of type I carcinomas consists of endometrioid carcinoma and its variants, and mucinous carcinoma. The prototypes of type II carcinomas are serous and clear cell carcinoma. Not all carcinomas, however, can be assigned to one of the two groups, because there are hybrid tumors and mixed carcinomas, e.g. endometrioid carcinoma with a serous component. The precursor lesions of the endometrioid carcinoma and the serous carcinoma are well characterized morphologically and by molecular pathology. Atypical hyperplasia is the precursor lesion of endometrioid carcinoma, whereas endometrial intraepithelial carcinoma (EIC) is the precursor lesion of serous carcinoma. No precursor lesion has as yet been identified for clear cell carcinoma. Immunohistochemical markers for endometrial carcinoma are CK7 and vimentin, for serous carcinoma markers are p53 and p16. Correct typing is of essential prognostic necessity in endometrial carcinoma. Of utmost importance is the detection of a serous component, because serous carcinoma leads to early tumor spread with the necessity of radical surgery, chemotherapy and radiotherapy.

  13. Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.

    PubMed

    Massberg, Steffen; Konrad, Ildiko; Schürzinger, Katrin; Lorenz, Michael; Schneider, Simon; Zohlnhoefer, Dietlind; Hoppe, Katharina; Schiemann, Matthias; Kennerknecht, Elisabeth; Sauer, Susanne; Schulz, Christian; Kerstan, Sandra; Rudelius, Martina; Seidl, Stefan; Sorge, Falko; Langer, Harald; Peluso, Mario; Goyal, Pankaj; Vestweber, Dietmar; Emambokus, Nikla R; Busch, Dirk H; Frampton, Jon; Gawaz, Meinrad

    2006-05-15

    The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow-derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1alpha, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.

  14. Dual role for Drosophila lethal of scute in CNS midline precursor formation and dopaminergic neuron and motoneuron cell fate

    PubMed Central

    Stagg, Stephanie B.; Guardiola, Amaris R.; Crews, Stephen T.

    2011-01-01

    Dopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities. PMID:21558367

  15. Structural phenotyping of stem cell-derived cardiomyocytes.

    PubMed

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-03-10

    Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  17. Benzoxazole derivatives suppress lipopolysaccharide-induced mast cell activation.

    PubMed

    Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Choo, Hea-Young Park; Lee, Kyung Ho

    2018-05-01

    Mast cells are central regulators of allergic inflammation that function by releasing various proallergic inflammatory mediators, including histamine, eicosanoids and proinflammatory cytokines. Occasionally, bacterial infections may initiate or worsen allergic inflammation. A number of studies have indicated that activation of lipoxygenase in mast cells positive regulates allergic inflammatory responses by generating leukotrienes and proinflammatory cytokines. In the present study, the effects of benzoxazole derivatives on the lipopolysaccharide (LPS)‑induced expression of proinflammatory cytokines, production of histamine and surface expression of co‑stimulatory molecules on bone marrow-derived mast cells (BMMCs) were studied. The benzoxazole derivatives significantly reduced the expression of interleukin (IL)‑1β, IL‑6, IL‑13, tumor necrosis factor‑α, perilipin (PLIN) 2, and PLIN3 in BMMCs treated with LPS. Furthermore, histamine production was suppressed in BMMCs treated with LPS, or treated with phorbol-12-myristate-13-acetate/ionomycin. Benzoxazole derivatives marginally affected the surface expression of cluster of differentiation (CD)80 and CD86 on BMMCs in the presence of LPS, although LPS alone did not increase the expression of those proteins. Therefore, benzoxazole derivatives inhibited the secretion of proinflammatory cytokines in mast cells and may be potential candidate anti‑allergic agents to suppress mast cell activation.

  18. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

    PubMed

    Zhao, Dongxin; Chen, Song; Cai, Jun; Guo, Yushan; Song, Zhihua; Che, Jie; Liu, Chun; Wu, Chen; Ding, Mingxiao; Deng, Hongkui

    2009-07-31

    The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

  19. Langerhans cell precursors acquire RANK/CD265 in prenatal human skin.

    PubMed

    Schöppl, Alice; Botta, Albert; Prior, Marion; Akgün, Johnnie; Schuster, Christopher; Elbe-Bürger, Adelheid

    2015-01-01

    The skin is the first barrier against foreign pathogens and the prenatal formation of a strong network of various innate and adaptive cells is required to protect the newborn from perinatal infections. While many studies about the immune system in healthy and diseased adult human skin exist, our knowledge about the cutaneous prenatal/developing immune system and especially about the phenotype and function of antigen-presenting cells such as epidermal Langerhans cells (LCs) in human skin is still scarce. It has been shown previously that LCs in healthy adult human skin express receptor activator of NF-κB (RANK), an important molecule prolonging their survival. In this study, we investigated at which developmental stage LCs acquire this important molecule. Immunofluorescence double-labeling of cryostat sections revealed that LC precursors in prenatal human skin either do not yet [10-11 weeks of estimated gestational age (EGA)] or only faintly (13-15 weeks EGA) express RANK. LCs express RANK at levels comparable to adult LCs by the end of the second trimester. Comparable with adult skin, dermal antigen-presenting cells at no gestational age express this marker. These findings indicate that epidermal leukocytes gradually acquire RANK during gestation - a phenomenon previously observed also for other markers on LCs in prenatal human skin. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

  20. Methionine as a Precursor of Ethylene—Commentary

    USDA-ARS?s Scientific Manuscript database

    Lieberman et al. showed in a 1966 publication of Plant Physiology that methionine is a precursor of ethylene. It was the first paper that showed ethylene carbons are derived from carbons 3 and 4 of methionine. This paper catalyzed remarkable interest among plant biologists to elucidate the biosynth...

  1. Glutamine: Precursor or nitrogen donor for citrulline synthesis?

    USDA-ARS?s Scientific Manuscript database

    Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton, versus nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutami...

  2. Potential roles of cell-derived microparticles in ischemic brain disease.

    PubMed

    Horstman, Lawrence L; Jy, Wenche; Bidot, Carlos J; Nordberg, Mary L; Minagar, Alireza; Alexander, J Steven; Kelley, Roger E; Ahn, Yeon S

    2009-10-01

    The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases. An extensive PubMed search of literature pertaining to this study was performed in April 2009 using specific keyword search terms related to cell-derived microparticles and ischemic stroke. Some references are not cited here as it is not possible to be all inclusive or due to space limitation. Cell-derived microparticles are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. This review outlines evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. More specifically, cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions. If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

  3. Duct- and Acinar-Derived Pancreatic Ductal Adenocarcinomas Show Distinct Tumor Progression and Marker Expression.

    PubMed

    Ferreira, Rute M M; Sancho, Rocio; Messal, Hendrik A; Nye, Emma; Spencer-Dene, Bradley; Stone, Richard K; Stamp, Gordon; Rosewell, Ian; Quaglia, Alberto; Behrens, Axel

    2017-10-24

    The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial. Here, we show that identical oncogenic drivers trigger PDAC originating from both ductal and acinar cells with similar histology but with distinct pathophysiology and marker expression dependent on cell of origin. Whereas acinar-derived tumors exhibited low AGR2 expression and were preceded by pancreatic intraepithelial neoplasias (PanINs), duct-derived tumors displayed high AGR2 and developed independently of a PanIN stage via non-mucinous lesions. Using orthotopic transplantation and chimera experiments, we demonstrate that PanIN-like lesions can be induced by PDAC as bystanders in adjacent healthy tissues, explaining the co-existence of mucinous and non-mucinous lesions and highlighting the need to distinguish between true precursor PanINs and PanIN-like bystander lesions. Our results suggest AGR2 as a tool to stratify PDAC according to cell of origin, highlight that not all PanIN-like lesions are precursors of PDAC, and add an alternative progression route to the current model of PDAC development. Copyright © 2017 Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

  4. Efficient Generation of Human Embryonic Stem Cell-Derived Cardiac Progenitors Based on Tissue-Specific Enhanced Green Fluorescence Protein Expression

    PubMed Central

    Szebényi, Kornélia; Péntek, Adrienn; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I.; Sarkadi, Balázs

    2015-01-01

    Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFPhigh rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFPhigh rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFPhigh rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications. PMID:24734786

  5. Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

    PubMed

    Sajad, Mir; Zargan, Jamil; Zargar, Mohammad Afzal; Sharma, Jyoti; Umar, Sadiq; Arora, Rajesh; Khan, Haider A

    2013-05-01

    Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50μM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Polysiloxanes derived from the controlled hydrolysis of tetraethoxysilane as precursors to silica for use in ceramic processing

    NASA Technical Reports Server (NTRS)

    Philipp, Warren H.

    1990-01-01

    Synthesis, properties, and potential applications in ceramic processing for two polysiloxane silica precursors derived from the controlled hydrolysis of tetraethoxysilane (TEOS) are presented. The higher molecular weight TEOS-A is a thick adhesive liquid of viscosity 8000 to 12,000 c.p. having a SiO2 char yield of about 55 percent. The lower molecular weight TEOS-B is a more fluid liquid of viscosity 150 to 200 c.p. having a SiO2 char yield of about 52 percent. The acid catalyzed hydrolysis of TEOS to hydrated silica gel goes through a series of polysiloxane intermediates. The rate of this transition increases with the quantity of water added to the TEOS; thus, for ease of polymer isolation, the amount of water added must be carefully determined so as to produce the desired polymer in a reasonable time. The water to TEOS mole ratio falls in the narrow range of 1.05 for TEOS-A and 0.99 for TEOS-B. Further polymerization or gelation is prevented by storing at -5 C in a freezer. Both polysiloxanes thermoset to a glassy solid at 115 C. The liquid polymers are organic in nature in that they are miscible with toluene and ethanol, slightly souble in heptane, but immiscible with water. For both polymers, results on viscosity versus time are given at several temperatures and water additions. Based on these results, some examples of practical utilization of the precursors for ceramic fabrication are given.

  7. Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors.

    PubMed

    Prodon, François; Chenevert, Janet; Hébras, Céline; Dumollard, Rémi; Faure, Emmanuel; Gonzalez-Garcia, Jose; Nishida, Hiroki; Sardet, Christian; McDougall, Alex

    2010-06-01

    Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

  8. Single cell RNA sequencing of stem cell-derived retinal ganglion cells.

    PubMed

    Daniszewski, Maciej; Senabouth, Anne; Nguyen, Quan H; Crombie, Duncan E; Lukowski, Samuel W; Kulkarni, Tejal; Sluch, Valentin M; Jabbari, Jafar S; Chamling, Xitiz; Zack, Donald J; Pébay, Alice; Powell, Joseph E; Hewitt, Alex W

    2018-02-13

    We used single cell sequencing technology to characterize the transcriptomes of 1,174 human embryonic stem cell-derived retinal ganglion cells (RGCs) at the single cell level. The human embryonic stem cell line BRN3B-mCherry (A81-H7), was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells showed increased expression of genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature RGC phenotype.

  9. Mouse A6-positive hepatic oval cells derived from embryonic stem cells.

    PubMed

    Yin, Dong-zhi; Cai, Ji-ye; Zheng, Qi-chang; Chen, Zheng-wei; Zhao, Jing-xian; Yuan, You-neng

    2014-02-01

    Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

  10. Effect of extracorporeal shock wave on proliferation and differentiation of equine adipose tissue-derived mesenchymal stem cells in vitro

    PubMed Central

    Raabe, O; Shell, K; Goessl, A; Crispens, C; Delhasse, Y; Eva, A; Scheiner-Bobis, G; Wenisch, S; Arnhold, S

    2013-01-01

    Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nevertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was to investigate the influence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose tissue-derived mesenchymal stem cells (ASCs) and to explore its effects on gap junctional communication and the activation of signalling cascades associated with cell proliferation and differentiation. ASCs were treated with different pulses of focused ESWT. Treated cells showed increased proliferation and expression of Cx43, as detected by means of qRT-PCR, histological staining, immunocytochemistry and western blot. At the same time, cells responded to ESWT by significant activation (phosphorylation) of Erk1/2, detected in western blots. No significant effects on the differentiation potential of the ASCs were evident. Taken together, the present results show significant effects of shock waves on stem cells in vitro. PMID:23671817

  11. Chicken HOXA3 Gene: Its Expression Pattern and Role in Branchial Nerve Precursor Cell Migration

    PubMed Central

    Watari-Goshima, Natsuko; Chisaka, Osamu

    2011-01-01

    In vertebrates, the proximal and distal sensory ganglia of the branchial nerves are derived from neural crest cells (NCCs) and placodes, respectively. We previously reported that in Hoxa3 knockout mouse embryos, NCCs and placode-derived cells of the glossopharyngeal nerve were defective in their migration. In this report, to determine the cell-type origin for this Hoxa3 knockout phenotype, we blocked the expression of the gene with antisense morpholino oligonucleotides (MO) specifically in either NCCs/neural tube or placodal cells of chicken embryos. Our results showed that HOXA3 function was required for the migration of the epibranchial placode-derived cells and that HOXA3 regulated this cell migration in both NCCs/neural tube and placodal cells. We also report that the expression pattern of chicken HOXA3 was slightly different from that of mouse Hoxa3. PMID:21278919

  12. Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.

    PubMed

    Takikawa, Tetsuya; Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Kogure, Takayuki; Shimosegawa, Tooru

    2017-01-01

    Pancreatic stellate cells (PSCs) interact with pancreatic cancer cells in the tumor microenvironment. Cell constituents including microRNAs may be exported from cells within membranous nanovesicles termed exosomes. Exosomes might play a pivotal role in intercellular communication. This study aimed to clarify the microRNA signature of PSC-derived exosomes and their effects on pancreatic cancer cells. Exosomes were prepared from the conditioned medium of immortalized human PSCs. MicroRNAs were prepared from the exosomes and their source PSCs, and the microRNA expression profiles were compared by microarray. The effects of PSC-derived exosomes on proliferation, migration, and the mRNA expression profiles were examined in pancreatic cancer cells. Pancreatic stellate cell-derived exosomes contained a variety of microRNAs including miR-21-5p. Several microRNAs such as miR-451a were enriched in exosomes compared to their source PSCs. Pancreatic stellate cell-derived exosomes stimulated the proliferation, migration and expression of mRNAs for chemokine (C - X - C motif) ligands 1 and 2 in pancreatic cancer cells. The stimulation of proliferation, migration, and chemokine gene expression by the conditioned medium of PSCs was suppressed by GW4869, an exosome inhibitor. We clarified the microRNA expression profile in PSC-derived exosomes. Pancreatic stellate cell-derived exosomes might play a role in the interactions between PSCs and pancreatic cancer cells.

  13. Precursor B-cell lymphoblastic lymphoma of oral cavity: A case report with its diagnostic workup

    PubMed Central

    Talreja, Komal Ladharam; Barpande, Suresh Ramchandra; Bhavthankar, Jyoti Dilip; Mandale, Mandakini S

    2016-01-01

    Lymphoblastic lymphoma (LBL), seen primarily in children or young adults, is a malignant neoplasia that originates from B or T lymphocyte precursors and rarely occurs in the oral cavity. In this localization, neither the clinical features nor the radiologic appearances are pathognomic and can pose significant diagnostic problems. Histopathologically, it presents as a round blue cell tumor. An early and accurate diagnosis of this entity is very important due to its high cure rate. We report a case of B-cell LBL involving oral cavity in a 10-year-old child. The purpose of this report is to explore the diagnostic workup. PMID:27194876

  14. Crypteins derived from the mouse neuropeptide FF (NPFF)A precursor display NPFF-like effects in nociceptive tests in mice.

    PubMed

    Kotlinska, Jolanta H; Gibula-Bruzda, Ewa; Suder, Piotr; Wasielak, Magdalena; Bray, Lauriane; Raoof, Hana; Bodzon-Kulakowska, Anna; Silberring, Jerzy

    2012-07-01

    NPFF precursor, pro-NPFF(A) contains three known bioactive sequences: NPFF (FLFQPQRF-NH(2)), neuropeptide AF (NPAF; AGEGLSSPFWSLAAPQRF-NH(2)) and neuropeptide SF (NPSF; SLAAPQRF-NH(2)). The key-feature of these fragments is their common PQRF-amidated sequence at their C termini. Here, we evaluated the biological activity of two other sequences derived from the mouse NPFF(A) precursor, that does not have PQRF-amidated C-terminus. One peptide was residing between positions 85 and 99 in the mice pro-NPFF(A). This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA), assigned due to its flanking amino acids. Another sequence used in the experiments was N-terminal fragment of NPSA, here referred to as neuropeptide SS (NPSS; SAWGSWS). These two peptides, classified as crypteins, were synthesized and tested in the hot-plate and tail immersion tests in mice for their pharmacological activity in morphine-induced antinociception. The effects of both crypteins were compared to NPFF. Our experiments indicated that both crypteins inhibited morphine antinociception and their effects were reversed by RF9, an antagonist of NPFF receptors. These data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these behavioral tests. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor acute lymphoblastic leukemia in the bone marrow niche

    PubMed Central

    Shiozawa, Yusuke; Pedersen, Elisabeth A.; Taichman, Russell S.

    2009-01-01

    Despite improvements in current combinational chemotherapy regimens, the prognosis of the (1;19)(q23;p13) translocation (E2A/PBX1) positive B-cell precursor acute lymphoblastic leukemia (ALL) is poor in pediatric leukemia patients. In this study, we examined the roles of GAS6/Mer axis in the interactions between E2A/PBX1 positive B-cell precursor ALL cells and the osteoblastic niche in the bone marrow. The data show that primary human osteoblasts secrete GAS6 in response to the Mer-over-expressed E2A/PBX1 positive ALL cells through MAPK signaling pathway and that leukemia cells migrate toward GAS6 using pathways activated by Mer. Importantly, GAS6 supports the survival and prevents apoptosis from chemotherapy of E2A/PBX1 positive ALL cells by inducing dormancy. Together, these data suggest that GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor ALL in the bone marrow niche. PMID:19922767

  16. CuInSe₂ thin-film solar cells with 7.72 % efficiency prepared via direct coating of a metal salts/alcohol-based precursor solution.

    PubMed

    Ahn, Sejin; Son, Tae Hwa; Cho, Ara; Gwak, Jihye; Yun, Jae Ho; Shin, Keeshik; Ahn, Seoung Kyu; Park, Sang Hyun; Yoon, Kyunghoon

    2012-09-01

    A simple direct solution coating process for forming CuInSe₂ (CIS) thin films was described, employing a low-cost and environmentally friendly precursor solution. The precursor solution was prepared by mixing metal acetates, ethanol, and ethanolamine. The facile formation of a precursor solution without the need to prefabricate nanoparticles enables a rapid and easy processing, and the high stability of the solution in air further ensures the precursor preparation and the film deposition in ambient conditions without a glove box. The thin film solar cell fabricated with the absorber film prepared by this route showed an initial conversion efficiency of as high as 7.72 %. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    PubMed

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Investigation of relationship between precursor of miRNA-944 and its mature form in lung squamous-cell carcinoma - the diagnostic value.

    PubMed

    Powrózek, Tomasz; Mlak, Radosław; Dziedzic, Marcin; Małecka-Massalska, Teresa; Sagan, Dariusz

    2018-03-01

    MicroRNA (miRNA) are attractive markers of lung cancer, due to their regulatory role in cell cycle. However, we know more about function of miRNA in cancer development, there is still little known about role of their precursors (primary miRNA; pri-miRNA) in tumorgenesis. In present study we investigated potential role of miRNA-944 and its precursor pri-miRNA-944 in development of squamous-cell lung cancer (SCC) and explored interdependence between miRNA precursor and its mature form. This is a first available literature report analyzing pri-miRNA as a cancer diagnostic marker. Expression of miRNA-944 and its precursor was analyzed in 58 fresh-frozen tissues of non-small cell lung cancer and corresponding adjacent non-cancerous tissues using qRT-PCR. Expression of pri-miRNA-944 was correlated with TP63 and miRNA-944. Using ROC analysis diagnostic accuracy of studied markers was evaluated. miRNA-944 and its precursor were significantly overexspressed in SCC compared to adenocarcinoma (AC) and non-cancerous tissue. pri-miRNA-944 strongly and positively correlated with TP63 (r = 0.739, p < 0.001) and with mature miRNA-944 expression (r = 0.691, p < 0.001). Also, TP63 expression significantly correlated with mature miRNA (r = 0.785, p < 0.001). Combined analysis of pri-miRNA-944 and mature miRNA-944 allowed to distinguish SCC tissue form AC with sensitivity of 93.3% and specificity of 100% (AUC = 0.978), and SCC from non-cancerous tissue with 92.9% sensitivity and 100% specificity (AUC = 0.992). We assumed that pri-miRNA-944 and miRNA-944 may be involved in early squamous-type differentiation of lung tumors. Moreover, analysis of both markers provided high diagnostic accuracy for SCC detection. Copyright © 2018 Elsevier GmbH. All rights reserved.

  19. Melatonin and its precursors in Y79 human retinoblastoma cells: Effect of sodium butyrate

    NASA Technical Reports Server (NTRS)

    Deng, Mei Hua; Coviella, Ignacio Lopez G.; Lynch, Harry J.; Wurtman, Richard J.

    1991-01-01

    The release of melatonin and the production of its precursors, S-hydroxytryptophan and serotonin, in cultured Y79 human retinoblastoma cells were studied. This biosynthetic capability was found to be dependent on cell differentiation, which was initiated by culturing Y79 cells for 7 days in dishes coated with poly-D-lysine to promote cell adhesion to the surface of the culture dishes. Differentiation was further induced by exposing the cell monolayer to sodium butyrate (3 mM) for 3 days. This protocol dramatically increased the release of melatonin, and the syntheses of 5-hydroxytryptophan and serotonin in response to forskolin stimulation. Exposure to dopamine (10 micro-M) or L-DOPA (100 micro-M) markedly diminished the forskolin-stimulated release of melatonin, as well as the production of 5-hydroxytryptophan and serotonin. These observations indicate that Y79 cells represent a primitive cell line which, following appropriate differentiation (e.g. treatment with sodium butyrate) can display biochemical characteristics similar to those of the human retina. Moreover, serotonin synthesis and melatonin release appear to be coupled in Y79 cells. The inhibition of melatonin release by dopamine supports the hypothesis that in these cells, melatonin and dopamine are components of a retinal feedback loop.

  20. The Immunogenicity and Immune Tolerance of Pluripotent Stem Cell Derivatives

    PubMed Central

    Liu, Xin; Li, Wenjuan; Fu, Xuemei; Xu, Yang

    2017-01-01

    Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and differentiate into all cell types in human body, and therefore hold great potential for cell therapy of currently incurable diseases including neural degenerative diseases, heart failure, and macular degeneration. This potential is further underscored by the promising safety and efficacy data from the ongoing clinical trials of hESC-based therapy of macular degeneration. However, one main challenge for the clinical application of hESC-based therapy is the allogeneic immune rejection of hESC-derived cells by the recipient. The breakthrough of the technology to generate autologous-induced pluripotent stem cells (iPSCs) by nuclear reprogramming of patient’s somatic cells raised the possibility that autologous iPSC-derived cells can be transplanted into the patients without the concern of immune rejection. However, accumulating data indicate that certain iPSC-derived cells can be immunogenic. In addition, the genomic instability associated with iPSCs raises additional safety concern to use iPSC-derived cells in human cell therapy. In this review, we will discuss the mechanism underlying the immunogenicity of the pluripotent stem cells and recent progress in developing immune tolerance strategies of human pluripotent stem cell (hPSC)-derived allografts. The successful development of safe and effective immune tolerance strategy will greatly facilitate the clinical development of hPSC-based cell therapy. PMID:28626459

  1. Multiple Modes of Communication between Neurons and Oligodendrocyte Precursor Cells.

    PubMed

    Maldonado, Paloma P; Angulo, María Cecilia

    2015-06-01

    The surprising discovery of bona fide synapses between neurons and oligodendrocytes precursor cells (OPCs) 15 years ago placed these progenitors as real partners of neurons in the CNS. The role of these synapses has not been established yet, but a main hypothesis is that neuron-OPC synaptic activity is a signaling pathway controlling OPC proliferation/differentiation, influencing the myelination process. However, new evidences describing non-synaptic mechanisms of communication between neurons and OPCs have revealed that neuron-OPC interactions are more complex than expected. The activation of extrasynaptic receptors by ambient neurotransmitter or local spillover and the ability of OPCs to sense neuronal activity through a potassium channel suggest that distinct modes of communication mediate different functions of OPCs in the CNS. This review discusses different mechanisms used by OPCs to interact with neurons and their potential roles during postnatal development and in brain disorders. © The Author(s) 2014.

  2. An early thymic precursor phenotype predicts outcome exclusively in HOXA-overexpressing adult T-cell acute lymphoblastic leukemia: a Group for Research in Adult Acute Lymphoblastic Leukemia study.

    PubMed

    Bond, Jonathan; Marchand, Tony; Touzart, Aurore; Cieslak, Agata; Trinquand, Amélie; Sutton, Laurent; Radford-Weiss, Isabelle; Lhermitte, Ludovic; Spicuglia, Salvatore; Dombret, Hervé; Macintyre, Elizabeth; Ifrah, Norbert; Hamel, Jean-François; Asnafi, Vahid

    2016-06-01

    Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemias, but it is unclear whether these constitute a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA-positive cases among 209 patients with adult T-cell acute lymphoblastic leukemia uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA-positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% versus 14.5%, P=0.0004), chemoresistance (59.3% versus 40.8%, P=0.026) and positivity for minimal residual disease (48.5% versus 23.5%, P=0.01) than the HOXA-negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA-positive cases harboring fusion oncoproteins that transactivate HOXA Strikingly, the presence of an early thymic precursor-like immunophenotype was associated with marked outcome differences within the HOXA-positive group (5-year overall survival 31.2% in HOXA-positive early thymic precursor versus 66.7% in HOXA-positive non-early thymic precursor, P=0.03), but not in HOXA-negative cases (5-year overall survival 74.2% in HOXA-negative early thymic precursor versus 57.2% in HOXA-negative non-early thymic precursor, P=0.44). Multivariate analysis further revealed that HOXA positivity independently affected event-free survival (P=0.053) and relapse risk (P=0.039) of chemoresistant T-cell acute lymphoblastic leukemia. These results show that the underlying mechanism of HOXA deregulation dictates the clinico-biological phenotype, and that the negative prognosis of early thymic precursor acute lymphoblastic leukemia is exclusive to HOXA-positive patients, suggesting that early treatment intensification is currently

  3. Differential and directional estrogenic signaling pathways induced by enterolignans and their precursors

    PubMed Central

    Zhu, Yun; Kawaguchi, Kayoko; Kiyama, Ryoiti

    2017-01-01

    Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among lignans, have not been explored. We examined the estrogenic activity of enterolignans and their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle and chemokine secretion. We used DNA microarray-based gene expression profiling in human breast cancer MCF-7 cells to examine the similarities, as well as the differences, among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (R values: 0.44 to 0.81) with that of estrogen (17β-estradiol or E2). Significant correlations were observed among lignans (R values: 0.77 to 0.97), and the correlations were higher for cell functions related to enzymes, signaling, proliferation and transport. All the enterolignans/precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the involvement of rapid signaling through the non-genomic estrogen signaling pathway. However, when their effects on specific cell functions, cell cycle progression and chemokine (MCP-1) secretion were examined, positive effects were observed only for enterolactone, suggesting that signals are given in certain directions at a position closer to cell functions. We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors examined, their signals are differentially and directionally modulated later in the pathways, resulting in the differences at the cell function level. PMID:28152041

  4. Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe.

    PubMed

    Hara, Yusuke; Sudo, Tatsuya; Togane, Yu; Akagawa, Hiromi; Tsujimura, Hidenobu

    2018-04-01

    Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. To understand the development of the central nervous system, it is essential to reveal not only molecular mechanisms but also the role of neural cell death (Pinto-Teixeira et al., 2016). To understand the role of cell death in neural development, we investigated the effect of inhibition of cell death on optic lobe development. Our data demonstrate that, in the optic lobe of Drosophila, cell death occurs in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development-e.g., failures in the separation of the lamina and medulla cortices and the medulla rotation. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, we would also expect to observe them in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, we found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers

  5. Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration.

    PubMed

    Hackelberg, Sandra; Tuck, Samuel J; He, Long; Rastogi, Arjun; White, Christina; Liu, Liqian; Prieskorn, Diane M; Miller, Ryan J; Chan, Che; Loomis, Benjamin R; Corey, Joseph M; Miller, Josef M; Duncan, R Keith

    2017-01-01

    Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

  6. Manganite perovskite ceramics, their precursors and methods for forming

    DOEpatents

    Payne, David Alan; Clothier, Brent Allen

    2015-03-10

    Disclosed are a variety of ceramics having the formula Ln.sub.1-xM.sub.xMnO.sub.3, where 0.Itoreq.x.Itoreq.1 and where Ln is La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu or Y; M is Ca, Sr, Ba, Cd, or Pb; manganite precursors for preparing the ceramics; a method for preparing the precursors; and a method for transforming the precursors into uniform, defect-free ceramics having magnetoresistance properties. The manganite precursors contain a sol and are derived from the metal alkoxides: Ln(OR).sub.3, M(OR).sub.2 and Mn(OR).sub.2, where R is C.sub.2 to C.sub.6 alkyl or C.sub.3 to C.sub.9 alkoxyalkyl, or C.sub.6 to C.sub.9 aryl. The preferred ceramics are films prepared by a spin coating method and are particularly suited for incorporation into a device such as an integrated circuit device.

  7. Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies

    PubMed Central

    Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y.; Geron, Ifat; Strongin, Alex Y.; Itkin-Ansari, Pamela

    2009-01-01

    Background Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings. Methods Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Results Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response. Conclusions We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells. PMID:19352116

  8. Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

    PubMed

    Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

    2009-04-15

    Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

  9. In Situ Passivation for Efficient PbS Quantum Dot Solar Cells by Precursor Engineering.

    PubMed

    Wang, Yongjie; Lu, Kunyuan; Han, Lu; Liu, Zeke; Shi, Guozheng; Fang, Honghua; Chen, Si; Wu, Tian; Yang, Fan; Gu, Mengfan; Zhou, Sijie; Ling, Xufeng; Tang, Xun; Zheng, Jiawei; Loi, Maria Antonietta; Ma, Wanli

    2018-04-01

    Current efforts on lead sulfide quantum dot (PbS QD) solar cells are mostly paid to the device architecture engineering and postsynthetic surface modification, while very rare work regarding the optimization of PbS synthesis is reported. Here, PbS QDs are successfully synthesized using PbO and PbAc 2  · 3H 2 O as the lead sources. QD solar cells based on PbAc-PbS have demonstrated a high power conversion efficiency (PCE) of 10.82% (and independently certificated values of 10.62%), which is significantly higher than the PCE of 9.39% for PbO-PbS QD based ones. For the first time, systematic investigations are carried out on the effect of lead precursor engineering on the device performance. It is revealed that acetate can act as an efficient capping ligands together with oleic acid, providing better surface coverage and replace some of the harmful hydroxyl (OH) ligands during the synthesis. Then the acetate on the surface can be exchanged by iodide and lead to desired passivation. This work demonstrates that the precursor engineering has great potential in performance improvement. It is also pointed out that the initial synthesis is an often neglected but critical stage and has abundant room for optimization to further improve the quality of the resultant QDs, leading to breakthrough efficiency. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Low cost sol-gel derived SiC-SiO2 nanocomposite as anti reflection layer for enhanced performance of crystalline silicon solar cells

    NASA Astrophysics Data System (ADS)

    Jannat, Azmira; Lee, Woojin; Akhtar, M. Shaheer; Li, Zhen Yu; Yang, O.-Bong

    2016-04-01

    This paper describes the preparation, characterizations and the antireflection (AR) coating application in crystalline silicon solar cells of sol-gel derived SiC-SiO2 nanocomposite. The prepared SiC-SiO2 nanocomposite was effectively applied as AR layer on p-type Si-wafer via two step processes, where the sol-gel of precursor solution was first coated on p-type Si-wafer using spin coating at 2000 rpm and then subjected to annealing at 450 °C for 1 h. The crystalline, and structural observations revealed the existence of SiC and SiO2 phases, which noticeably confirmed the formation of SiC-SiO2 nanocomposite. The SiC-SiO2 layer on Si solar cells was found to be an excellent AR coating, exhibiting the low reflectance of 7.08% at wavelengths ranging from 400 to 1000 nm. The fabricated crystalline Si solar cell with SiC-SiO2 nanocomposite AR coating showed comparable power conversion efficiency of 16.99% to the conventional SixNx AR coated Si solar cell. New and effective sol-gel derived SiC-SiO2 AR layer would offer a promising technique to produce high performance Si solar cells with low-cost.

  11. Copy number profiling of adult relapsed B-cell precursor acute lymphoblastic leukemia reveals potential leukemia progression mechanisms.

    PubMed

    Ribera, Jordi; Zamora, Lurdes; Morgades, Mireia; Mallo, Mar; Solanes, Neus; Batlle, Montserrat; Vives, Susana; Granada, Isabel; Juncà, Jordi; Malinverni, Roberto; Genescà, Eulàlia; Guàrdia, Ramon; Mercadal, Santiago; Escoda, Lourdes; Martinez-Lopez, Joaquín; Tormo, Mar; Esteve, Jordi; Pratcorona, Marta; Martinez-Losada, Carmen; Solé, Francesc; Feliu, Evarist; Ribera, Josep-Maria

    2017-11-01

    The outcome of relapsed adult acute lymphoblastic leukemia (ALL) remains dismal despite new therapeutic approaches. Previous studies analyzing relapse samples have shown a high degree of heterogeneity regarding gene alterations without an evident relapse signature. Bone marrow or peripheral blood samples from 31 adult B-cell precursor ALL patients at first relapse, and 21 paired diagnostic samples were analyzed by multiplex ligation probe-dependent amplification (MLPA). Nineteen paired diagnostic and relapse samples of these 21 patients were also analyzed by SNP arrays. A trend to acquire homozygous CDKN2A/B deletions and a significant increase in the number of copy number alterations (CNA) was observed from diagnosis to first relapse. Evolution from an ancestral clone was the main pattern of clonal evolution. Relapse samples were extremely heterogeneous regarding CNA frequencies. However, CDKN2A/B, PAX5, ETV6, ATM, IKZF1, VPREB1, and TP53 deletions and duplications of 1q, 8q, 17q, 21, X/Y PAR1, and Xp were frequently detected at relapse. Duplications of genes involved in cell proliferation, drug resistance and stem cell homeostasis regulation, as well as deletions of KDM6A and STAG2 genes emerged as specific alterations at relapse. Genomics of relapsed adult B-cell precursor ALL is highly heterogeneous, although some recurrent lesions involved in essential pathways deregulation were frequently observed. Selective and simultaneous targeting of these deregulated pathways may improve the results of current salvage therapies. © 2017 Wiley Periodicals, Inc.

  12. Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

    PubMed

    Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

    2012-08-01

    The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

  13. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    PubMed Central

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  14. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita

    2010-03-12

    Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activitymore » in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.« less

  15. Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors Into Functional Islets Capable of Treating Pre-existing Diabetes in Mice

    PubMed Central

    Rezania, Alireza; Bruin, Jennifer E.; Riedel, Michael J.; Mojibian, Majid; Asadi, Ali; Xu, Jean; Gauvin, Rebecca; Narayan, Kavitha; Karanu, Francis; O’Neil, John J.; Ao, Ziliang; Warnock, Garth L.

    2012-01-01

    Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes. PMID:22740171

  16. Balancing Ethical Pros and Cons of Stem Cell Derived Gametes.

    PubMed

    Segers, Seppe; Mertes, Heidi; de Wert, Guido; Dondorp, Wybo; Pennings, Guido

    2017-07-01

    In this review we aim to provide an overview of the most important ethical pros and cons of stem cell derived gametes (SCD-gametes), as a contribution to the debate about reproductive tissue engineering. Derivation of gametes from stem cells holds promising applications both for research and for clinical use in assisted reproduction. We explore the ethical issues connected to gametes derived from embryonic stem cells (both patient specific and non-patient specific) as well as those related to gametes derived from induced pluripotent stem cells. The technology of SCD-gametes raises moral concerns of how reproductive autonomy relates to issues of embryo destruction, safety, access, and applications beyond clinical infertility.

  17. SYK as a New Therapeutic Target in B-Cell Precursor Acute Lymphoblastic Leukemia

    PubMed Central

    Uckun, Fatih M.; Qazi, Sanjive

    2014-01-01

    The identification of SYK as a master regulator of apoptosis controlling the activation of the PI3-K/AKT, NFκB, and STAT3 pathways—three major anti-apoptotic signaling pathways in B-lineage leukemia/lymphoma cells—prompts the hypothesis that rationally designed inhibitors targeting SYK may overcome the resistance of malignant B-lineage lymphoid cells to apoptosis and thereby provide the foundation for more effective multi-modality treatment regimens for poor prognosis B-precursor acute lymphoblastic leukemia (BPL). In recent preclinical proof-of-concept studies, a liposomal nanoparticle (LNP) formulation of a SYK substrate-binding site inhibitor, known as C61, has been developed as a nanomedicine candidate against poor prognosis and relapsed BPL. This nanoscale formulation of C61 exhibited a uniquely favorable pharmacokinetics and safety profile in mice, induced apoptosis in radiation-resistant primary leukemic cells taken directly from BPL patients as well as in vivo clonogenic BPL xenograft cells, destroyed the leukemic stem cell fraction of BPL blasts, and exhibited potent in vivo anti-leukemic activity in xenograft models of aggressive BPL. Further development of C61-LNP may provide the foundation for new and effective treatment strategies against therapy-refractory BPL. PMID:24851191

  18. Allogeneic mesenchymal precursor cells (MPCs): an innovative approach to treating advanced heart failure.

    PubMed

    Westerdahl, Daniel E; Chang, David H; Hamilton, Michele A; Nakamura, Mamoo; Henry, Timothy D

    2016-09-01

    Over 37 million people worldwide are living with Heart Failure (HF). Advancements in medical therapy have improved mortality primarily by slowing the progression of left ventricular dysfunction and debilitating symptoms. Ultimately, heart transplantation, durable mechanical circulatory support (MCS), or palliative care are the only options for patients with end-stage HF. Regenerative therapies offer an innovative approach, focused on reversing myocardial dysfunction and restoring healthy myocardial tissue. Initial clinical trials using autologous (self-donated) bone marrow mononuclear cells (BMMCs) demonstrated excellent safety, but only modest efficacy. Challenges with autologous stem cells include reduced quality and efficacy with increased patient age. The use of allogeneic mesenchymal precursor cells (MPCs) offers an "off the shelf" therapy, with consistent potency and less variability than autologous cells. Preclinical and initial clinical trials with allogeneic MPCs have been encouraging, providing the support for a large ongoing Phase III trial-DREAM-HF. We provide a comprehensive review of preclinical and clinical data supporting MPCs as a therapeutic option for HF patients. The current data suggest allogeneic MPCs are a promising therapy for HF patients. The results of DREAM-HF will determine whether allogeneic MPCs can decrease major adverse clinical events (MACE) in advanced HF patients.

  19. Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

    PubMed Central

    Bidard, J N; de Nadai, F; Rovere, C; Moinier, D; Laur, J; Martinez, J; Cuber, J C; Kitabgi, P

    1993-01-01

    Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at

  20. Adipose-derived stem cells and periodontal tissue engineering.

    PubMed

    Tobita, Morikuni; Mizuno, Hiroshi

    2013-01-01

    Innovative developments in the multidisciplinary field of tissue engineering have yielded various implementation strategies and the possibility of functional tissue regeneration. Technologic advances in the combination of stem cells, biomaterials, and growth factors have created unique opportunities to fabricate tissues in vivo and in vitro. The therapeutic potential of human multipotent mesenchymal stem cells (MSCs), which are harvested from bone marrow and adipose tissue, has generated increasing interest in a wide variety of biomedical disciplines. These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, and nerve tissue. Adipose-derived stem cells have some advantages compared with other sources of stem cells, most notably that a large number of cells can be easily and quickly isolated from adipose tissue. In current clinical therapy for periodontal tissue regeneration, several methods have been developed and applied either alone or in combination, such as enamel matrix proteins, guided tissue regeneration, autologous/allogeneic/xenogeneic bone grafts, and growth factors. However, there are various limitations and shortcomings for periodontal tissue regeneration using current methods. Recently, periodontal tissue regeneration using MSCs has been examined in some animal models. This method has potential in the regeneration of functional periodontal tissues because the various secreted growth factors from MSCs might not only promote the regeneration of periodontal tissue but also encourage neovascularization of the damaged tissues. Adipose-derived stem cells are especially effective for neovascularization compared with other MSC sources. In this review, the possibility and potential of adipose-derived stem cells for regenerative medicine are introduced. Of particular interest, periodontal tissue regeneration with adipose-derived stem cells is discussed.

  1. Dental pulp stem cell-derived chondrogenic cells demonstrate differential cell motility in type I and type II collagen hydrogels.

    PubMed

    Yao, Li; Flynn, Nikol

    2018-06-01

    Advances in the development of biomaterials and stem cell therapy provide a promising approach to regenerating degenerated discs. The normal nucleus pulposus (NP) cells exhibit similar phenotype to chondrocytes. Because dental pulp stem cells (DPSCs) can be differentiated into chondrogenic cells, the DPSCs and DPSCs-derived chondrogenic cells encapsulated in type I and type II collagen hydrogels can potentially be transplanted into degenerated NP to repair damaged tissue. The motility of transplanted cells is critical because the cells need to migrate away from the hydrogels containing the cells of high density and disperse through the NP tissue after implantation. The purpose of this study was to determine the motility of DPSC and DPSC-derived chondrogenic cells in type I and type II collagen hydrogels. The time lapse imaging that recorded cell migration was analyzed to quantify the cell migration velocity and distance. The cell viability of DPSCs in native or poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG)-crosslinked type I and type II collagen hydrogels was determined using LIVE/DEAD cell viability assay and AlamarBlue assay. DPSCs were differentiated into chondrogenic cells. The migration of DPSCs and DPSC-derived chondrogenic cells in these hydrogels was recorded using a time lapse imaging system. This study was funded by the Regional Institute on Aging and Wichita Medical Research and Education Foundation, and the authors declare no competing interest. DPSCs showed high cell viability in non-crosslinked and crosslinked collagen hydrogels. DPSCs migrated in collagen hydrogels, and the cell migration speed was not significantly different in either type I collagen or type II collagen hydrogels. The migration speed of DPSC-derived chondrogenic cells was higher in type I collagen hydrogel than in type II collagen hydrogel. Crosslinking of type I collagen with 4S-StarPEG significantly reduced the cell migration speed of DPSC-derived

  2. Dendritic Cells Pulsed with Leukemia Cell-Derived Exosomes More Efficiently Induce Antileukemic Immunities

    PubMed Central

    Wei, Wei; Shen, Chang; Deng, Xiaohui; Chen, Linjun; Ma, Liyuan; Hao, Siguo

    2014-01-01

    Dendritic cells (DCs) and tumor cell-derived exosomes have been used to develop antitumor vaccines. However, the biological properties and antileukemic effects of leukemia cell-derived exosomes (LEXs) are not well described. In this study, the biological properties and induction of antileukemic immunity of LEXs were investigated using transmission electron microscopy, western blot analysis, cytotoxicity assays, and animal studies. Similar to other tumor cells, leukemia cells release exosomes. Exosomes derived from K562 leukemia cells (LEXK562) are membrane-bound vesicles with diameters of approximately 50–100 μm and harbor adhesion molecules (e.g., intercellular adhesion molecule-1) and immunologically associated molecules (e.g., heat shock protein 70). In cytotoxicity assays and animal studies, LEXs-pulsed DCs induced an antileukemic cytotoxic T-lymphocyte immune response and antileukemic immunity more effectively than did LEXs and non-pulsed DCs (P<0.05). Therefore, LEXs may harbor antigens and immunological molecules associated with leukemia cells. As such, LEX-based vaccines may be a promising strategy for prolonging disease-free survival in patients with leukemia after chemotherapy or hematopoietic stem cell transplantation. PMID:24622345

  3. The Influence of Water Vapor on the Stability and Processing of Hybrid Perovskite Solar Cells Made from Non-Stoichiometric Precursor Mixtures.

    PubMed

    Petrus, Michiel L; Hu, Yinghong; Moia, Davide; Calado, Philip; Leguy, Aurélien M A; Barnes, Piers R F; Docampo, Pablo

    2016-09-22

    We investigated the influence of moisture on methylammonium lead iodide perovskite (MAPbI 3 ) films and solar cells derived from non-stoichiometric precursor mixtures. We followed both the structural changes under controlled air humidity through in situ X-ray diffraction, and the electronic behavior of devices prepared from these films. A small PbI 2 excess in the films improved the stability of the perovskite compared to stoichiometric samples. We assign this to excess PbI 2 layers at the perovskite grain boundaries or to the termination of the perovskite crystals with Pb and I. In contrast, the MAI-excess films composed of smaller perovskite crystals showed increased electronic disorder and reduced device performance owing to poor charge collection. Upon exposure to moisture followed by dehydration (so-called solvent annealing), these films recrystallized to form larger, highly oriented crystals with fewer electronic defects and a remarkable improvement in photocurrent and photovoltaic efficiency. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Enhanced Performance of PbS-quantum-dot-sensitized Solar Cells via Optimizing Precursor Solution and Electrolytes

    NASA Astrophysics Data System (ADS)

    Tian, Jianjun; Shen, Ting; Liu, Xiaoguang; Fei, Chengbin; Lv, Lili; Cao, Guozhong

    2016-03-01

    This work reports a PbS-quantum-dot-sensitized solar cell (QDSC) with power conversion efficiency (PCE) of 4%. PbS quantum dots (QDs) were grown on mesoporous TiO2 film using a successive ion layer absorption and reaction (SILAR) method. The growth of QDs was found to be profoundly affected by the concentration of the precursor solution. At low concentrations, the rate-limiting factor of the crystal growth was the adsorption of the precursor ions, and the surface growth of the crystal became the limiting factor in the high concentration solution. The optimal concentration of precursor solution with respect to the quantity and size of synthesized QDs was 0.06 M. To further increase the performance of QDSCs, the 30% deionized water of polysulfide electrolyte was replaced with methanol to improve the wettability and permeability of electrolytes in the TiO2 film, which accelerated the redox couple diffusion in the electrolyte solution and improved charge transfer at the interfaces between photoanodes and electrolytes. The stability of PbS QDs in the electrolyte was also improved by methanol to reduce the charge recombination and prolong the electron lifetime. As a result, the PCE of QDSC was increased to 4.01%.

  5. Enhanced Performance of PbS-quantum-dot-sensitized Solar Cells via Optimizing Precursor Solution and Electrolytes.

    PubMed

    Tian, Jianjun; Shen, Ting; Liu, Xiaoguang; Fei, Chengbin; Lv, Lili; Cao, Guozhong

    2016-03-15

    This work reports a PbS-quantum-dot-sensitized solar cell (QDSC) with power conversion efficiency (PCE) of 4%. PbS quantum dots (QDs) were grown on mesoporous TiO2 film using a successive ion layer absorption and reaction (SILAR) method. The growth of QDs was found to be profoundly affected by the concentration of the precursor solution. At low concentrations, the rate-limiting factor of the crystal growth was the adsorption of the precursor ions, and the surface growth of the crystal became the limiting factor in the high concentration solution. The optimal concentration of precursor solution with respect to the quantity and size of synthesized QDs was 0.06 M. To further increase the performance of QDSCs, the 30% deionized water of polysulfide electrolyte was replaced with methanol to improve the wettability and permeability of electrolytes in the TiO2 film, which accelerated the redox couple diffusion in the electrolyte solution and improved charge transfer at the interfaces between photoanodes and electrolytes. The stability of PbS QDs in the electrolyte was also improved by methanol to reduce the charge recombination and prolong the electron lifetime. As a result, the PCE of QDSC was increased to 4.01%.

  6. High-efficiency perovskite solar cells prepared by using a sandwich structure MAI-PbI2-MAI precursor film.

    PubMed

    Zhang, Xuhui; Ye, Jiajiu; Zhu, Liangzheng; Zheng, Haiying; Liu, Guozhen; Liu, Xuepeng; Duan, Bin; Pan, Xu; Dai, Songyuan

    2017-04-06

    Two-step deposition has been widely used in the perovskite layer preparation for perovskite solar cells due to its attractive morphology controllability. However, the limited diffusivity of CH 3 NH 3 I (MAI) might cause some PbI 2 to remain in the perovskite film. The residual PbI 2 in the perovskite film would lead to inferior performance of devices, such as, low power conversion efficiency (PCE), poor reproducibility and weak air stability. In this work, we developed a sandwich structure MAI-PbI 2 -MAI precursor film to prepare a PbI 2 -free CH 3 NH 3 PbI 3 perovskite film. In comparison to the two-step approach, the MAI-PbI 2 -MAI precursor film with a typical sandwich structure formed a uniform and pinhole-free perovskite film without any PbI 2 residue, which could significantly improve the performance of the devices. Moreover, the bottom MAI layer of the MAI-PbI 2 -MAI precursor film could improve the interfacial contact of the porous TiO 2 layer, leading to the promotion of the charge transfer and reduction of the recombination rate. Therefore, the devices fabricated from the sandwich structure MAI-PbI 2 -MAI precursor films showed dramatic improvements of open-circuit voltage (V oc ), short-circuit current density (J sc ), fill factor (FF) and PCE. As a result, a promising PCE of 17.8% with good long-term air stability was achieved for the MAI-PbI 2 -MAI precursor film based PSC, which is better than that prepared by a two-step approach.

  7. Large grained perovskite solar cells derived from single-crystal perovskite powders with enhanced ambient stability

    DOE PAGES

    Yen, Hung -Ju; Liang, Po -Wei; Chueh, Chu -Chen; ...

    2016-05-25

    In this study, we demonstrate the large grained perovskite solar cells prepared from precursor solution comprising single-crystal perovskite powders for the first time. Here, the resultant large grained perovskite thin film possesses negligible physical (structural) gap between each large grain and are highly crystalline as evidenced by its fan-shaped birefringence observed under polarized light, which is very different to the thin film prepared from the typical precursor route (MAI + PbI 2).

  8. Sol-Gel Deposited Double Layer TiO₂ and Al₂O₃ Anti-Reflection Coating for Silicon Solar Cell.

    PubMed

    Jung, Jinsu; Jannat, Azmira; Akhtar, M Shaheer; Yang, O-Bong

    2018-02-01

    In this work, the deposition of double layer ARC on p-type Si solar cells was carried out by simple spin coating using sol-gel derived Al2O3 and TiO2 precursors for the fabrication of crystalline Si solar cells. The first ARC layer was created by freshly prepared sol-gel derived Al2O3 precursor using spin coating technique and then second ARC layer of TiO2 was deposited with sol-gel derived TiO2 precursor, which was finally annealed at 400 °C. The double layer Al2O3/TiO2 ARC on Si wafer exhibited the low average reflectance of 4.74% in the wavelength range of 400 and 1000 nm. The fabricated solar cells based on double TiO2/Al2O3 ARC attained the conversion efficiency of ~13.95% with short circuit current (JSC) of 35.27 mA/cm2, open circuit voltage (VOC) of 593.35 mV and fill factor (FF) of 66.67%. Moreover, the fabricated solar cells presented relatively low series resistance (Rs) as compared to single layer ARCs, resulting in the high VOC and FF.

  9. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takahara, Kiyoshi; Ii, Masaaki, E-mail: masaii@art.osaka-med.ac.jp; Inamoto, Teruo

    2014-04-18

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferationmore » of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.« less

  10. Derivation, propagation and differentiation of human embryonic stem cells.

    PubMed

    Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

    2004-04-01

    Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug

  11. Turnover of bone marrow-derived cells in the irradiated mouse cornea

    PubMed Central

    Chinnery, Holly R; Humphries, Timothy; Clare, Adam; Dixon, Ariane E; Howes, Kristen; Moran, Caitlin B; Scott, Danielle; Zakrzewski, Marianna; Pearlman, Eric; McMenamin, Paul G

    2008-01-01

    In light of an increasing awareness of the presence of bone marrow (BM)-derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte-derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM-derived cells from transgenic eGFP mice were transplanted into whole-body irradiated wild-type recipients. Additionally, wild-type BM-derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8 weeks post-reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid-derived cells in the normal Cx3cr1+/gfp cornea was 366 cells/mm2. In BM chimeras 2 weeks post-reconstitution, 24% of the myeloid-derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post-reconstitution 75% of the myeloid-derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid-derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non-haematopoietic cells using BM chimeric mice in models of corneal inflammation. PMID:18540963

  12. Stimulation of Mucosal Mast Cell Growth in Normal and Nude Rat Bone Marrow Cultures

    NASA Astrophysics Data System (ADS)

    Haig, David M.; McMenamin, Christine; Gunneberg, Christian; Woodbury, Richard; Jarrett, Ellen E. E.

    1983-07-01

    Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.

  13. Fetal Membranes-Derived Stem Cells Microenvironment.

    PubMed

    Favaron, Phelipe Oliveira; Miglino, Maria Angelica

    2017-01-01

    Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

  14. Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism

    NASA Astrophysics Data System (ADS)

    Patel, Bhavesh H.; Percivalle, Claudia; Ritson, Dougal J.; Duffy, Colm D.; Sutherland, John D.

    2015-04-01

    A minimal cell can be thought of as comprising informational, compartment-forming and metabolic subsystems. To imagine the abiotic assembly of such an overall system, however, places great demands on hypothetical prebiotic chemistry. The perceived differences and incompatibilities between these subsystems have led to the widely held assumption that one or other subsystem must have preceded the others. Here we experimentally investigate the validity of this assumption by examining the assembly of various biomolecular building blocks from prebiotically plausible intermediates and one-carbon feedstock molecules. We show that precursors of ribonucleotides, amino acids and lipids can all be derived by the reductive homologation of hydrogen cyanide and some of its derivatives, and thus that all the cellular subsystems could have arisen simultaneously through common chemistry. The key reaction steps are driven by ultraviolet light, use hydrogen sulfide as the reductant and can be accelerated by Cu(I)-Cu(II) photoredox cycling.

  15. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    PubMed

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

  16. Immunomodulatory Properties of Induced Pluripotent Stem Cell-Derived Mesenchymal Cells.

    PubMed

    Ng, Jia; Hynes, Kim; White, Gregory; Sivanathan, Kisha Nandini; Vandyke, Kate; Bartold, Peter Mark; Gronthos, Stan

    2016-12-01

    MSC-like populations derived from induced pluripotent stem cells (iPSC-MSC) serve as an alternative stem cell source due to their high proliferative capacity. In this study, we assessed the immunomodulatory potential of iPSC-MSC generated from periodontal ligament (PDL) and gingival (GF) tissue. The iPSC-MSC lines exhibited a similar level of suppression of mitogen-stimulated peripheral blood mononuclear cells (PBMNC) proliferation compared to their respective parental fibroblast populations in vitro. Moreover, iPSC-MSC demonstrated the ability to suppress T-cells effector cells, Th1/Th2/Th17 populations, and increase levels of Treg cells. In order to investigate the mechanisms involved, expression of common MSC-derived soluble factors known to supress lymphocyte proliferation were assessed in iPSC-MSC cultured with PBMNC with direct cell-cell contact or separated in transwells. Real-time PCR analysis of factors known to be involved in MSC mediated immune regulation, found a general trend of elevated IDO1 and IL6 transcript levels in iPSC-MSC lines and their respective primary cells co-cultured with activated PBMNC, with a wide range of gene expression levels between the different mesenchymal cell types. The results suggest that different iPSC-MSC may be useful as a potential alternative source of cells for future clinical use in therapeutic applications because of their potent immunosuppressive properties. J. Cell. Biochem. 117: 2844-2853, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Microglia modulate hippocampal neural precursor activity in response to exercise and aging.

    PubMed

    Vukovic, Jana; Colditz, Michael J; Blackmore, Daniel G; Ruitenberg, Marc J; Bartlett, Perry F

    2012-05-09

    Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor, CX(3)CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process.

  18. Human Adipose-Derived Stem Cells Labeled with Plasmonic Gold Nanostars for Cellular Tracking and Photothermal Cancer Cell Ablation.

    PubMed

    Shammas, Ronnie L; Fales, Andrew M; Crawford, Bridget M; Wisdom, Amy J; Devi, Gayathri R; Brown, David A; Vo-Dinh, Tuan; Hollenbeck, Scott T

    2017-04-01

    Gold nanostars are unique nanoplatforms that can be imaged in real time and transform light energy into heat to ablate cells. Adipose-derived stem cells migrate toward tumor niches in response to chemokines. The ability of adipose-derived stem cells to migrate and integrate into tumors makes them ideal vehicles for the targeted delivery of cancer nanotherapeutics. To test the labeling efficiency of gold nanostars, undifferentiated adipose-derived stem cells were incubated with gold nanostars and a commercially available nanoparticle (Qtracker), then imaged using two-photon photoluminescence microscopy. The effects of gold nanostars on cell phenotype, proliferation, and viability were assessed with flow cytometry, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide metabolic assay, and trypan blue, respectively. Trilineage differentiation of gold nanostar-labeled adipose-derived stem cells was induced with the appropriate media. Photothermolysis was performed on adipose-derived stem cells cultured alone or in co-culture with SKBR3 cancer cells. Efficient uptake of gold nanostars occurred in adipose-derived stem cells, with persistence of the luminescent signal over 4 days. Labeling efficiency and signal quality were greater than with Qtracker. Gold nanostars did not affect cell phenotype, viability, or proliferation, and exhibited stronger luminescence than Qtracker throughout differentiation. Zones of complete ablation surrounding the gold nanostar-labeled adipose-derived stem cells were observed following photothermolysis in both monoculture and co-culture models. Gold nanostars effectively label adipose-derived stem cells without altering cell phenotype. Once labeled, photoactivation of gold nanostar-labeled adipose-derived stem cells ablates neighboring cancer cells, demonstrating the potential of adipose-derived stem cells as a vehicle for the delivery of site-specific cancer therapy.

  19. Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.

    PubMed

    Martin, Colin H; Woll, Petter S; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos; Kaufman, Dan S

    2008-10-01

    Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34(+) cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34(+) cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34(+) cells also did not produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

  20. Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

    PubMed

    Fortuna, Vitor; Pardanaud, Luc; Brunet, Isabelle; Ola, Roxana; Ristori, Emma; Santoro, Massimo M; Nicoli, Stefania; Eichmann, Anne

    2015-06-23

    The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chu, Yijing; Tang, Huijuan; Guo, Yan

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOCmore » cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.« less

  2. Autologous Pluripotent Stem Cell-Derived β-Like Cells for Diabetes Cellular Therapy.

    PubMed

    Millman, Jeffrey R; Pagliuca, Felicia W

    2017-05-01

    Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into β-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach. © 2017 by the American Diabetes Association.

  3. Ovary-derived precursor gibberellin A9 is essential for female flower development in cucumber.

    PubMed

    Pimenta Lange, Maria João; Lange, Theo

    2016-12-01

    Gibberellins (GAs) are hormones that control many aspects of plant development, including flowering. It is well known that stamen is the source of GAs that regulate male and bisexual flower development. However, little is known about the role of GAs in female flower development. In cucumber, high levels of GA precursors are present in ovaries and high levels of bioactive GA 4 are identified in sepals/petals, reflecting the expression of GA 20-oxidase and 3-oxidase in these organs, respectively. Here, we show that the biologically inactive precursor GA 9 moves from ovaries to sepal/petal tissues where it is converted to the bioactive GA 4 necessary for female flower development. Transient expression of a catabolic GA 2-oxidase from pumpkin in cucumber ovaries decreases GA 9 and GA 4 levels and arrests the development of female flowers, and this can be restored by application of GA 9 to petals thus confirming its function. Given that bioactive GAs can promote sex reversion of female flowers, movement of biologically inactive precursors, instead of the hormone itself, might help to maintain floral organ identity, ensuring fruit and seed production. © 2016. Published by The Company of Biologists Ltd.

  4. On the difficulties of detecting PP precursors

    NASA Astrophysics Data System (ADS)

    Lessing, Stephan; Thomas, Christine; Saki, Morvarid; Schmerr, Nicholas; Vanacore, Elizabeth

    2015-06-01

    the velocity and density changes with depth across the transition zone. We also compare the observed precursors' amplitudes with seismic models from calculations of phase equilibria and find that a seismic velocity model derived from a pyrolite composition reproduces the data better than the currently available 1-D earth models. This largely owes to the pyrolite models producing a stronger minimum in the reflection coefficient across the epicentral distances where the reduction in amplitudes of the PP precursors is observed. To suppress the precursors entirely in a small subset of earthquakes, other effects, such as localized discontinuity topography and seismic signal processing effects are required in addition to the changed velocity model.

  5. DNA methylation in schizophrenia in different patient-derived cell types.

    PubMed

    Vitale, Alejandra M; Matigian, Nicholas A; Cristino, Alexandre S; Nones, Katia; Ravishankar, Sugandha; Bellette, Bernadette; Fan, Yongjun; Wood, Stephen A; Wolvetang, Ernst; Mackay-Sim, Alan

    2017-01-01

    DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a "ground state" upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.

  6. Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shafarman, William N.

    This project “Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells”, completed by the Institute of Energy Conversion (IEC) at the University of Delaware in collaboration with the Department of Chemical Engineering at the University of Florida, developed the fundamental understanding and technology to increase module efficiency and improve the manufacturability of Cu(InGa)(SeS)2 films using the precursor reaction approach currently being developed by a number of companies. Key results included: (1) development of a three-step H2Se/Ar/H2S reaction process to control Ga distribution through the film and minimizes back contact MoSe2 formation; (2) Ag-alloying to improve precursor homogeneity by avoiding In phasemore » agglomeration, faster reaction and improved adhesion to allow wider reaction process window; (3) addition of Sb, Bi, and Te interlayers at the Mo/precursor junction to produce more uniform precursor morphology and improve adhesion with reduced void formation in reacted films; (4) a precursor structure containing Se and a reaction process to reduce processing time to 5 minutes and eliminate H2Se usage, thereby increasing throughput and reducing costs. All these results were supported by detailed characterization of the film growth, reaction pathways, thermodynamic assessment and device behavior.« less

  7. Umbilical Cord Blood-Derived Mesenchymal Stem Cells Inhibit, But Adipose Tissue-Derived Mesenchymal Stem Cells Promote, Glioblastoma Multiforme Proliferation

    PubMed Central

    Akimoto, Keiko; Kimura, Kenichi; Nagano, Masumi; Takano, Shingo; To'a Salazar, Georgina; Yamashita, Toshiharu

    2013-01-01

    Mesenchymal stem cells (MSCs) possess self-renewal and multipotential differentiation abilities, and they are thought to be one of the most reliable stem cell sources for a variety of cell therapies. Recently, cell therapy using MSCs has been studied as a novel therapeutic approach for cancers that show refractory progress and poor prognosis. MSCs from different tissues have different properties. However, the effect of different MSC properties on their application in anticancer therapies has not been thoroughly investigated. In this study, to characterize the anticancer therapeutic application of MSCs from different sources, we established two different kinds of human MSCs: umbilical cord blood-derived MSCs (UCB-MSCs) and adipose-tissue-derived MSCs (AT-MSCs). We used these MSCs in a coculture assay with primary glioblastoma multiforme (GBM) cells to analyze how MSCs from different sources can inhibit GBM growth. We found that UCB-MSCs inhibited GBM growth and caused apoptosis, but AT-MSCs promoted GBM growth. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay clearly demonstrated that UCB-MSCs promoted apoptosis of GBM via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL was expressed more highly by UCB-MSCs than by AT-MSCs. Higher mRNA expression levels of angiogenic factors (vascular endothelial growth factor, angiopoietin 1, platelet-derived growth factor, and insulin-like growth factor) and stromal-derived factor-1 (SDF-1/CXCL12) were observed in AT-MSCs, and highly vascularized tumors were developed when AT-MSCs and GBM were cotransplanted. Importantly, CXCL12 inhibited TRAIL activation of the apoptotic pathway in GBM, suggesting that AT-MSCs may support GBM development in vivo by at least two distinct mechanisms—promoting angiogenesis and inhibiting apoptosis. The opposite effects of AT-MSCs and UCB-MSCs on GBM clearly demonstrate that differences must be considered when choosing a stem cell source

  8. Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

    PubMed

    Kim, So-Yeon; Kim, Ye-Ryung; Park, Woo-Jae; Kim, Han Su; Jung, Sung-Chul; Woo, So-Youn; Jo, Inho; Ryu, Kyung-Ha; Park, Joo-Won

    2015-01-01

    Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  9. Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene.

    PubMed Central

    Schultz, A M; Rabin, E H; Oroszlan, S

    1979-01-01

    Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [35S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP94gag. Both shared identical [35S]methionine peptides with Pr65gag and p30. Of the two nonglycosylated precursors, Pr65gag and Pr75gag, only Pr65gag was found to be detectably phosphorylated, and Pr75gag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr75gag, by two divergent pathways: one leading through the intermediate Pr65gag to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr80gag, which is further glycosylated to yield cell surface polyprotein gP94gag. Images PMID:480454

  10. Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

    PubMed Central

    Anderson, Claire; Williams, Victoria C.; Moyon, Benjamin; Daubas, Philippe; Tajbakhsh, Shahragim; Buckingham, Margaret E.; Shiroishi, Toshihiko; Hughes, Simon M.; Borycki, Anne-Gaëlle

    2012-01-01

    How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles. PMID:22987640

  11. Carbon-Nanodot Solar Cells from Renewable Precursors.

    PubMed

    Marinovic, Adam; Kiat, Lim S; Dunn, Steve; Titirici, Maria-Magdalena; Briscoe, Joe

    2017-03-09

    It has recently been shown that waste biomass can be converted into a wide range of functional materials, including those with desirable optical and electronic properties, offering the opportunity to find new uses for these renewable resources. Photovoltaics is one area in which finding the combination of abundant, low-cost and non-toxic materials with the necessary functionality can be challenging. In this paper the performance of carbon nanodots derived from a wide range of biomaterials obtained from different biomass sources as sensitisers for TiO 2 -based nanostructured solar cells was compared; polysaccharides (chitosan and chitin), monosaccharide (d-glucose), amino acids (l-arginine and l-cysteine) and raw lobster shells were used to produce carbon nanodots through hydrothermal carbonisation. The highest solar power conversion efficiency (PCE) of 0.36 % was obtained by using l-arginine carbon nanodots as sensitisers, whereas lobster shells, as a model source of chitin from actual food waste, showed a PCE of 0.22 %. By comparing this wide range of materials, the performance of the solar cells was correlated with the materials characteristics by carefully investigating the structural and optical properties of each family of carbon nanodots, and it was shown that the combination of amine and carboxylic acid functionalisation is particularly beneficial for the solar-cell performance. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Persistence of perfluoroalkyl acid precursors in AFFF-impacted groundwater and soil.

    PubMed

    Houtz, Erika F; Higgins, Christopher P; Field, Jennifer A; Sedlak, David L

    2013-08-06

    Several classes of polyfluorinated chemicals that are potential precursors to the perfluorinated carboxylates and sulfonates are present in aqueous film-forming foams (AFFF). To assess the persistence of these AFFF-derived precursors, groundwater, soil, and aquifer solids were obtained in 2011 from an unlined firefighter training area at a U.S. Air Force Base where AFFF was regularly used between 1970 and 1990. To measure the total concentration of perfluorinated carboxylate and sulfonate precursors in archived AFFF formulations and AFFF-impacted environmental samples, a previously developed assay that uses hydroxyl radical to oxidize precursors to perfluorinated carboxylates was adapted for these media. This assay was employed along with direct measurement of 22 precursors found in AFFF and a suite of other poly- and perfluoroalkyl substances (PFASs). On a molar basis, precursors accounted for 41-100% of the total concentration of PFASs in archived AFFF formulations. In the training area, precursors measured by the oxidation assay accounted for an average of 23% and 28% of total PFASs (i.e., precursors and perfluorinated carboxylates and sulfonates) in groundwater and solids samples, respectively. One precursor in AFFF, perfluorohexane sulfonamide amine, was observed on several highly contaminated soil and aquifer solids samples, but no other precursors present in AFFF formulations were detected in any samples at this field site. Suspected intermediate transformation products of precursors in AFFF that were directly measured accounted for approximately half of the total precursor concentration in samples from the training site. The fraction of PFASs consisting of perfluorinated carboxylates and sulfonates was greater in groundwater and solid samples than in any archived AFFF formulations, suggesting that much of the mass of precursors released at the site was converted to perfluorinated carboxylates and sulfonates. The precursors that have persisted at this site

  13. Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

    PubMed Central

    Martin, Colin H.; Woll, Petter S.; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos

    2008-01-01

    Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs. PMID:18621931

  14. Platinum-free catalysts for low temperature fuel cells

    NASA Astrophysics Data System (ADS)

    Lastovina, Tatiana; Pimonova, Julia; Budnyk, Andriy

    2017-04-01

    In this work, we have successfully prepared Zn/Co-N/C and Zn/Co-Fe/N/C composites, both derived from single zeolitic imidazolate framework (ZIF) precursor Zn/Co-ZIF containing equivalent quantities of Zn and Co metal sites. The composites were formed by pyrolysis of the precursor at 700 °C in inert gas atmosphere as such and after mixing it with Fe(II) salt and 1,10-phenontraline in ethanol. Catalytic tests for oxygen reduction reaction (ORR) in electrochemical cell demonstrated promising results allowing us to consider these composites as potential Pt-free catalysts for low temperature fuel cells.

  15. Distinct MicroRNA Expression Profile and Targeted Biological Pathways in Functional Myeloid-derived Suppressor Cells Induced by Δ9-Tetrahydrocannabinol in Vivo

    PubMed Central

    Hegde, Venkatesh L.; Tomar, Sunil; Jackson, Austin; Rao, Roshni; Yang, Xiaoming; Singh, Udai P.; Singh, Narendra P.; Nagarkatti, Prakash S.; Nagarkatti, Mitzi

    2013-01-01

    Δ9-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b+Gr-1+ MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (∼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b+Ly6G+Ly6C+ and CD11b+Ly6G−Ly6C+ purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in myeloid

  16. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

    PubMed

    Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

    2017-11-15

    BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Immune surveillance properties of human NK cell-derived exosomes.

    PubMed

    Lugini, Luana; Cecchetti, Serena; Huber, Veronica; Luciani, Francesca; Macchia, Gianfranco; Spadaro, Francesca; Paris, Luisa; Abalsamo, Laura; Colone, Marisa; Molinari, Agnese; Podo, Franca; Rivoltini, Licia; Ramoni, Carlo; Fais, Stefano

    2012-09-15

    Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

  18. PLC-beta2 monitors the drug-induced release of differentiation blockade in tumoral myeloid precursors.

    PubMed

    Brugnoli, Federica; Bovolenta, Matteo; Benedusi, Mascia; Miscia, Sebastianó; Capitani, Silvano; Bertagnolo, Valeria

    2006-05-01

    The differentiation therapy in treatment of acute promyelocytic leukemia (APL), based on the administration of all-trans retinoic acid (ATRA), is currently flanked with the use of As2O3, a safe and effective agent for patients showing a resistance to ATRA treatment. A synergy between ATRA and As3O3 was also reported in inducing granulocytic differentiation of APL-derived cells. We have demonstrated that phospholipase C-beta2 (PLC-beta2), highly expressed in neutrophils and nearly absent in tumoral promyelocytes, largely increases during ATRA treatment of APL-derived cells and strongly correlates with the responsiveness of APL patients to ATRA-based differentiating therapies. Here we report that, in APL-derived cells, low doses of As3O3 induce a slight increase of PLC-beta2 together with a moderate maturation, and cooperate with ATRA to provoke a significant increase of PLC-beta2 expression. Remarkably, the amounts of PLC-beta2 draw a parallel with the differentiation levels reached by both ATRA-responsive and -resistant cells treated with ATRA/As2O3 combinations. PLC-beta2 is not necessary for the progression of tumoral promyelocytes along the granulocytic lineage and is unable to overcome the differentiation block or to potentiate the agonist-induced maturation. On the other hand, since its expression closely correlates with the differentiation level reached by APL-derived cells induced to maturate by drugs presently employed in APL therapies, PLC-beta2 represents indeed a specific marker to test the ability of differentiation agents to induce the release of the maturation blockade of tumoral myeloid precursors.

  19. Single cell cultures of Drosophila neuroectodermal and mesectodermal central nervous system progenitors reveal different degrees of developmental autonomy.

    PubMed

    Lüer, Karin; Technau, Gerhard M

    2009-08-03

    The Drosophila embryonic central nervous system (CNS) develops from two sets of progenitor cells, neuroblasts and ventral midline progenitors, which behave differently in many respects. Neuroblasts derive from the neurogenic region of the ectoderm and form the lateral parts of the CNS. Ventral midline precursors are formed by two rows of mesectodermal cells and build the CNS midline. There is plenty of evidence that individual identities are conferred to precursor cells by positional information in the ectoderm. It is unclear, however, how far the precursors can maintain their identities and developmental properties in the absence of normal external signals. To separate the respective contributions of autonomous properties versus extrinsic signals during their further development, we isolated individual midline precursors and neuroectodermal precursors at the pre-mitotic gastrula stage, traced their development in vitro, and analyzed the characteristics of their lineages in comparison with those described for the embryo. Although individually cultured mesectodermal cells exhibit basic characteristics of CNS midline progenitors, the clones produced by these progenitors differ from their in situ counterparts with regard to cell numbers, expression of molecular markers, and the separation of neuronal and glial fate. In contrast, clones derived from individually cultured precursors taken from specific dorsoventral zones of the neuroectoderm develop striking similarities to the lineages of neuroblasts that normally delaminate from these zones and develop in situ. This in vitro analysis allows for the first time a comparison of the developmental capacities in situ and in vitro of individual neural precursors of defined spatial and temporal origin. The data reveal that cells isolated at the pre-mitotic and pre-delamination stage express characteristics of the progenitor type appropriate to their site of origin in the embryo. However, presumptive neuroblasts, once

  20. Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

    PubMed

    Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

    2016-12-01

    Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na +  current (I Na ), nifedipine, a blocker of L-type Ca 2+  current (I CaL ), and E4031, a blocker of the rapid component of delayed rectifier K +  current (I Kr ). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K +  current (I Ks ). In hiPSC-derived cardiomyocytes of cardiac origin, I Na , I CaL , I Kr , and I Ks were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic

  1. Coelomic epithelium-derived cells in visceral morphogenesis.

    PubMed

    Ariza, Laura; Carmona, Rita; Cañete, Ana; Cano, Elena; Muñoz-Chápuli, Ramón

    2016-03-01

    Coelomic cavities of vertebrates are lined by a mesothelium which develops from the lateral plate mesoderm. During development, the coelomic epithelium is a highly active cell layer, which locally is able to supply mesenchymal cells that contribute to the mesodermal elements of many organs and provide signals which are necessary for their development. The relevance of this process of mesenchymal cell supply to the developing organs is becoming clearer because genetic lineage tracing techniques have been developed in recent years. Body wall, heart, liver, lungs, gonads, and gastrointestinal tract are populated by cells derived from the coelomic epithelium which contribute to their connective and vascular tissues, and sometimes to specialized cell types such as the stellate cells of the liver, the Cajal interstitial cells of the gut or the Sertoli cells of the testicle. In this review we collect information about the contribution of coelomic epithelium derived cells to visceral development, their developmental fates and signaling functions. The common features displayed by all these processes suggest that the epithelial-mesenchymal transition of the embryonic coelomic epithelium is an underestimated but key event of vertebrate development, and probably it is shared by all the coelomate metazoans. © 2015 Wiley Periodicals, Inc.

  2. A sintering study on the β-spodumene-based glass ceramics prepared from gel-derived precursor powders with LiF additive

    NASA Astrophysics Data System (ADS)

    Wang, Moo-Chin; Wu, Nan-Chung; Yang, Sheng; Wen, Shaw-Bing

    2002-01-01

    Beta-spodumene (Li2O·Al2O3·4SiO2, LAS) powders were prepared by a sol-gel process using Si(OC2H5)4, Al(OC4H9)3, and LiNO3 as precursors and LiF as a sintering aid agent. Dilatometry, X-ray diffraction (XRD), scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), and electron diffraction (ED) were utilized to study the sintering, phase transformation, microstructure, and properties of the β-spodumene glass-ceramics prepared from the gel-derived precursor powders with and without LiF additives. For the LAS precursor powders containing no LiF, the only crystalline phase obtained was β-spodumene. For the pellets containing less than 4 wt pct LiF and sintered at 1050 °C for 5 hours the crystalline phases were β-spodumene and β-eucryptite (Li2O·Al2O3·2SiO2). When the LiF content was 5 wt pct and the sintering process was carried out at 1050 °C for 5 hours, the crystalline phases were β-spodumene, β-eucryptite (triclinic), and eucryptite (rhombohedral (hex.)) phases. With the LiF additive increased from 0.5 to 4 wt pct and sintering at 1050 °C for 5 hours, the open porosity of the sintered bodies decrease from 30 to 2.1 pct. The grains size is about to 4 to 5 µm when pellect LAS compact contains LiF 3 wt pct as sintered at 1050 °C for 5 hours. The grains size grew to 8 to 25 µm with a remarkable discontinuous grain growth for pellet LAS compact contain LiF 5 wt pct sintered at 1050 °C for 5 hours. Relative densities greater than 90 pct could be obtained for the LAS precursor powders with LiF > 2 wt pct when sintered at 1050 °C for 5 hours. The coefficient of thermal expansion of the sintered bodies decreased from 8.3 × 10-7 to 5.2 × 10-7/°C (25 °C to 900 °C) as the LiF addition increased from 0 to 5 wt pct.

  3. The Hippo Pathway Regulates Homeostatic Growth of Stem Cell Niche Precursors in the Drosophila Ovary

    PubMed Central

    Sarikaya, Didem P.; Extavour, Cassandra G.

    2015-01-01

    The Hippo pathway regulates organ size, stem cell proliferation and tumorigenesis in adult organs. Whether the Hippo pathway influences establishment of stem cell niche size to accommodate changes in organ size, however, has received little attention. Here, we ask whether Hippo signaling influences the number of stem cell niches that are established during development of the Drosophila larval ovary, and whether it interacts with the same or different effector signaling pathways in different cell types. We demonstrate that canonical Hippo signaling regulates autonomous proliferation of the soma, while a novel hippo-independent activity of Yorkie regulates autonomous proliferation of the germ line. Moreover, we demonstrate that Hippo signaling mediates non-autonomous proliferation signals between germ cells and somatic cells, and contributes to maintaining the correct proportion of these niche precursors. Finally, we show that the Hippo pathway interacts with different growth pathways in distinct somatic cell types, and interacts with EGFR and JAK/STAT pathways to regulate non-autonomous proliferation of germ cells. We thus provide evidence for novel roles of the Hippo pathway in establishing the precise balance of soma and germ line, the appropriate number of stem cell niches, and ultimately regulating adult female reproductive capacity. PMID:25643260

  4. Fatty Aldehydes in Cyanobacteria Are a Metabolically Flexible Precursor for a Diversity of Biofuel Products

    PubMed Central

    Kaiser, Brett K.; Carleton, Michael; Hickman, Jason W.; Miller, Cameron; Lawson, David; Budde, Mark; Warrener, Paul; Paredes, Angel; Mullapudi, Srinivas; Navarro, Patricia; Cross, Fred; Roberts, James M.

    2013-01-01

    We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis. PMID:23505484

  5. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    USDA-ARS?s Scientific Manuscript database

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  6. Tumorigenicity studies for human pluripotent stem cell-derived products.

    PubMed

    Kuroda, Takuya; Yasuda, Satoshi; Sato, Yoji

    2013-01-01

    Human pluripotent stem cells (hPSCs), i.e. human embryonic stem cells and human induced pluripotent stem cells, are able to self-renew and differentiate into multiple cell types. Because of these abilities, numerous attempts have been made to utilize hPSCs in regenerative medicine/cell therapy. hPSCs are, however, also tumorigenic, that is, they can give rise to the progressive growth of tumor nodules in immunologically unresponsive animals. Therefore, assessing and managing the tumorigenicity of all final products is essential in order to prevent ectopic tissue formation, tumor development, and/or malignant transformation elicited by residual pluripotent stem cells after implantation. No detailed guideline for the tumorigenicity testing of hPSC-derived products has yet been issued for regenerative medicine/cell therapy, despite the urgent necessity. Here, we describe the current situations and issues related to the tumorigenicity testing of hPSC-derived products and we review the advantages and disadvantages of several types of tumorigenicity-associated tests. We also refer to important considerations in the execution and design of specific studies to monitor the tumorigenicity of hPSC-derived products.

  7. Stem Cell-Derived Exosome in Cardiovascular Diseases: Macro Roles of Micro Particles.

    PubMed

    Yuan, Ye; Du, Weijie; Liu, Jiaqi; Ma, Wenya; Zhang, Lai; Du, Zhimin; Cai, Benzhi

    2018-01-01

    The stem cell-based therapy has emerged as the promising therapeutic strategies for cardiovascular diseases (CVDs). Recently, increasing evidence suggest stem cell-derived active exosomes are important communicators among cells in the heart via delivering specific substances to the adjacent/distant target cells. These exosomes and their contents such as certain proteins, miRNAs and lncRNAs exhibit huge beneficial effects on preventing heart damage and promoting cardiac repair. More importantly, stem cell-derived exosomes are more effective and safer than stem cell transplantation. Therefore, administration of stem cell-derived exosomes will expectantly be an alternative stem cell-based therapy for the treatment of CVDs. Furthermore, modification of stem cell-derived exosomes or artificial synthesis of exosomes will be the new therapeutic tools for CVDs in the future. In addition, stem cell-derived exosomes also have been implicated in the diagnosis and prognosis of CVDs. In this review, we summarize the current advances of stem cell-derived exosome-based treatment and prognosis for CVDs, including their potential benefits, underlying mechanisms and limitations, which will provide novel insights of exosomes as a new tool in clinical therapeutic translation in the future.

  8. Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko

    Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APPmore » has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.« less

  9. History of myeloid-derived suppressor cells.

    PubMed

    Talmadge, James E; Gabrilovich, Dmitry I

    2013-10-01

    Tumour-induced granulocytic hyperplasia is associated with tumour vasculogenesis and escape from immunity via T cell suppression. Initially, these myeloid cells were identified as granulocytes or monocytes; however, recent studies have revealed that this hyperplasia is associated with populations of multipotent progenitor cells that have been identified as myeloid-derived suppressor cells (MDSCs). The study of MDSCs has provided a wealth of information regarding tumour pathobiology, has extended our understanding of neoplastic progression and has modified our approaches to immune adjuvant therapy. In this Timeline article, we discuss the history of MDSCs, their influence on tumour progression and metastasis, and the crosstalk between tumour cells, MDSCs and the host macroenvironment.

  10. Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

    PubMed Central

    Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

    2009-01-01

    BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

  11. Amnion-derived stem cells: in quest of clinical applications

    PubMed Central

    2011-01-01

    In the promising field of regenerative medicine, human perinatal stem cells are of great interest as potential stem cells with clinical applications. Perinatal stem cells could be isolated from normally discarded human placentae, which are an ideal cell source in terms of availability, the fewer number of ethical concerns, less DNA damage, and so on. Numerous studies have demonstrated that some of the placenta-derived cells possess stem cell characteristics like pluripotent differentiation ability, particularly in amniotic epithelial (AE) cells. Term human amniotic epithelium contains a relatively large number of stem cell marker-positive cells as an adult stem cell source. In this review, we introduce a model theory of why so many AE cells possess stem cell characteristics. We also describe previous work concerning the therapeutic applications and discuss the pluripotency of the AE cells and potential pitfalls for amnion-derived stem cell research. PMID:21596003

  12. Natural attenuation of NDMA precursors in an urban, wastewater-dominated wash.

    PubMed

    Woods, Gwen C; Dickenson, Eric R V

    2016-02-01

    N-Nitrosodimethylamine (NDMA) is a disinfection by-product (DBP) that is potentially carcinogenic and has been found to occur in drinking water treatment systems impacted with treated wastewater. A major gap in NDMA research is an understanding of the persistence of wastewater-derived precursors within the natural environment. This research sought to fill this knowledge gap by surveying NDMA precursors across the length of a wastewater effluent-dominated wash. Significant precursor reduction (17%) was found to occur from introduction into the wash to a point 9 h downstream. This reduction translates into a half-life of roughly 32 h for bulk NDMA precursors. Further laboratory experiments examining rates of photolysis, biodegradation and loss to sediments, illustrated that both photolytic and biological degradation were effective removal mechanisms for NDMA precursors. Loss to sediments that were acquired from the wash did not appear to reduce NDMA precursors in the water column, although a control conducted with DI water provided evidence that significant NDMA precursors could be released from autoclaved sediments (suggesting that sorption does occur). Microbial experiments revealed that microbes associated with sediments were much more effective at degrading precursors than microbes within the water column. Overall, this study demonstrated that natural processes are capable of attenuating NDMA precursors relatively quickly within the environment, and that utilities might benefit from maximizing source water residency time in the environment, prior to introduction into treatment plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Circulating red cell-derived microparticles in human malaria.

    PubMed

    Nantakomol, Duangdao; Dondorp, Arjen M; Krudsood, Srivicha; Udomsangpetch, Rachanee; Pattanapanyasat, Kovit; Combes, Valery; Grau, Georges E; White, Nicholas J; Viriyavejakul, Parnpen; Day, Nicholas P J; Chotivanich, Kesinee

    2011-03-01

    In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell-derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13-4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281-503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127-200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3-166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.

  14. Nuclear localization of foamy virus Gag precursor protein.

    PubMed Central

    Schliephake, A W; Rethwilm, A

    1994-01-01

    All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

  15. DNA precursor pool: a significant target for N-methyl-N-nitrosourea in C3H/10T1/2 clone 8 cells.

    PubMed Central

    Topal, M D; Baker, M S

    1982-01-01

    Synchronized C3H/10T1/2 clone 8 cells were treated in vitro with a nontoxic dose of N-methyl-N-nitrosourea during their S phase. Chromatographic isolation of the deoxyribonucleotide DNA precursor pool and measurement of the precursor content per cell showed that a nucleic acid residue in the precursor pool is 190-13,000 times more susceptible to methylation than a residue in the DNA duplex, depending on the site of methylation. This conclusion comes from measurements indicating that, for example, the N-1 position of adenine in dATP is 6.3 times more methylated than the same position in the DNA, even though the adenine content of the pool is only a fraction (0.0005) of the adenine content of the DNA helix. The comparative susceptibility between pool and DNA was found to vary with the site of methylation in the order the N-1 position of adenine greater than phosphate greater than the N-3 position of adenine greater than the O6 position of guanine greater than the N-7 position of guanine. The significance of these results for chemical mutagenesis and carcinogenesis is discussed. PMID:6954535

  16. Base Composition Differences between Avian Myeloblastosis Virus Transfer RNA and Transfer RNA Isolated from Host Cells

    PubMed Central

    Randerath, Kurt; Rosenthal, Leonard J.; Zamecnik, Paul C.

    1971-01-01

    Using a novel chemical tritium derivative method, we have determined the base composition of 4S RNA isolated from an RNA tumor virus, the avian myeloblastosis virus, and from normal and neoplastic host cells. Extensive differences were detected, particularly with respect to the amount of methylated bases in the viral RNA. The viral 4S RNA, which fulfills the criteria for designation as transfer RNA, appears to be derived from a precursor pool that is different from the precursor population of host-cell 4S RNA. These results are discussed in regard to the possible relationship between transfer RNA of avian mycoblastosis virus and cellular transfer RNA. Images PMID:4332019

  17. Donor-derived stem-cells and epithelial mesenchymal transition in squamous cell carcinoma in transplant recipients.

    PubMed

    Verneuil, Laurence; Leboeuf, Christophe; Bousquet, Guilhem; Brugiere, Charlotte; Elbouchtaoui, Morad; Plassa, Louis-François; Peraldi, Marie-Noelle; Lebbé, Celeste; Ratajczak, Philippe; Janin, Anne

    2015-12-08

    Skin squamous-cell-carcinoma (SCC), is the main complication in long-term kidney-transplant recipients, and it can include donor-derived cells. Preclinical models demonstrated the involvement of epithelial mesenchymal transition (EMT) in the progression of skin SCC, and the role of Snail, an EMT transcription factor, in cancer stem-cell survival and expansion.Here, we studied stem-cells and EMT expression in SCCs and concomitant actinic keratoses (AK) in kidney-transplant recipients. In SCC and AK in 3 female recipients of male kidney-transplants, donor-derived Y chromosome in epidermal stem cells was assessed using combined XY-FISH/CD133 immunostaining, and digital-droplet-PCR on laser-microdissected CD133 expressing epidermal cells.For EMT study, double immunostainings of CD133 with vimentin or snail and slug, electron microscopy and immunostainings of keratinocytes junctions were performed. Digital droplet PCR was used to check CDH1 (E-cadherin) expression level in laser-microdissected cells co-expressing CD133 and vimentin or snail and slug.The numbers of Y-chromosome were assessed using digital droplet PCR in laser-microdissected cells co-expressing CD133 and vimentin, or snail and slug, and in CD133 positive cells not expressing any EMT maker. We identified donor-derived stem-cells in basal layers and invasive areas in all skin SCCs and in concomitant AKs, but not in surrounding normal skin.The donor-derived stem-cells expressed the EMT markers, vimentin, snail and slug in SCCs but not in AKs. The expression of the EMT transcription factor, SNAI1, was higher in stem-cells when they expressed vimentin. They were located in invasive areas of SCCs. In these areas, the expressions of claudin-1 and desmoglein 1 were reduced or absent, and within the basal layer there were features of basal membrane disappearance.Donor-derived stem cells were in larger numbers in stem cells co-expressing vimentin or snail and slug than in stem cells not expressing any EMT marker

  18. Treatment of hypoxic-ischemic encephalopathy in mouse by transplantation of embryonic stem cell-derived cells.

    PubMed

    Ma, Jie; Wang, Yu; Yang, Jianhua; Yang, Min; Chang, Keun-A; Zhang, Linhua; Jiang, Feng; Li, Yi; Zhang, Zhonggong; Heo, Chaejeong; Suh, Yoo-Hun

    2007-07-01

    A 7-day-old hypoxic-ischemic encephalopathy (HIE) mouse model was used to study the effect of transplantation of embryonic stem (ES) cell-derived cells on the HIE. After the inducement in vitro, the ES cell-derived cells expressed Nestin and MAP-2, rather than GFAP mRNA. After transplantation, ES cell-derived cells can survive, migrate into the injury site, and specifically differentiate into neurons, showing improvement of the learning ability and memory of the HIE mouse at 8 months post-transplantation. The non-grafted HIE mouse brain showed typical pathological changes in the hippocampus and cerebral cortex, where the number of neurons was reduced, while in the cell graft group, number of the neurons increased in the same regions. Although further study is necessary to elucidate the precise mechanisms responsible for this functional recovery, we believe that ES cells have advantages for use as a donor source in HIE.

  19. Myeloid derived suppressor cells enhance IgE-mediated mast cell responses

    USDA-ARS?s Scientific Manuscript database

    We previously demonstrated that enhanced development of myeloid derived suppressor cells (MDSC) in ADAM10 transgenic mice yielded resistance to infection with Nippostrongylus brasiliensis infection, and that co-culturing MDSC with IgE-activated mast cells enhanced cytokine production. In the current...

  20. Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

    PubMed

    Weber, Marlen; Apostolova, Galina; Widera, Darius; Mittelbronn, Michel; Dechant, Georg; Kaltschmidt, Barbara; Rohrer, Hermann

    2015-02-01

    Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage. © 2014 AlphaMed Press.

  1. LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration.

    PubMed

    Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

    2015-11-09

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1(Atoh1) CKO) to investigate the function of LKB1 in cerebellar development. The LKB1(Atoh1) CKO mice displayed motor dysfunction. In the LKB1(Atoh1) CKO cerebellum, the overall structure had a larger volume and more lobules. LKB1 inactivation led to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1(Atoh1) CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development.

  2. LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

    PubMed Central

    Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

  3. Alternative group V precursors for CVD applications

    NASA Astrophysics Data System (ADS)

    Lum, R. M.; Klingert, J. K.

    1991-01-01

    The chemical vapor deposition (CVD) techniques used to grow III/V semiconductors films, such as metalorganic vapor phase epitaxy (MOVPE), hydride VPE, chemical beam epitaxy (CBE) and gas source molecular beam epitaxy (GS-MBE), all use hydrides (AsH 3 and PH 3) as the Group V source. However, the hydrides are extremely toxic gases which are stored under high pressure (200-2000 psi). To reduce the safety hazards associated with these gases, alternative Group V precursors have been investigated. Organoarsenic and phosphorous compounds have received the most attention as replacements for AsH 3 and PH 3 because they are typically low vapor pressure liquids, and thus present significantly lower exposure risks than the hydrides. For AsH 3 these have included the methyl, ethyl and butyl-based derivatives RnAsH 3- n, with varying degrees ( n = 1-3) of hydrogen atom substitution. In this paper the growth properties, thermochemistry and toxicity of the various alkylarsine precursors are compared with arsine. Data are presented on the impact of the thermochemistry of these compounds on film electrical properties, and on the effects of precursor composition and purity on overall film quality. The suitability of alternative As-precursors for device applications is demonstrated, and selection criteria are presented for the most effective alkylarsine compound for a particular CVD growth process.

  4. Sex Differences in Maturation of Human Embryonic Stem Cell-Derived β Cells in Mice.

    PubMed

    Saber, Nelly; Bruin, Jennifer E; O'Dwyer, Shannon; Schuster, Hellen; Rezania, Alireza; Kieffer, Timothy J

    2018-04-01

    Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate that pancreatic progenitor cells can mature into a mixed population of endocrine cells, including glucose-responsive β cells several months after implantion. However, it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these hESC-derived cells. Here, we investigated the effects of (1) pregnancy on the maturation of human stage 4 (S4) pancreatic progenitor cells and (2) the impact of host sex on both S4 cells and more mature stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-beige mice. Pregnancy led to increased proliferation of endogenous pancreatic β cells, but did not appear to affect proliferation or maturation of S4 cells at midgestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males, whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells before implantation.

  5. Hematopoietic Stem Cells in Neural-crest Derived Bone Marrow.

    PubMed

    Jiang, Nan; Chen, Mo; Yang, Guodong; Xiang, Lusai; He, Ling; Hei, Thomas K; Chotkowski, Gregory; Tarnow, Dennis P; Finkel, Myron; Ding, Lei; Zhou, Yanheng; Mao, Jeremy J

    2016-12-21

    Hematopoietic stem cells (HSCs) in the endosteum of mesoderm-derived appendicular bones have been extensively studied. Neural crest-derived bones differ from appendicular bones in developmental origin, mode of bone formation and pathological bone resorption. Whether neural crest-derived bones harbor HSCs is elusive. Here, we discovered HSC-like cells in postnatal murine mandible, and benchmarked them with donor-matched, mesoderm-derived femur/tibia HSCs, including clonogenic assay and long-term culture. Mandibular CD34 negative, LSK cells proliferated similarly to appendicular HSCs, and differentiated into all hematopoietic lineages. Mandibular HSCs showed a consistent deficiency in lymphoid differentiation, including significantly fewer CD229 + fractions, PreProB, ProB, PreB and B220 + slgM cells. Remarkably, mandibular HSCs reconstituted irradiated hematopoietic bone marrow in vivo, just as appendicular HSCs. Genomic profiling of osteoblasts from mandibular and femur/tibia bone marrow revealed deficiencies in several HSC niche regulators among mandibular osteoblasts including Cxcl12. Neural crest derived bone harbors HSCs that function similarly to appendicular HSCs but are deficient in the lymphoid lineage. Thus, lymphoid deficiency of mandibular HSCs may be accounted by putative niche regulating genes. HSCs in craniofacial bones have functional implications in homeostasis, osteoclastogenesis, immune functions, tumor metastasis and infections such as osteonecrosis of the jaw.

  6. Detailed immunophenotyping of B-cell precursors in regenerating bone marrow of acute lymphoblastic leukaemia patients: implications for minimal residual disease detection.

    PubMed

    Theunissen, Prisca M J; Sedek, Lukasz; De Haas, Valerie; Szczepanski, Tomasz; Van Der Sluijs, Alita; Mejstrikova, Ester; Nováková, Michaela; Kalina, Tomas; Lecrevisse, Quentin; Orfao, Alberto; Lankester, Arjan C; van Dongen, Jacques J M; Van Der Velden, Vincent H J

    2017-07-01

    Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34 high pre-B-I cell immunophenotype. These CD34 -dim pre-B-I cells were relatively abundant in regenerating BM (11-85% within pre-B-I subset), while hardly present in healthy control BM (9-13% within pre-B-I subset; P = 0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34 -dim pre-B-I cells. Our results indicate that newly identified CD34 -dim pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34 -dim pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements. © 2017 John Wiley & Sons Ltd.

  7. Tannins and terpenoids as major precursors of Suwannee River fulvic acid

    USGS Publications Warehouse

    Leenheer, Jerry A.; Rostad, Colleen E.

    2004-01-01

    Suwannee River fulvic acid (SRFA) was fractionated into 7 fractions by normal-phase chromatography on silica gel followed by reverse-phase fractionation on XAD-8 resin that produced 18 subfractions. Selected major subfractions were characterized by 13C-nuclear magnetic resonance (NMR), infrared spectrometry, and elemental analyses. 13C-NMR spectra of the subfractions were more indicative of precursor structures than unfractionated SRFA, and gave spectral profiles that indicated SRFA mass was about equally split between tannin precursors and terpenoid precursors. Lignin precursors were minor components. Synthesis of 13C-NMR data with elemental data for subfractions derived from both tannin and terpenoid precursors revealed high ring contents and low numbers of carbon per rings which is indicative of fused ring structures that are extensively substituted with carboxyl and methyl groups. These results ruled out extended chain structures for SRFA. This information is useful for determining sources and properties of fulvic acid in drinking water supplies as tannins are more reactive with chlorine to produce undesirable disinfection by-products than are terpenoids.

  8. Adipose‑derived stem cells and hyaluronic acid based gel compatibility, studied in vitro.

    PubMed

    Guo, Jiayan; Guo, Shu; Wang, Yuxin; Yu, Yanqiu

    2017-10-01

    Minimally invasive aesthetic and cosmetic procedures have increased in popularity. Injectable dermal fillers provide soft tissue augmentation, improve facial rejuvenation and wrinkles, and correct tissue defects. To investigate the use of adipose‑derived stem cells integrated with a hyaluronic acid based gel as a dermal filler, the present study used cytotoxicity studies, proliferation studies, adipogenic and osteogenic differentiation, apoptosis assays and scanning electron microscopy. Although hyaluronic acid induced low levels of apoptosis in adipose‑derived stem cells, its significantly promoted proliferation of adipose‑derived stem cells. Hyaluronic acid demonstrates little toxicity against adipose‑derived stem cells. Adipose‑derived stem cells were able to differentiate into adipocytes and osteoblasts. Furthermore, scanning electron microscopy revealed that adipose‑derived stem cells maintained intact structures on the surface of hyaluronic acid as well as in it, and demonstrated abundant cell attachments. The present study demonstrated the compatibility of adipose‑derived stem cells and hyaluronic acid based gels in vitro.

  9. Differentiation and Application of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

    PubMed

    Maguire, Eithne Margaret; Xiao, Qingzhong; Xu, Qingbo

    2017-11-01

    Vascular smooth muscle cells (VSMCs) play a role in the development of vascular disease, for example, neointimal formation, arterial aneurysm, and Marfan syndrome caused by genetic mutations in VSMCs, but little is known about the mechanisms of the disease process. Advances in induced pluripotent stem cell technology have now made it possible to derive VSMCs from several different somatic cells using a selection of protocols. As such, researchers have set out to delineate key signaling processes involved in triggering VSMC gene expression to grasp the extent of gene regulatory networks involved in phenotype commitment. This technology has also paved the way for investigations into diseases affecting VSMC behavior and function, which may be treatable once an identifiable culprit molecule or gene has been repaired. Moreover, induced pluripotent stem cell-derived VSMCs are also being considered for their use in tissue-engineered blood vessels as they may prove more beneficial than using autologous vessels. Finally, while several issues remains to be clarified before induced pluripotent stem cell-derived VSMCs can become used in regenerative medicine, they do offer both clinicians and researchers hope for both treating and understanding vascular disease. In this review, we aim to update the recent progress on VSMC generation from stem cells and the underlying molecular mechanisms of VSMC differentiation. We will also explore how the use of induced pluripotent stem cell-derived VSMCs has changed the game for regenerative medicine by offering new therapeutic avenues to clinicians, as well as providing researchers with a new platform for modeling of vascular disease. © 2017 American Heart Association, Inc.

  10. Vitrification of mouse embryo-derived ICM cells: a tool for preserving embryonic stem cell potential?

    PubMed

    Desai, Nina; Xu, Jing; Tsulaia, Tamara; Szeptycki-Lawson, Julia; AbdelHafez, Faten; Goldfarb, James; Falcone, Tommaso

    2011-02-01

    Vitrification technology presents new opportunities for preservation of embryo derived stem cells without first establishing a viable ESC line. This study tests the feasibility of cryopreserving ICM cells using vitrification. ICMs from mouse embryos were isolated and vitrified in HSV straws or on cryoloops. Upon warming, the vitrified ICMs were cultured and observed for attachment and morphology. Colonies were passaged every 3-6 days. ICMs and ICM-derived ESC colonies were tested for expression of stem cell specific markers. ICMs vitrified on both the cryoloop and the HSV straw had high survival rates. ICM derived ESCs remained undifferentiated for several passages and demonstrated expression of typical stem cell markers; SSEA-1, Sox-2, Oct 4 and alkaline phosphatase. This is the first report on successful vitrification of isolated ICMs and the subsequent derivation of ESC colonies. Vitrification of isolated ICMs is a novel approach for preservation of the "stem cell source" material.

  11. Generation of amyloid-β is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

    PubMed

    Stemmer, Nina; Strekalova, Elena; Djogo, Nevena; Plöger, Frank; Loers, Gabriele; Lutz, David; Buck, Friedrich; Michalak, Marek; Schachner, Melitta; Kleene, Ralf

    2013-01-01

    Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with

  12. Comparative examination of titania nanocrystals synthesized by peroxo titanic acid approach from different precursors.

    PubMed

    Liu, Yong-Jun; Aizawa, Mami; Wang, Zheng-Ming; Hatori, Hiroaki; Uekawa, Naofumi; Kanoh, Hirofumi

    2008-06-15

    Titanium dioxide nanocrystalline particles were synthesized by peroxo titanium acid (PTA) approach from titanium alkoxide and inorganic salt precursors, and their structural and surface properties, porosities, and photocatalytic activities were comparatively examined by XRD, TG/DTA, DRIFT, UV-vis, low temperature N(2) adsorption, and methyl orange (MO) degradation. It was found that nanoparticles with single anatase phase can be obtained from alkoxide precursor even near room temperature if synthesis conditions are appropriately controlled. PTA-derived anatase nanoparticles from titanium alkoxide precursor have smaller crystalline sizes and better porosities, and contain less amount of peroxo group and no organic impurities as compared to those from TiCl(4) precursor. The advantages in structural property, porosity, and surface properties (few deficiencies) lead to a much better photocatalytic activity for TiO(2) nanoparticles from titanium alkoxide precursor in comparison with those from TiCl(4) precursor.

  13. CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol

    PubMed Central

    Ghazavi, Farzaneh; Clappier, Emmanuelle; Lammens, Tim; Suciu, Stefan; Caye, Aurélie; Zegrari, Samira; Bakkus, Marleen; Grardel, Nathalie; Benoit, Yves; Bertrand, Yves; Minckes, Odile; Costa, Vitor; Ferster, Alina; Mazingue, Françoise; Plat, Geneviève; Plouvier, Emmanuel; Poirée, Marilyne; Uyttebroeck, Anne; van der Werff-ten Bosch, Jutte; Yakouben, Karima; Helsmoortel, Hetty; Meul, Magali; Van Roy, Nadine; Philippé, Jan; Speleman, Frank; Cavé, Hélène; Van Vlierberghe, Pieter; De Moerloose, Barbara

    2015-01-01

    DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23–3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728. PMID:26137961

  14. CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol.

    PubMed

    Ghazavi, Farzaneh; Clappier, Emmanuelle; Lammens, Tim; Suciu, Stefan; Caye, Aurélie; Zegrari, Samira; Bakkus, Marleen; Grardel, Nathalie; Benoit, Yves; Bertrand, Yves; Minckes, Odile; Costa, Vitor; Ferster, Alina; Mazingue, Françoise; Plat, Geneviève; Plouvier, Emmanuel; Poirée, Marilyne; Uyttebroeck, Anne; van der Werff-Ten Bosch, Jutte; Yakouben, Karima; Helsmoortel, Hetty; Meul, Magali; Van Roy, Nadine; Philippé, Jan; Speleman, Frank; Cavé, Hélène; Van Vlierberghe, Pieter; De Moerloose, Barbara

    2015-10-01

    DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728. Copyright© Ferrata Storti Foundation.

  15. The compartmentation of phosphorylated thiamine derivatives in cultured neuroblastoma cells.

    PubMed

    Bettendorff, L

    1994-05-26

    Thiamine transport in cultured neuroblastoma cells is mediated by a high-affinity carrier (KM = 40 nM). In contrast, the uptake of the more hydrophobic sulbutiamine (isobutyrylthiamine disulfide) is unsaturable and its initial transport rate is 20-times faster than for thiamine. In the cytoplasm, sulbutiamine is rapidly hydrolyzed and reduced to free thiamine, the overall process resulting in a rapid and concentrative thiamine accumulation. Incorporation of radioactivity from [14C]thiamine or [14C]sulbutiamine into intracellular thiamine diphosphate is slow in both cases. Despite the fact that the diphosphate is probably the direct precursor for both thiamine monophosphate and triphosphate, the specific radioactivity increased much faster for the latter two compounds than for thiamine diphosphate. This suggests the existence of two pools of thiamine diphosphate, the larger one having a very slow turnover (about 17 h); a much smaller, rapidly turning over pool would be the precursor of thiamine mono- and triphosphate. The turnover time for thiamine triphosphate could be estimated to be 1-2 h. When preloading the cells with [14C]sulbutiamine was followed by a chase with the same concentration of the unlabeled compound, the specific radioactivities of thiamine and thiamine monophosphate decreased exponentially as expected, but labeling of the diphosphate continued to increase slowly. Specific radioactivity of thiamine triphosphate increased first, but after 30 min it began to slowly decrease. These results show for the first time the existence of distinct thiamine diphosphate pools in the same homogeneous cell population. They also suggest a complex compartmentation of thiamine metabolism.

  16. Making Single-Source Precursors of Ternary Semiconductors

    NASA Technical Reports Server (NTRS)

    Hepp, Aloysius; Banger, Kulbindre K.

    2007-01-01

    A synthesis route has been developed for the commercial manufacture of single- source precursors of chalcopyrite semiconductor absorber layers of thin-film solar photovoltaic cells. A closely related class of single-source precursors of these semiconductors, and their synthesis routes, were reported in "Improved Single-Source Precursors for Solar-Cell Absorbers" (LEW-17445-1), NASA Tech Briefs, Vol. 31, No. 6 (June 2007), page 56. The present synthesis route is better suited to commercialization because it is simpler and involves the use of commercially available agents, yet offers the flexibility needed for synthesis of a variety of precursors. A single-source precursor of the type of interest here is denoted by the general formula L2M'(mu-ER)2M(ER)2, where L signifies a Lewis base; M signifies Al, In, or Ga; M' signifies Ag or Cu; R signifies an alkyl, aryl, silyl, or perfluorocarbon group; E signifies O, S, Se, or Te; and mu signifies a bridging ligand. This compound can be synthesized in a "one-pot" procedure from ingredients that are readily available from almost any chemical supplier. In a demonstration, the following synthesis was performed: Under anaerobic conditions, InCl3 was reacted with sodium ethanethiolate in methanol in a 1:4 molar ratio to afford the ionic stable intermediate compound Na+[In(SEt)4]- (where Et signifies ethyl group). After approximately 15 minutes, a heterogeneous solution of CuCl and the Lewis base PPh3 (where Ph signifies phenyl) in a 1:2 ratio in a mixture of CH3CN and CH2Cl2 was added directly to the freshly prepared Na+[In(SEt)4]-. After 24 hours, the reaction was essentially complete. The methanolic solution was concentrated, then the product was extracted with CH2Cl2, then the product was washed with dry ether and pentane. The product in its final form was a creamy white solid. Spectroscopic and elemental analysis confirmed that the product was (PPh3)2Cu(mu-SEt)2In(mu-SEt)2, which is known to be a precursor of the ternary

  17. Goat red blood cells as precursor for iron oxide nanocrystal synthesis to develop nuclear targeted lung cancer therapy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sreevani, Vellingiri; Shanthi, Krishnamurthy; Kannan, Soundarapandian, E-mail: sk_protein@buc.edu.in

    Graphical abstract: - Highlights: • Molecular approach of synthesis of Fe{sub 2}O{sub 3}-NC using goat blood as a bio-precursor. • The method is simple, efficient and environment friendly. • Synthesized nanocrystals were characterized by UV–vis spectroscopy, XRD, SEM, TEM, DLS and EDS. • Nanocrystals exhibited potent cytotoxicity against A549 cancer cell. • Nuclear targeting with expression of caspase-3, caspase-7 and Bcl-2 in A549 cancer cells. - Abstract: In this study, we synthesised iron oxide nanocrystals (Fe{sub 2}O{sub 3}-NC) from goat blood (bio-precursor) using red blood cells (RBC) lysis method (a molecular level approach) for the first time. The formation ofmore » Fe{sub 2}O{sub 3}-NC was achieved through a single-phase chemical reduction method. The size distribution of Fe{sub 2}O{sub 3}-NC falls between 20–30 nm for pellet and 100–200 nm for lysate and were found to be crystalline. Fe{sub 2}O{sub 3}-NC demonstrated significant cytotoxicity on A549. We report the direct visualization of interactions between Fe{sub 2}O{sub 3}-NC and the cancer cell nucleus. The active transport of Fe{sub 2}O{sub 3}-NC to the nucleus induces major changes to nuclear phenotype via nuclear envelope invaginations. We further examined the root cause for the involvement of Fe{sub 2}O{sub 3}-NC on the expression of caspase-3, caspase-7 and Bcl-2 in A549 cancer cells. This functional proteomic analysis clearly implies that the lung cancer cell proliferation is perfectly targeted by the biosynthesised Fe{sub 2}O{sub 3}-NC which could provide new insight for nuclear-targeted cancer therapy.« less

  18. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wei, Yan; Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou; Li, Yuan

    2011-04-15

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined usingmore » reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.« less

  19. In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

    NASA Astrophysics Data System (ADS)

    Luchinat, Enrico; Barbieri, Letizia; Rubino, Jeffrey T.; Kozyreva, Tatiana; Cantini, Francesca; Banci, Lucia

    2014-11-01

    Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone.

  20. Chronic intake of high fish oil diet induces myeloid-derived suppressor cells to promote tumor growth

    PubMed Central

    Li, Xiaoping; Cheng, Lu; Han, Mutian; Zhang, Miaomiao; Liu, Xia; Xu, Huaxi; Zhang, Minghui; Shao, Qixiang; Qi, Ling

    2014-01-01

    Omega-3 polyunsaturated fatty acids enriched fish oil exerts beneficial anti-inflammatory effects in animal models with acute and chronic inflammatory diseases. Myeloid-derived suppressor cells (MDSCs), comprised of myeloid progenitors and precursors of myeloid cells, play vital roles in cancer. How fish oil affects the generation of MDSCs and the tumor development remains largely unexplored. Here, we show that dietary intake of high fish oil diet suppresses CD8+ T cells activation and proliferation in vivo via elevated levels of MDSCs. Mechanistically, high fish oil diet induces the expression of immunosuppressive cytokine IL-10 and promotes myelopoiesis in the spleen as well as other peripheral tissues. The immature myeloid cells in the spleen exhibit morphological and functional characteristics of MDSCs with the capability to downregulate CD8+ T cells activation. Depletion of MDSCs using anti-Gr-1 antibody decreases the growth of subcutaneously transferred B16 melanoma in mice on high fish oil diet. Interestingly, diet-induced production of MDSCs is not solely dependent of the spleen, as splenectomy has no effect on the tumor progress. Our data show that the liver functions as an alternative extramedullary hematopoiesis organ to support MDSCs differentiation and maintain tumor growth. Taken together, our study provides a novel insight into the physiological effects of fish oil and points to MDSCs as a possible mediator linking dietary fish oil intake and immunosuppression in cancer immunosurveillance. PMID:24691944

  1. Nuclear NF-κB contributes to chlorpyrifos-induced apoptosis through p53 signaling in human neural precursor cells.

    PubMed

    Lee, Jeong Eun; Lim, Mi Sun; Park, Jae Hyeon; Park, Chang Hwan; Koh, Hyun Chul

    2014-05-01

    Chlorpyrifos (CPF) is one of the most widely used organophosphate insecticides with several harmful effects, including neurotoxicity. Although many studies have addressed the neurotoxicity induced by CPF, most data on neurodevelopmental damage was obtained from animal models. We are the first group to use human neural precursor cells (hNPCs) derived from human embryonic stem cells (hESCs) as a developing neuron model to evaluate the mechanisms involved in CPF-induced neurotoxicity. CPF was cytotoxic to these cells in a concentration-dependent manner, as shown by decreased cell viability and increased lactate dehydrogenase release. Furthermore, CPF reduced the expression of AKT and ERK proteins which are involved in intracellular survival pathways. Exposure of hNPCs to CPF led to the production of reactive oxygen species (ROS), and the antioxidant N-acetyl-cystein (NAC) attenuated ROS production induced by CPF. In addition, CPF increased cytochrome c release into the cytosol and activated caspase-9 and -3, indicating that cell death induced by CPF was due to apoptosis in hNPCs. Consistent with these findings, CPF treatment reduced the level of Bcl-2 protein and increased the level of Bax protein. Especially, CPF increased the translocation of BAX into the mitochondria. CPF also induced nuclear accumulation of NF-κB and p53 proteins in a concentration-dependent manner, and their inhibitors attenuated CPF-induced cytotoxicity. In addition, an inhibitor of NF-κB nuclear translocation blocked the increase of p53 in CPF-treated hNPCs. These findings show that CPF induced hNPCs death in part through NF-κB activation via ROS generation, enabling the interaction of p53 with Bcl-2 and Bax and subsequent release of cytochrome c. Collectively, these results represent a unique molecular characterization of CPF-induced cytotoxicity in hNPCs. These data suggest that CPF may affect neurodevelopment in a manner similar to that of several known and suspected neurotoxicants. Crown

  2. Generation of avian cells resembling osteoclasts from mononuclear phagocytes

    NASA Technical Reports Server (NTRS)

    Alvarez, J. I.; Teitelbaum, S. L.; Blair, H. C.; Greenfield, E. M.; Athanasou, N. A.; Ross, F. P.

    1991-01-01

    Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.

  3. The chemokine receptor CCR1 is identified in mast cell-derived exosomes

    PubMed Central

    Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

    2018-01-01

    Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses. PMID:29511430

  4. The chemokine receptor CCR1 is identified in mast cell-derived exosomes.

    PubMed

    Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

    2018-01-01

    Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses.

  5. Fas-L promotes the stem cell potency of adipose-derived mesenchymal cells.

    PubMed

    Solodeev, Inna; Meilik, Benjamin; Volovitz, Ilan; Sela, Meirav; Manheim, Sharon; Yarkoni, Shai; Zipori, Dov; Gur, Eyal; Shani, Nir

    2018-06-11

    Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.

  6. Perspectives on testicular sex cord-stromal tumors and those composed of both germ cells and sex cord-stromal derivatives with a comparison to corresponding ovarian neoplasms.

    PubMed

    Roth, Lawrence M; Lyu, Bingjian; Cheng, Liang

    2017-07-01

    Sex cord-stromal tumors (SCSTs) are the second most frequent category of testicular neoplasms, accounting for approximately 2% to 5% of cases. Both genetic and epigenetic factors account for the differences in frequency and histologic composition between testicular and ovarian SCSTs. For example, large cell calcifying Sertoli cell tumor and intratubular large cell hyalinizing Sertoli cell neoplasia occur in the testis but have not been described in the ovary. In this article, we discuss recently described diagnostic entities as well as inconsistencies in nomenclature used in the recent World Health Organization classifications of SCSTs in the testis and ovary. We also thoroughly review the topic of neoplasms composed of both germ cells and sex cord derivatives with an emphasis on controversial aspects. These include "dissecting gonadoblastoma" and testicular mixed germ cell-sex cord stromal tumor (MGC-SCST). The former is a recently described variant of gonadoblastoma that sometimes is an immediate precursor of germinoma in the dysgenetic gonads of patients with a disorder of sex development. Although the relationship of dissecting gonadoblastoma to the previously described undifferentiated gonadal tissue is complex and not entirely resolved, we believe that it is preferable to continue to use the term undifferentiated gonadal tissue for those cases that are not neoplastic and are considered to be the precursor of classical gonadoblastoma. Although the existence of testicular MGC-SCST has been challenged, the most recent evidence supports its existence; however, testicular MGC-SCST differs significantly from ovarian examples due to both genetic and epigenetic factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Cell-derived microparticles in haemostasis and vascular medicine.

    PubMed

    Burnier, Laurent; Fontana, Pierre; Kwak, Brenda R; Angelillo-Scherrer, Anne

    2009-03-01

    Considerable interest for cell-derived microparticles has emerged, pointing out their essential role in haemostatic response and their potential as disease markers, but also their implication in a wide range of physiological and pathological processes. They derive from different cell types including platelets - the main source of microparticles - but also from red blood cells, leukocytes and endothelial cells, and they circulate in blood. Despite difficulties encountered in analyzing them and disparities of results obtained with a wide range of methods, microparticle generation processes are now better understood. However, a generally admitted definition of microparticles is currently lacking. For all these reasons we decided to review the literature regarding microparticles in their widest definition, including ectosomes and exosomes, and to focus mainly on their role in haemostasis and vascular medicine.

  8. Comparative study of adipose-derived stem cells and bone marrow-derived stem cells in similar microenvironmental conditions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guneta, Vipra; Tan, Nguan Soon; KK Research Centre, KK Women's and Children Hospital, 100 Bukit Timah Road, Singapore 229899

    Mesenchymal stem cells (MSCs), which were first isolated from the bone marrow, are now being extracted from various other tissues in the body, including the adipose tissue. The current study presents systematic evidence of how the adipose tissue-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (Bm-MSCs) behave when cultured in specific pro-adipogenic microenvironments. The cells were first characterized and identified as MSCs in terms of their morphology, phenotypic expression, self-renewal capabilities and multi-lineage potential. Subsequently, the proliferation and gene expression profiles of the cell populations cultured on two-dimensional (2D) adipose tissue extracellular matrix (ECM)-coated tissue culture plastic (TCP)more » and in three-dimensional (3D) AlgiMatrix® microenvironments were analyzed. Overall, it was found that adipogenesis was triggered in both cell populations due to the presence of adipose tissue ECM. However, in 3D microenvironments, ASCs and Bm-MSCs were predisposed to the adipogenic and osteogenic lineages respectively. Overall, findings from this study will contribute to ongoing efforts in adipose tissue engineering as well as provide new insights into the role of the ECM and cues provided by the immediate microenvironment for stem cell differentiation. - Highlights: • Native adipose tissue ECM coated on 2D TCP triggers adipogenesis in both ASCs and Bm-MSCs. • A 3D microenvironment with similar stiffness to adipose tissue induces adipogenic differentiation of ASCs. • ASCs cultured in 3D alginate scaffolds exhibit predisposition to adipogenesis. • Bm-MSCs cultured in 3D alginate scaffolds exhibit predisposition to osteogenesis. • The native microenvironment of the cells affects their differentiation behaviour in vitro.« less

  9. Tumor and Endothelial Cell-Derived Microvesicles Carry Distinct CEACAMs and Influence T-Cell Behavior

    PubMed Central

    Muturi, Harrison T.; Dreesen, Janine D.; Nilewski, Elena; Jastrow, Holger; Giebel, Bernd; Ergun, Suleyman; Singer, Bernhard B.

    2013-01-01

    Normal and malignant cells release a variety of different vesicles into their extracellular environment. The most prominent vesicles are the microvesicles (MVs, 100-1 000 nm in diameter), which are shed of the plasma membrane, and the exosomes (70-120 nm in diameter), derivates of the endosomal system. MVs have been associated with intercellular communication processes and transport numerous proteins, lipids and RNAs. As essential component of immune-escape mechanisms tumor-derived MVs suppress immune responses. Additionally, tumor-derived MVs have been found to promote metastasis, tumor-stroma interactions and angiogenesis. Since members of the carcinoembryonic antigen related cell adhesion molecule (CEACAM)-family have been associated with similar processes, we studied the distribution and function of CEACAMs in MV fractions of different human epithelial tumor cells and of human and murine endothelial cells. Here we demonstrate that in association to their cell surface phenotype, MVs released from different human epithelial tumor cells contain CEACAM1, CEACAM5 and CEACAM6, while human and murine endothelial cells were positive for CEACAM1 only. Furthermore, MVs derived from CEACAM1 transfected CHO cells carried CEACAM1. In terms of their secretion kinetics, we show that MVs are permanently released in low doses, which are extensively increased upon cellular starvation stress. Although CEACAM1 did not transmit signals into MVs it served as ligand for CEACAM expressing cell types. We gained evidence that CEACAM1-positive MVs significantly increase the CD3 and CD3/CD28-induced T-cell proliferation. All together, our data demonstrate that MV-bound forms of CEACAMs play important roles in intercellular communication processes, which can modulate immune response, tumor progression, metastasis and angiogenesis. PMID:24040308

  10. Free and Conjugated Benzoic Acid in Tobacco Plants and Cell Cultures. Induced Accumulation upon Elicitation of Defense Responses and Role as Salicylic Acid Precursors1

    PubMed Central

    Chong, Julie; Pierrel, Marie-Agnès; Atanassova, Rossitza; Werck-Reichhart, Danièle; Fritig, Bernard; Saindrenan, Patrick

    2001-01-01

    Salicylic acid (SA) is a key endogenous component of local and systemic disease resistance in plants. In this study, we investigated the role of benzoic acid (BA) as precursor of SA biosynthesis in tobacco (Nicotiana tabacum cv Samsun NN) plants undergoing a hypersensitive response following infection with tobacco mosaic virus or in tobacco cell suspensions elicited with β-megaspermin, an elicitor from Phytophthora megasperma. We found a small pool of conjugated BA in healthy leaves and untreated cell suspensions of tobacco, whereas free BA levels were barely detectable. Infection of plants with tobacco mosaic virus or elicitation of cells led to a rapid de novo synthesis and accumulation of conjugated BA, whereas free BA was weakly induced. In presence of diphenylene iodonium, an inhibitor of superoxide anion formation, SA accumulation was abolished in elicited cells and much higher BA levels were concomitantly induced, mainly as a conjugated form. Furthermore, piperonylic acid, an inhibitor of cinnamate-4-hydroxylase was used as a powerful tool to redirect the metabolic flow from the main phenylpropanoid pathway into the SA biosynthetic branch. Under these conditions, in vivo labeling and radioisotope dilution experiments with [14C]trans-cinnamic acid as precursor clearly indicated that the free form of BA produced in elicited tobacco cells is not the major precursor of SA biosynthesis. The main conjugated form of BA accumulating after elicitation of tobacco cells was identified for the first time as benzoyl-glucose. Our data point to the likely role of conjugated forms of BA in SA biosynthesis. PMID:11154339

  11. Modeling precursor diffusion and reaction of atomic layer deposition in porous structures

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Keuter, Thomas, E-mail: t.keuter@fz-juelich.de; Menzler, Norbert Heribert; Mauer, Georg

    2015-01-01

    Atomic layer deposition (ALD) is a technique for depositing thin films of materials with a precise thickness control and uniformity using the self-limitation of the underlying reactions. Usually, it is difficult to predict the result of the ALD process for given external parameters, e.g., the precursor exposure time or the size of the precursor molecules. Therefore, a deeper insight into ALD by modeling the process is needed to improve process control and to achieve more economical coatings. In this paper, a detailed, microscopic approach based on the model developed by Yanguas-Gil and Elam is presented and additionally compared with themore » experiment. Precursor diffusion and second-order reaction kinetics are combined to identify the influence of the porous substrate's microstructural parameters and the influence of precursor properties on the coating. The thickness of the deposited film is calculated for different depths inside the porous structure in relation to the precursor exposure time, the precursor vapor pressure, and other parameters. Good agreement with experimental results was obtained for ALD zirconiumdioxide (ZrO{sub 2}) films using the precursors tetrakis(ethylmethylamido)zirconium and O{sub 2}. The derivation can be adjusted to describe other features of ALD processes, e.g., precursor and reactive site losses, different growth modes, pore size reduction, and surface diffusion.« less

  12. Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

    PubMed

    Zhang, Jue; Chu, Li-Fang; Hou, Zhonggang; Schwartz, Michael P; Hacker, Timothy; Vickerman, Vernella; Swanson, Scott; Leng, Ning; Nguyen, Bao Kim; Elwell, Angela; Bolin, Jennifer; Brown, Matthew E; Stewart, Ron; Burlingham, William J; Murphy, William L; Thomson, James A

    2017-07-25

    Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

  13. Potential differentiation of islet-like cells from pregnant cow-derived placental stem cells.

    PubMed

    Peng, Shao-Yu; Chou, Chien-Wen; Kuo, Yu-Hsuan; Shen, Perng-Chih; Shaw, S W Steven

    2017-06-01

    Type 1 diabetes is an autoimmune disease that destroys islet cells and results in insufficient insulin secretion by pancreatic β-cells. Islet transplantation from donors is an approach used for treating patients with diabetes; however, this therapy is difficult to implement because of the lack of donors. Nevertheless, several stem cells have the potential to differentiate from islet-like cells and enable insulin secretion for treating diabetes in animal models. For example, placenta is considered a waste material and can be harvested noninvasively during delivery without ethical or moral concerns. To date, the differentiation of islet-like cells from cow-derived placental stem cells (CPSCs) has yet to be demonstrated. The investigation of potential differentiation of islet-like cells from CPSCs was conducted by supplementation with nicotinamide, exendin-4, glucose, and poly-d-lysine and was detected through reverse transcription polymerase chain reaction, dithizone staining, and immunocytochemical methods. Our results indicated that CPSCs are established and express mesenchymal stem cell surface antigen markers, such as CD73, CD166, β-integrin, and Oct-4, but not hematopoietic stem cell surface antigen markers, such as CD45. After induction, the CPSCs successfully differentiated into islet-like cells. The CPSC-derived islet-like cells expressed islet cell development-related genes, such as insulin, glucagon, pax-4, Nkx6.1, pax-6, and Fox. Moreover, CPSC-derived islet-like cells can be stained with zinc ions, which are widely distributed in the islet cells and enable insulin secretion. Altogether, islet-like cells have the potential to be differentiated from CPSCs without gene manipulation, and can be used in diabetic animal models in the future for preclinical and drug testing trial investigations. Copyright © 2017. Published by Elsevier B.V.

  14. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

    PubMed

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-04-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

  15. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells

    PubMed Central

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-01-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

  16. Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jensen, Pia; Department of Neurosurgery, University of Bern, CH-3010 Bern; Gramsbergen, Jan-Bert

    Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in high oxygen cultures. Low oxygen during FGF2-mediated expansion resulted also in a significant increase in tyrosine hydroxylase-immunoreactivemore » (TH-ir) dopaminergic neurons as compared to high oxygen tension, but no corresponding effect was observed for dopamine release into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells.« less

  17. Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea.

    PubMed

    Chai, Renjie; Kuo, Bryan; Wang, Tian; Liaw, Eric J; Xia, Anping; Jan, Taha A; Liu, Zhiyong; Taketo, Makoto M; Oghalai, John S; Nusse, Roeland; Zuo, Jian; Cheng, Alan G

    2012-05-22

    Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5(+) cells were enriched precursors to myo7a(+) cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition, overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.

  18. Characterization of Cancer Stem-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Transformed by Tumor-Derived Extracellular Vesicles

    PubMed Central

    Yan, Ting; Mizutani, Akifumi; Chen, Ling; Takaki, Mai; Hiramoto, Yuki; Matsuda, Shuichi; Shigehiro, Tsukasa; Kasai, Tomonari; Kudoh, Takayuki; Murakami, Hiroshi; Masuda, Junko; Hendrix, Mary J. C.; Strizzi, Luigi; Salomon, David S.; Fu, Li; Seno, Masaharu

    2014-01-01

    Several studies have shown that cancer niche can perform an active role in the regulation of tumor cell maintenance and progression through extracellular vesicles-based intercellular communication. However, it has not been reported whether this vesicle-mediated communication affects the malignant transformation of normal stem cells/progenitors. We have previously reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into cancer stem cells (CSCs), indicating that normal stem cells when placed in an aberrant microenvironment can give rise to functionally active CSCs. Here, we focused on the contribution of tumor-derived extracellular vesicles (tEVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. We isolated tEVs from the conditioned medium of LLC cells, and then the differentiating miPSCs were exposed to tEVs for 4 weeks. The resultant tEV treated cells (miPS-LLCev) expressed Nanog and Oct3/4 proteins comparable to miPSCs. The frequency of sphere formation of the miPS-LLCev cells in suspension culture indicated that the self-renewal capacity of the miPS-LLCev cells was significant. When the miPS-LLCev cells were subcutaneously transplanted into Balb/c nude mice, malignant liposarcomas with extensive angiogenesis developed. miPS-LLCevPT and miPS-LLCevDT, the cells established from primary site and disseminated liposarcomas, respectively, showed their capacities to self-renew and differentiate into adipocytes and endothelial cells. Moreover, we confirmed the secondary liposarcoma development when these cells were transplanted. Taken together, these results indicate that miPS-LLCev cells possess CSC properties. Thus, our current study provides the first evidence that tEVs have the potential to induce CSC properties in normal tissue stem cells/progenitors. PMID:25057308

  19. The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson's Disease.

    PubMed

    Schöndorf, David C; Ivanyuk, Dina; Baden, Pascale; Sanchez-Martinez, Alvaro; De Cicco, Silvia; Yu, Cong; Giunta, Ivana; Schwarz, Lukas K; Di Napoli, Gabriele; Panagiotakopoulou, Vasiliki; Nestel, Sigrun; Keatinge, Marcus; Pruszak, Jan; Bandmann, Oliver; Heimrich, Bernd; Gasser, Thomas; Whitworth, Alexander J; Deleidi, Michela

    2018-06-05

    While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

    PubMed Central

    van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

    2012-01-01

    The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699