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Sample records for cell-derived embryoid bodies

  1. Profiling ethanol-targeted transcription factors in human carcinoma cell-derived embryoid bodies.

    PubMed

    Mandal, Chanchal; Halder, Debasish; Chai, Jin Choul; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2016-01-15

    Fetal alcohol spectrum disorder is a collective term that represents fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not yet understood. Few insights have been gained from genetic and epigenetic studies of fetal alcohol spectrum disorder. Our aim was to profile the important molecular regulators of ethanol-related alterations of the genome. For this purpose, we have analyzed the gene expression pattern of human carcinoma cell-derived embryoid bodies in the absence or presence of ethanol. A cDNA microarray analysis was used to profile mRNA expression in embryoid bodies at day 7 with or without ethanol treatment. A total of 493 differentially expressed genes were identified in response to 50 mM ethanol exposure. Of these, 111 genes were up-regulated, and 382 were down-regulated. Gene ontology term enrichment analysis revealed that these genes are involved in important biological processes: neurological system processes, cognition, behavior, sensory perception of smell, taste and chemical stimuli and synaptic transmission. Similarly, the enrichment of disease-related genes included relevant categories such as neurological diseases, developmental disorders, skeletal and muscular disorders, and connective tissue disorders. Furthermore, we have identified a group of 26 genes that encode transcription factors. We validated the relative gene expression of several transcription factors using quantitative real time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanisms underlying the pathology of alcohol-mediated anomalies and facilitates further research.

  2. Properties of embryoid bodies.

    PubMed

    Brickman, Joshua M; Serup, Palle

    2017-03-01

    Embryoid bodies (EBs) have been popular in vitro differentiation models for pluripotent stem cells for more than five decades. Initially, defined as aggregates formed by embryonal carcinoma cells, EBs gained more prominence after the derivation of karyotypically normal embryonic stem cells from early mouse blastocysts. In many cases, formation of EBs constitutes an important initial step in directed differentiation protocols aimed at generated specific cell types from undifferentiated stem cells. Indeed state-of-the-art protocols for directed differentiation of cardiomyocytes still rely on this initial EB step. Analyses of spontaneous differentiation of embryonic stem cells in EBs have yielded important insights into the molecules that direct primitive endoderm differentiation and many of the lessons we have learned about the signals and transcription factors governing this differentiation event is owed to EB models, which later were extensively validated in studies of early mouse embryos. EBs show a degree of self-organization that mimics some aspects of early embryonic development, but with important exceptions. Recent studies that employ modern signaling reporters and tracers of lineage commitment have revealed both the strengths and the weaknesses of EBs as a model of embryonic axis formation. In this review, we discuss the history, application, and future potential of EBs as an experimental model. WIREs Dev Biol 2017, 6:e259. doi: 10.1002/wdev.259 For further resources related to this article, please visit the WIREs website.

  3. Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

    SciTech Connect

    Gu, Angel; Tsark, Walter; Holmes, Kathryn V.; Shively, John E.

    2009-06-10

    CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (- 8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as - 5 to - 3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased

  4. Graphene induces spontaneous cardiac differentiation in embryoid bodies

    NASA Astrophysics Data System (ADS)

    Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

    2016-03-01

    Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac

  5. Neural induced embryoid bodies present high levels of metals detected by x-ray microfluorescence

    NASA Astrophysics Data System (ADS)

    Stelling, Mariana P.; Cardoso, Simone C.; Paulsen, Bruna S.; Rehen, Stevens K.

    2012-05-01

    Molecular mechanisms driving neural differentiation in human embryonic stem cells are not completely elucidated, specially, the role of atomic elements within this process. In this work, we described the distribution of trace elements in those stem cells growing as embryoid bodies by using synchrotron radiation X-ray microfluorescence (SR-XRF). Naive and neural induced embryoid bodies derived from embryonic stem cells were irradiated with a spatial resolution of 20 μm to make elemental maps and qualitative chemical analyses. We consistently detected metallic elements content raise on neural induced embryoid bodies, mimicking characteristic brain development. The use of SR-XRF reveals that human embryoid bodies exhibit self-organization at the atomic level, which is enhanced during neurogenesis triggered in vitro.

  6. Neural induced embryoid bodies present high levels of metals detected by x-ray microfluorescence

    SciTech Connect

    Stelling, Mariana P.; Cardoso, Simone C.; Paulsen, Bruna S.; Rehen, Stevens K.

    2012-05-17

    Molecular mechanisms driving neural differentiation in human embryonic stem cells are not completely elucidated, specially, the role of atomic elements within this process. In this work, we described the distribution of trace elements in those stem cells growing as embryoid bodies by using synchrotron radiation X-ray microfluorescence (SR-XRF). Naive and neural induced embryoid bodies derived from embryonic stem cells were irradiated with a spatial resolution of 20 {mu}m to make elemental maps and qualitative chemical analyses. We consistently detected metallic elements content raise on neural induced embryoid bodies, mimicking characteristic brain development. The use of SR-XRF reveals that human embryoid bodies exhibit self-organization at the atomic level, which is enhanced during neurogenesis triggered in vitro.

  7. Matrix Rigidity-Modulated Cardiovascular Organoid Formation from Embryoid Bodies

    PubMed Central

    Shkumatov, Artem; Baek, Kwanghyun; Kong, Hyunjoon

    2014-01-01

    Stem cell clusters, such as embryoid bodies (EBs) derived from embryonic stem cells, are extensively studied for creation of multicellular clusters and complex functional tissues. It is common to control phenotypes of ES cells with varying molecular compounds; however, there is still a need to improve the controllability of cell differentiation, and thus, the quality of created tissue. This study demonstrates a simple but effective strategy to promote formation of vascularized cardiac muscle - like tissue in EBs and form contracting cardiovascular organoids by modulating the stiffness of a cell adherent hydrogel. Using collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we discovered that cellular organization in a form of vascularized cardiac muscle sheet was maximal on the gel with the stiffness similar to cardiac muscle. We envisage that the results of this study will greatly contribute to better understanding of emergent behavior of stem cells in developmental and regeneration process and will also expedite translation of EB studies to drug-screening device assembly and clinical treatments. PMID:24732893

  8. Graphene induces spontaneous cardiac differentiation in embryoid bodies.

    PubMed

    Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

    2016-04-07

    Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.

  9. Functional maintenance of differentiated embryoid bodies in microfluidic systems: a platform for personalized medicine.

    PubMed

    Guven, Sinan; Lindsey, Jennifer S; Poudel, Ishwari; Chinthala, Sireesha; Nickerson, Michael D; Gerami-Naini, Behzad; Gurkan, Umut A; Anchan, Raymond M; Demirci, Utkan

    2015-03-01

    Hormone replacement therapies have become important for treating diseases such as premature ovarian failure or menopausal complications. The clinical use of bioidentical hormones might significantly reduce some of the potential risks reportedly associated with the use of synthetic hormones. In the present study, we demonstrate the utility and advantage of a microfluidic chip culture system to enhance the development of personalized, on-demand, treatment modules using embryoid bodies (EBs). Functional EBs cultured on microfluidic chips represent a platform for personalized, patient-specific treatment cassettes that can be cryopreserved until required for treatment. We assessed the viability, differentiation, and functionality of EBs cultured and cryopreserved in this system. During extended microfluidic culture, estradiol, progesterone, testosterone, and anti-müllerian hormone levels were measured, and the expression of differentiated steroidogenic cells was confirmed by immunocytochemistry assay for the ovarian tissue markers anti-müllerian hormone receptor type II, follicle-stimulating hormone receptor, and inhibin β-A and the estrogen biosynthesis enzyme aromatase. Our studies showed that under microfluidic conditions, differentiated steroidogenic EBs continued to secrete estradiol and progesterone at physiologically relevant concentrations (30-120 pg/ml and 150-450 pg/ml, respectively) for up to 21 days. Collectively, we have demonstrated for the first time the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived EBs, the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality on thawing. These results present a platform for the development of a new therapeutic system for personalized medicine.

  10. Modelling human embryoid body cell adhesion to a combinatorial library of polymer surfaces.

    PubMed

    Epa, V Chandana; Yang, Jing; Mei, Ying; Hook, Andrew L; Langer, Robert; Anderson, Daniel G; Davies, Martyn C; Alexander, Morgan R; Winkler, David A

    2012-09-18

    Designing materials to control biology is an intense focus of biomaterials and regenerative medicine research. Discovering and designing materials with appropriate biological compatibility or active control of cells and tissues is being increasingly undertaken using high throughput synthesis and assessment methods. We report a relatively simple but powerful machine-learning method of generating models that link microscopic or molecular properties of polymers or other materials to their biological effects. We illustrate the potential of these methods by developing the first robust, predictive, quantitative, and purely computational models of adhesion of human embryonic stem cell embryoid bodies (hEB) to the surfaces of a 496-member polymer micro array library.

  11. Modelling human embryoid body cell adhesion to a combinatorial library of polymer surfaces

    PubMed Central

    Epa, V. Chandana; Yang, Jing; Mei, Ying; Hook, Andrew L.; Langer, Robert; Anderson, Daniel G.; Davies, Martyn C.; Alexander, Morgan R.; Winkler, David A.

    2013-01-01

    Designing materials to control biology is an intense focus of biomaterials and regenerative medicine research. Discovering and designing materials with appropriate biological compatibility or active control of cells and tissues is being increasingly undertaken using high throughput synthesis and assessment methods. We report a relatively simple but powerful machine-learning method of generating models that link microscopic or molecular properties of polymers or other materials to their biological effects. We illustrate the potential of these methods by developing the first robust, predictive, quantitative, and purely computational models of adhesion of human embryonic stem cell embryoid bodies (hEB) to the surfaces of a 496-member polymer micro array library. PMID:24092955

  12. Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

    PubMed Central

    Pettinato, Giuseppe

    2015-01-01

    Human pluripotent stem cells (hPSCs) are powerful tools for regenerative therapy and studying human developmental biology, attributing to their ability to differentiate into many functional cell types in the body. The main challenge in realizing hPSC potential is to guide their differentiation in a well-controlled manner. One way to control the cell differentiation process is to recapitulate during in vitro culture the key events in embryogenesis to obtain the three developmental germ layers from which all cell types arise. To achieve this goal, many techniques have been tested to obtain a cellular cluster, an embryoid body (EB), from both mouse and hPSCs. Generation of EBs that are homogeneous in size and shape would allow directed hPSC differentiation into desired cell types in a more synchronous manner and define the roles of cell–cell interaction and spatial organization in lineage specification in a setting similar to in vivo embryonic development. However, previous success in uniform EB formation from mouse PSCs cannot be extrapolated to hPSCs possibly due to the destabilization of adherens junctions on cell surfaces during the dissociation into single cells, making hPSCs extremely vulnerable to cell death. Recently, new advances have emerged to form uniform human embryoid bodies (hEBs) from dissociated single cells of hPSCs. In this review, the existing methods for hEB production from hPSCs and the results on the downstream differentiation of the hEBs are described with emphases on the efficiency, homogeneity, scalability, and reproducibility of the hEB formation process and the yield in terminal differentiation. New trends in hEB production and directed differentiation are discussed. PMID:25900308

  13. Embryoid Body-Explant Outgrowth Cultivation from Induced Pluripotent Stem Cells in an Automated Closed Platform

    PubMed Central

    Tone, Hiroshi; Yoshioka, Saeko; Akiyama, Hirokazu; Nishimura, Akira; Ichimura, Masaki; Nakatani, Masaru; Kiyono, Tohru

    2016-01-01

    Automation of cell culture would facilitate stable cell expansion with consistent quality. In the present study, feasibility of an automated closed-cell culture system “P 4C S” for an embryoid body- (EB-) explant outgrowth culture was investigated as a model case for explant culture. After placing the induced pluripotent stem cell- (iPSC-) derived EBs into the system, the EBs successfully adhered to the culture surface and the cell outgrowth was clearly observed surrounding the adherent EBs. After confirming the outgrowth, we carried out subculture manipulation, in which the detached cells were simply dispersed by shaking the culture flask, leading to uniform cell distribution. This enabled continuous stable cell expansion, resulting in a cell yield of 3.1 × 107. There was no evidence of bacterial contamination throughout the cell culture experiments. We herewith developed the automated cultivation platform for EB-explant outgrowth cells. PMID:27648449

  14. Phenotype characterization of embryoid body structures generated by a crystal comet effect tail in an intercellular cancer collision scenario

    PubMed Central

    Diaz, Jairo A; Murillo, Mauricio F

    2012-01-01

    Cancer is, by definition, the uncontrolled growth of autonomous cells that eventually destroy adjacent tissues and generate architectural disorder. However, this concept cannot be totally true. In three well documented studies, we have demonstrated that cancer tissues produce order zones that evolve over time and generate embryoid body structures in a space-time interval. The authors decided to revise the macroscopic and microscopic material in well-developed malignant tumors in which embryoid bodies were identified to determine the phenotype characterization that serves as a guideline for easy recognition. The factors responsible for this morphogenesis are physical, bioelectric, and magnetic susceptibilities produced by crystals that act as molecular designers for the topographic gradients that guide the surrounding silhouette and establish tissue head-tail positional identities. The structures are located in amniotic-like cavities and show characteristic somite-like embryologic segmentation. Immunophenotypic study has demonstrated exclusion factor positional identity in relation to enolase-immunopositive expression of embryoid body and human chorionic gonadotropin immunopositivity exclusion factor expression in the surrounding tissues. The significance of these observations is that they can also be predicted by experimental image data collected by the Large Hadron Collider (LHC) accelerator at the European Organization for Nuclear Research, in which two-beam subatomic collision particles in the resulting debris show hyperorder domains similar to those identified by us in intercellular cancer collisions. Our findings suggest that we are dealing with true reverse biologic system information in an activated collective cancer stem cell memory, in which physics participates in the elaboration of geometric complexes and chiral biomolecules that serve to build bodies with embryoid print as it develops during gestation. Reversal mechanisms in biology are intimately

  15. Cell death activation during cavitation of embryoid bodies is mediated by hydrogen peroxide.

    PubMed

    Hernández-García, David; Castro-Obregón, Susana; Gómez-López, Sandra; Valencia, Concepción; Covarrubias, Luis

    2008-06-10

    The formation of the proamniotic cavity is the first indication of programmed cell death associated to a morphogenetic process in mammals. Although some growth factors have been implicated in proamniotic cavitation, very little is known about the intracellular mechanisms that control the cell death process itself. Reactive oxygen species (ROS) are potent activators of cell death, thus, in the present work we evaluated the role of ROS during the cavitation of embryoid bodies (EBs), a common model to study proamniotic cavitation. During cavitation, ROS concentration increases in the inner cells of EBs, and this ROS accumulation appears to be associated with the mitochondrial respiratory activity. In agreement with a role of ROS in cavitation, EBs derived from ES cells that overproduce catalase, an enzyme that specifically degrades hydrogen peroxide, do not cavitate, and caspase activation and cell death is markedly decreased. Notably, cell death, but not the rise in ROS, during EB cavitation is caspase-dependent. The apoptosis-inducing factor (Aif) is released from the mitochondria during cavitation, but EBs derived from Aif(-/y) ES cells cavitate and ROS levels in the inner cells remain high. We conclude that hydrogen peroxide is a cell death activating signal essential for EB cavitation, suggesting that cell death during proamniotic cavitation is mediated by ROS.

  16. Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation

    PubMed Central

    Boxman, Jonathan; Sagy, Naor; Achanta, Sirisha; Vadigepalli, Rajanikanth; Nachman, Iftach

    2016-01-01

    Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live two-photon imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a ‘developmental clock’ using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model. PMID:27530599

  17. Tunable shrink-induced honeycomb microwell arrays for uniform embryoid bodies.

    PubMed

    Nguyen, Diep; Sa, Silin; Pegan, Jonathan D; Rich, Brent; Xiang, Guangxin; McCloskey, Kara E; Manilay, Jennifer O; Khine, Michelle

    2009-12-07

    Embryoid body (EB) formation closely recapitulates early embryonic development with respect to lineage commitment. Because it is greatly affected by cell-cell and cell-substrate interactions, the ability to control the initial number of cells in the aggregates and to provide an appropriate substrate are crucial parameters for uniform EB formation. Here we report of an ultra-rapid fabrication and culture method utilizing a laser-jet printer to generate closely arrayed honeycomb microwells of tunable sizes for the induction of uniform EBs from single cell suspension. By printing various microwell patterns onto pre-stressed polystyrene sheets, and through heat induced shrinking, high aspect micromolds are generated. Notably, we achieve rounded bottom polydimethylsiloxane (PDMS) wells not easily achievable with standard microfabrication methods, but critical to achieve spherical EBs. Furthermore, by simply controlling the size of the microwells and the concentration of the cell suspension we can control the initial size of the cell aggregate, thus influencing lineage commitment. In addition, these microwells are easily adaptable and scalable to most standard well plates and easily integrated into commercial liquid handling systems to provide an inexpensive and easy high throughput compound screening platform.

  18. A standardized human embryoid body platform for the detection and analysis of teratogens

    PubMed Central

    Flamier, Anthony; Singh, Supriya; Rasmussen, Theodore P.

    2017-01-01

    Teratogens are compounds that can induce birth defects upon exposure of the developing fetus. To date, most teratogen studies utilize pregnant rodents to determine compound teratogenicity in vivo. However, this is a low throughput approach that cannot easily meet the need for comprehensive high-volume teratogen assessment, a goal of the US Environmental Protection Agency. In addition, rodent and human development differ substantially, and therefore the use of assays using relevant human cells has utility. For these reasons, interest has recently focused on the use of human embryonic stem cells for teratogen assessment. Here we present a highly standardized and quantitative system for the detection and analysis of teratogens that utilizes well-characterized and purified highly pluripotent stem cells. We have devised strategies to mass-produce thousands of uniformly sized spheroids of human ESCs (hESCs) that can be caused to undergo synchronous differentiation to yield embryoid bodies (EBs) in the presence and absence of suspected teratogens. The system uses all human cells and rigorously controlled and standardized EB culture conditions. Furthermore, the approach has been made quantitative by using high-content imaging approaches. Our system offers distinct advantages over earlier EB systems that rely heavily on the use on mouse ESCs and EB aggregates of stochastic sizes. Together, our results show that thousands of suspected teratogens could be assessed using human EB-based approaches. PMID:28182681

  19. Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation.

    PubMed

    Liu, Yarong; Fox, Victoria; Lei, Yuning; Hu, Biliang; Joo, Kye-Il; Wang, Pin

    2014-07-01

    The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications.

  20. Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies.

    PubMed

    Moon, Sung-Hwan; Ju, Jongil; Park, Soon-Jung; Bae, Daekyeong; Chung, Hyung-Min; Lee, Sang-Hoon

    2014-07-01

    Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.

  1. Establishment of cell polarity by afadin during the formation of embryoid bodies.

    PubMed

    Komura, Hitomi; Ogita, Hisakazu; Ikeda, Wataru; Mizoguchi, Akira; Miyoshi, Jun; Takai, Yoshimi

    2008-01-01

    Afadin directly links nectin, an immunoglobulin-like cell-cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin-afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell-cell junctions. However, the molecular mechanism of cell-cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell-cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin alpha(6) and integrin beta(1). These results indicate that afadin organizes the formation of cell-cell junctions by regulating cell polarization in early embryonic development.

  2. Cell adhesion and sorting in embryoid bodies derived from N- or E-cadherin deficient murine embryonic stem cells

    PubMed Central

    Moore, Robert; Tao, Wensi; Meng, Yue; Smith, Elizabeth R.; Xu, Xiang-Xi

    2014-01-01

    Summary The primitive endoderm epithelial structure in mouse blastocysts forms following cell differentiation and subsequent sorting, and this two-step process can be reproduced in vitro using an embryoid body model. We found that in the chimeric embryoid bodies consisting of paired wildtype and E-cadherin null ES cells, the wildtype sorted to the center and were enveloped by the less adhesive E-cadherin null cells, in accord with Steinberg's hypothesis. However, wildtype and N-cadherin null ES cells intermixed and did not segregate, a situation that may be explained by Albert Harris' modified principle, which incorporates the unique properties of living cells. Furthermore, in chimeric embryoid bodies composed of N-cadherin and E-cadherin null ES cells, the two weakly interacting cell types segregated but did not envelop one another. Lastly, the most consistent and striking observation was that differentiated cells sorted to the surface and formed an enveloping layer, regardless of the relative cell adhesive affinity of any cell combination, supporting the hypothesis that the ability of the differentiated cells to establish apical polarity is the determining factor in surface sorting and positioning. PMID:24414205

  3. Cell adhesion and sorting in embryoid bodies derived from N- or E-cadherin deficient murine embryonic stem cells.

    PubMed

    Moore, Robert; Tao, Wensi; Meng, Yue; Smith, Elizabeth R; Xu, Xiang-Xi

    2014-02-15

    The primitive endoderm epithelial structure in mouse blastocysts forms following cell differentiation and subsequent sorting, and this two-step process can be reproduced in vitro using an embryoid body model. We found that in the chimeric embryoid bodies consisting of paired wildtype and E-cadherin null ES cells, the wildtype sorted to the center and were enveloped by the less adhesive E-cadherin null cells, in accord with Steinberg's hypothesis. However, wildtype and N-cadherin null ES cells intermixed and did not segregate, a situation that may be explained by Albert Harris' modified principle, which incorporates the unique properties of living cells. Furthermore, in chimeric embryoid bodies composed of N-cadherin and E-cadherin null ES cells, the two weakly interacting cell types segregated but did not envelop one another. Lastly, the most consistent and striking observation was that differentiated cells sorted to the surface and formed an enveloping layer, regardless of the relative cell adhesive affinity of any cell combination, supporting the hypothesis that the ability of the differentiated cells to establish apical polarity is the determining factor in surface sorting and positioning.

  4. Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

    SciTech Connect

    Taru Sharma, G.; Dubey, Pawan K.; Verma, Om Prakash; Pratheesh, M.D.; Nath, Amar; Sai Kumar, G.

    2012-08-03

    Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it

  5. Synthesis and Organization of Hyaluronan and Versican by Embryonic Stem Cells Undergoing Embryoid Body Differentiation

    PubMed Central

    Shukla, Shreya; Nair, Rekha; Rolle, Marsha W.; Braun, Kathleen R.; Chan, Christina K.; Johnson, Pamela Y.; Wight, Thomas N.; McDevitt, Todd C.

    2010-01-01

    Embryonic stem cells (ESCs) provide a convenient model to probe the molecular and cellular dynamics of developmental cell morphogenesis. ESC differentiation in vitro via embryoid bodies (EBs) recapitulates many aspects of early stages of development, including the epithelial–mesenchymal transition (EMT) of pluripotent cells into more differentiated progeny. Hyaluronan and versican are important extracellular mediators of EMT processes, yet the temporal expression and spatial distribution of these extracellular matrix (ECM) molecules during EB differentiation remains undefined. Thus, the objective of this study was to evaluate the synthesis and organization of hyaluronan and versican by using murine ESCs during EB differentiation. Hyaluronan and versican (V0 and V1 isoforms), visualized by immunohistochemistry and evaluated biochemically, accumulated within EBs during the course of differentiation. Interestingly, increasing amounts of a 70-kDa proteolytic fragment of versican were also detected over time, along with ADAMTS-1 and -5 protein expression. ESCs expressed each of the hyaluronan synthases (HAS) -1, -2, and -3 and versican splice variants (V0, V1, V2, and V3) throughout EB differentiation, but HAS-2, V0, and V1 were expressed at significantly increased levels at each time point examined. Hyaluronan and versican exhibited overlapping expression patterns within EBs in regions of low cell density, and versican expression was excluded from clusters of epithelial (cytokeratin-positive) cells but was enriched within the vicinity of mesenchymal (N-cadherin-positive) cells. These results indicate that hyaluronan and versican synthesized by ESCs within EB microenvironments are associated with EMT processes and furthermore suggest that endogenously produced ECM molecules play a role in ESC differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58

  6. Dual gene expression in embryoid bodies derived from human induced pluripotent stem cells using episomal vectors.

    PubMed

    Tomizawa, Minoru; Shinozaki, Fuminobu; Motoyoshi, Yasufumi; Sugiyama, Takao; Yamamoto, Shigenori; Sueishi, Makoto

    2014-12-01

    Transcription factors are essential for the differentiation of human induced pluripotent stem cells (iPS) into specialized cell types. Embryoid body (EB) formation promotes the differentiation of iPS cells. We sought to establish an efficient method of transfection and rotary culture to generate EBs that stably express two genes. The pMetLuc2-Reporter vector was transfected using FuGENE HD (FuGENE), Lipofectamine LTX (LTX), X-tremeGENE, or TransIT-2020 transfection reagents. The media was analyzed using a Metridia luciferase (MetLuc) assay. Transfections were performed on cells adherent to plates/dishes (adherent method) or suspended in the media (suspension method). The 201B7 cells transfected with episomal vectors were selected using G418 (200 μg/mL) or hygromycin B (300 μg/mL). Rotary culture was performed at 2.5 or 9.9 rpm. Efficiency of EB formation was compared among plates and dishes. Cell density was compared at 1.6×10(3),×10(4), and×10(5) cells/mL. The suspended method of transfection using the FuGENE HD reagent was the most efficient. The expression of pEBMulti/Met-Hyg was detected 11 days posttransfection. Double transformants were selected 6 days posttransfection with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg. Both EGFP and CherryPicker were expressed in all of the surviving cells. EBs were formed most efficiently from cells cultured at a density of 1.6×10(5) cells/mL in six-well plates or 6 cm dishes. The selected cells formed EBs. FuGENE-mediated transfection of plasmids using the suspension method was effective in transforming iPS cells. Furthermore, the episomal vectors enabled us to perform a stable double transfection of EB-forming iPS cells.

  7. Characterization of a subpopulation of developing cortical interneurons from human iPSCs within serum-free embryoid bodies

    PubMed Central

    Jacob, Samson; Sun, Bruce; Prè, Deborah; Sproul, Andrew A.; Hong, Seong Im; Woodard, Chris; Zimmer, Matthew; Chinchalongporn, Vorapin; Arancio, Ottavio; Noggle, Scott A.

    2014-01-01

    Production and isolation of forebrain interneuron progenitors are essential for understanding cortical development and developing cell-based therapies for developmental and neurodegenerative disorders. We demonstrate production of a population of putative calretinin-positive bipolar interneurons that express markers consistent with caudal ganglionic eminence identities. Using serum-free embryoid bodies (SFEBs) generated from human inducible pluripotent stem cells (iPSCs), we demonstrate that these interneuron progenitors exhibit morphological, immunocytochemical, and electrophysiological hallmarks of developing cortical interneurons. Finally, we develop a fluorescence-activated cell-sorting strategy to isolate interneuron progenitors from SFEBs to allow development of a purified population of these cells. Identification of this critical neuronal cell type within iPSC-derived SFEBs is an important and novel step in describing cortical development in this iPSC preparation. PMID:25394470

  8. A local redox cycling-based electrochemical chip device with nanocavities for multi-electrochemical evaluation of embryoid bodies.

    PubMed

    Kanno, Yusuke; Ino, Kosuke; Shiku, Hitoshi; Matsue, Tomokazu

    2015-12-07

    An electrochemical device, which consists of electrode arrays, nanocavities, and microwells, was developed for multi-electrochemical detection with high sensitivity. A local redox cycling-based electrochemical (LRC-EC) system was used for multi-electrochemical detection and signal amplification. The LRC-EC system consists of n(2) sensors with only 2n bonding pads for external connection. The nanocavities fabricated in the sensor microwells enable significant improvement of the signal amplification compared with the previous devices we have developed. The present device was successfully applied for evaluation of embryoid bodies (EBs) from embryonic stem (ES) cells via electrochemical measurements of alkaline phosphatase (ALP) activity in the EBs. In addition, the EBs were successfully trapped in the sensor microwells of the device using dielectrophoresis (DEP) manipulation, which led to high-throughput cell analysis. This device is considered to be useful for multi-electrochemical detection and imaging for bioassays including cell analysis.

  9. Morphological observation of embryoid bodies completes the in vitro evaluation of nanomaterial embryotoxicity in the embryonic stem cell test (EST).

    PubMed

    Corradi, Sara; Dakou, Eleni; Yadav, Ajay; Thomassen, Leen C J; Kirsch-Volders, Micheline; Leyns, Luc

    2015-10-01

    The wide and frequent use of engineered nanomaterials (NMs) raises serious concerns about their safety for human health. Our aim is to evaluate the embryotoxic potential of silver, uncoated and coated zinc oxide, titanium dioxide and silica NMs through the embryonic stem cell test (EST). EST is a validated in vitro assay that permits classification of chemicals into three classes (non, weakly or strongly embryotoxic). Because of the peculiar physico-chemical characteristics of NMs, we first adapted and simplified the differentiation protocol. To verify the efficiency of this adapted protocol we screened 3 well-characterized chemicals (5-fluorouracil, hydroxyurea and saccharin). Next, we assessed the embryotoxic potential of NMs. Our data showed that silver NM is classified as a strong embryotoxic compound, while coated and uncoated zinc oxide, titanium and silica NMs as weak embryotoxic compounds. In addition, we observed daily the formation and growth of embryoid bodies (EBs). We showed that multiple EBs formed in each well starting from 50 μg/ml of SiO2 while EB formation was inhibited starting from 20 μg/ml of ZnO NMs. This has never been reported with chemicals and could pose a risk of wrongly evaluating the NMs embryotoxic potential. For NMs, morphological observation of EBs can provide valuable information on early differentiation effects. Finally, we suggest that the prediction model should be revised for the assessment of NMs embryotoxicity.

  10. Positioning of the mouse Hox gene clusters in the nuclei of developing embryos and differentiating embryoid bodies

    SciTech Connect

    Lanctot, Christian . E-mail: christian.lanctot@lrz.uni-muenchen.de; Kaspar, Cornelius; Cremer, Thomas

    2007-04-15

    Expression of Hox genes located on different chromosomes is precisely regulated and synchronized during development. In order to test the hypothesis that the Hox loci might cluster in nuclear space in order to share regulatory components, we performed 3D FISH on cryosections of developing mouse embryos and differentiating embryoid bodies. We did not observe any instances of co-localization of 4 different Hox alleles. Instances of 2 different alleles touching each other were found in 20-47% of nuclei depending on the tissue. The frequency of such 'kissing' events was not significantly different in cells expressing a high proportion of the Hox clusters when compared to cells expressing none or only a few Hox genes. We found that the HoxB and HoxC clusters, which are located in gene-rich regions, were involved more frequently in gene kissing in embryonic nuclei. In the case of HoxB, this observation correlated with the positioning of the corresponding chromosome towards the interior of the nucleus. Our results indicate that co-regulation of the different Hox clusters is not associated with co-localization of the loci at a single regulatory compartment and that the chromosomal context may influence the extent to which they contact each other in the nucleus.

  11. Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.

    PubMed

    Francis, Sheila E; Goh, Keow Lin; Hodivala-Dilke, Kairbaan; Bader, Bernhard L; Stark, Margaret; Davidson, Duncan; Hynes, Richard O

    2002-06-01

    Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the alpha5 integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on alpha(v) or beta3 integrin subunits. In mouse embryos lacking alpha5 integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in alpha5-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for alpha5 integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking alpha5 integrin or FN compared with wild-type EBs or EBs lacking alpha(v) or beta3 integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for alpha5 and FN in early blood vessel development not dependent on embryo nutrition or alpha(v) or beta3 integrin subunits. Thus, successful early vasculogenesis and angiogenesis require alpha5-FN interactions.

  12. Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development.

    PubMed

    Takei, Shunsuke; Ichikawa, Hinako; Johkura, Kohei; Mogi, Akimi; No, Heesung; Yoshie, Susumu; Tomotsune, Daihachiro; Sasaki, Katsunori

    2009-06-01

    Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5-25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.

  13. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori.

    PubMed

    Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G

    2013-01-01

    In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+ (pe) females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+ (pe) ) of the mother. Because the polar body nuclei had + (pe) genes in the white eggs laid by a pe/+ (pe) female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos.

  14. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

    PubMed Central

    Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G.

    2013-01-01

    In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+pe) of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/+pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos. PMID:24027530

  15. Transcript profiling and lipidomic analysis of ceramide subspecies in mouse embryonic stem cells and embryoid bodies.

    PubMed

    Park, Hyejung; Haynes, Christopher A; Nairn, Alison V; Kulik, Michael; Dalton, Stephen; Moremen, Kelley; Merrill, Alfred H

    2010-03-01

    Ceramides (Cers) are important in embryogenesis, but no comprehensive analysis of gene expression for Cer metabolism nor the Cer amounts and subspecies has been conducted with an often used model: mouse embryonic stem cells (mESCs) versus embroid bodies (EBs). Measuring the mRNA levels by quantitative RT-PCR and the amounts of the respective metabolites by LC-ESI/MS/MS, notable differences between R1 mESCs and EBs were: EBs have higher mRNAs for CerS1 and CerS3, which synthesize C18- and C>or=24-carbons dihydroceramides (DH)Cer, respectively; EBs have higher CerS2 (for C24:0- and C24:1-); and EBs have lower CerS5 + CerS6 (for C16-). In agreement with these findings, EBs have (DH)Cer with higher proportions of C18-, C24- and C26- and less C16-fatty acids, and longer (DH)Cer are also seen in monohexosyl Cers and sphingomyelins. EBs had higher mRNAs for fatty acyl-CoA elongases that produce C18-, C24-, and C26-fatty acyl-CoAs (Elovl3 and Elovl6), and higher amounts of these cosubstrates for CerS. Thus, these studies have found generally good agreement between genomic and metabolomic data in defining that conversion of mESCs to EBs is accompanied by a large number of changes in gene expression and subspecies distributions for both sphingolipids and fatty acyl-CoAs.

  16. Development of a multi-layer microfluidic array chip to culture and replate uniform-sized embryoid bodies without manual cell retrieval.

    PubMed

    Kang, Edward; Choi, Yoon Young; Jun, Yesl; Chung, Bong Geun; Lee, Sang-Hoon

    2010-10-21

    We have developed a multi-layer, microfluidic array platform containing concave microwells and flat cell culture chambers to culture embryonic stem (ES) cells and regulate uniform-sized embryoid body (EB) formation. The main advantage of this platform was that EBs cultured within the concave microwells of a bottom layer were automatically replated into flat cell culture chambers of a top layer, following inversion of the multi-layer microfluidic array platform. This allowed EB formation and EB replating to be controlled simultaneously inside a single microfluidic device without pipette-based manual cell retrieval, a drawback of previous EB culture methods.

  17. Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays.

    PubMed

    Yang, Jing; Mei, Ying; Hook, Andrew L; Taylor, Michael; Urquhart, Andrew J; Bogatyrev, Said R; Langer, Robert; Anderson, Daniel G; Davies, Martyn C; Alexander, Morgan R

    2010-12-01

    High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterization (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates

  18. Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

    NASA Astrophysics Data System (ADS)

    Shinde, Vaibhav; Brungs, Sonja; Hescheler, Jürgen; Hemmersbach, Ruth; Sachinidis, Agapios

    2016-06-01

    The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.

  19. Establishment and characterization of lymphoid and myeloid mixed-cell populations from mouse late embryoid bodies, "embryonic-stem-cell fetuses".

    PubMed Central

    Chen, U; Kosco, M; Staerz, U

    1992-01-01

    Mouse embryonic stem (ES) cells have the potential to differentiate into embryoid bodies in vitro and mimic normal embryonic development. The "ES fetus" is a specific development at a late stage seen under our culture conditions. We have established several mixed populations from ES fetuses by using combinations of retroviruses carrying different oncogenes (v-abl, v-raf, c-myc), interleukins 2 and 3, and Con A. Six groups of mixed populations were characterized by immunophenotyping. For some groups, transfer of cells into sublethally irradiated mice resulted in the development of macrophages, mature T and B lymphocytes, and plasma cells of donor origin. Thus, these mixed populations may contain immortalized precursors of hematopoietic lineages. These mixed populations should be valuable for defining hematopoietic stem cells and their committed progenitors. Images PMID:1557357

  20. Development of cystic embryoid bodies with visceral yolk-sac-like structures from mouse embryonic stem cells using low-adherence 96-well plate.

    PubMed

    Yasuda, Emiko; Seki, Yuji; Higuchi, Takatoshi; Nakashima, Fumio; Noda, Tomozumi; Kurosawa, Hiroshi

    2009-04-01

    Cystic embryoid bodies with visceral yolk-sac-like structure (cystic EB-Vs) are used as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives. In this study, we optimized the cell density of embryonic stem (ES) cells for developing cystic EB-Vs in a low-adherence 96-well plate. When ES cells were seeded at a density of 4000 cells/well, the cystic EB-Vs were most efficiently developed from ES cells via forming multicellular spherical aggregates called embryoid bodies (EBs). The suspension culture in the low-adherence plate was preferable for developing EBs into cystic EB-Vs rather than the attachment culture in the plate coated with 0.1% gelatin. The seeding cell density of 4000 cells/well was always superior to 1000 cells/well in the efficiency of cystic EB-V development. Because the high-cell density culture generally raises the limitation of oxygen and nutrient supplies, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. It was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. It was suggested that a low-nutrient medium is preferred for developing cystic EB-Vs rather than a sufficient-nutrient medium. In conclusion, the suspension culture in the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. The low-nutrient condition may be one of the factors for promoting the development of cystic EB-Vs.

  1. TGF-β1 induces the formation of vascular-like structures in embryoid bodies derived from human embryonic stem cells.

    PubMed

    Wang, Yan; Qian, DE-Jian; Zhong, Wen-Yu; Lu, Jun-Hong; Guo, Xiang-Kai; Cao, Yi-Lin; Liu, Ju

    2014-07-01

    Human embryonic stem cells (ESCs) can differentiate into endothelial cells in response to stimuli from extracellular cytokines. Transforming growth factor (TGF)-β1 signaling is involved in stem cell renewal and vascular development. Previously, human ESCs were isolated from inner cell mass and a stable ESC line was developed. In the present study, the effects of extracellular TGF-β1 were investigated on human ESC-derived embryoid bodies (EB) in suspension. The structures of the EBs were analyzed with light and electron microscopy, while the cellular composition of the EBs was examined via the expression levels of specific markers. Vascular-like tubular structures and cardiomyocyte-like beating cells were observed in the EBs at day 3 and 8, respectively. The frequencies of vascular-like structures and beating cells in the TGF-β1 treated group were significantly higher compared with the control group (84.31 vs. 12.77%; P<0.001; 37.25 vs. 8.51%; P<0.001, respectively). Electron microscopy revealed the presence of lumens and gap junctions in the sections of the tubular structures. Semiquantitative polymerase chain reaction revealed elevated expression levels of CD31 and fetal liver kinase-1 in EBs cultured with TGF-β1. In addition, extensive staining of von Willebrand factor was observed in the vascular-like structures of TGF-β1-treated EBs. Therefore, the results of the present study may aid the understanding of the underlying mechanisms of human ESC differentiation and improve the methods of propagating specific cell types for the clinical therapy of cardiovascular diseases.

  2. Remyelination Is Correlated with Regulatory T Cell Induction Following Human Embryoid Body-Derived Neural Precursor Cell Transplantation in a Viral Model of Multiple Sclerosis

    PubMed Central

    Plaisted, Warren C.; Zavala, Angel; Hingco, Edna; Tran, Ha; Coleman, Ronald; Lane, Thomas E.; Loring, Jeanne F.; Walsh, Craig M.

    2016-01-01

    We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement. PMID:27310015

  3. Transcript profiling and lipidomic analysis of ceramide subspecies in mouse embryonic stem cells and embryoid bodies[S

    PubMed Central

    Park, Hyejung; Haynes, Christopher A.; Nairn, Alison V.; Kulik, Michael; Dalton, Stephen; Moremen, Kelley; Merrill, Alfred H.

    2010-01-01

    Ceramides (Cers) are important in embryogenesis, but no comprehensive analysis of gene expression for Cer metabolism nor the Cer amounts and subspecies has been conducted with an often used model: mouse embryonic stem cells (mESCs) versus embroid bodies (EBs). Measuring the mRNA levels by quantitative RT-PCR and the amounts of the respective metabolites by LC-ESI/MS/MS, notable differences between R1 mESCs and EBs were: EBs have higher mRNAs for CerS1 and CerS3, which synthesize C18- and C≥24-carbons dihydroceramides (DH)Cer, respectively; EBs have higher CerS2 (for C24:0- and C24:1-); and EBs have lower CerS5 + CerS6 (for C16-). In agreement with these findings, EBs have (DH)Cer with higher proportions of C18-, C24- and C26- and less C16-fatty acids, and longer (DH)Cer are also seen in monohexosylCers and sphingomyelins. EBs had higher mRNAs for fatty acyl-CoA elongases that produce C18-, C24-, and C26-fatty acyl-CoAs (Elovl3 and Elovl6), and higher amounts of these cosubstrates for CerS. Thus, these studies have found generally good agreement between genomic and metabolomic data in defining that conversion of mESCs to EBs is accompanied by a large number of changes in gene expression and subspecies distributions for both sphingolipids and fatty acyl-CoAs. PMID:19786568

  4. Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1α

    PubMed Central

    Berda-Haddad, Yaël; Robert, Stéphane; Salers, Paul; Zekraoui, Leila; Farnarier, Catherine; Dinarello, Charles A.; Dignat-George, Françoise; Kaplanski, Gilles

    2011-01-01

    Sterile inflammation resulting from cell death is due to the release of cell contents normally inactive and sequestered within the cell; fragments of cell membranes from dying cells also contribute to sterile inflammation. Endothelial cells undergoing stress-induced apoptosis release membrane microparticles, which become vehicles for proinflammatory signals. Here, we show that stress-activated endothelial cells release two distinct populations of particles: One population consists of membrane microparticles (<1 μm, annexin V positive without DNA and no histones) and another larger (1–3 μm) apoptotic body-like particles containing nuclear fragments and histones, representing apoptotic bodies. Contrary to present concepts, endothelial microparticles do not contain IL-1α and do not induce neutrophilic chemokines in vitro. In contrast, the large apoptotic bodies contain the full-length IL-1α precursor and the processed mature form. In vitro, these apoptotic bodies induce monocyte chemotactic protein-1 and IL-8 chemokine secretion in an IL-1α–dependent but IL-1β–independent fashion. Injection of these apoptotic bodies into the peritoneal cavity of mice induces elevated serum neutrophil-inducing chemokines, which was prevented by cotreatment with the IL-1 receptor antagonist. Consistently, injection of these large apoptotic bodies into the peritoneal cavity induced a neutrophilic infiltration that was prevented by IL-1 blockade. Although apoptosis is ordinarily considered noninflammatory, these data demonstrate that nonphagocytosed endothelial apoptotic bodies are inflammatory, providing a vehicle for IL-1α and, therefore, constitute a unique mechanism for sterile inflammation. PMID:22143786

  5. Engineering musculoskeletal tissues with human embryonic germ cell derivatives.

    PubMed

    Varghese, Shyni; Hwang, Nathaniel S; Ferran, Angela; Hillel, Alexander; Theprungsirikul, Parnduangjai; Canver, Adam C; Zhang, Zijun; Gearhart, John; Elisseeff, Jennifer

    2010-04-01

    The cells derived from differentiating embryoid bodies of human embryonic germ (hEG) cells express a broad spectrum of gene markers and have been induced toward ecto- and endodermal lineages. We describe here in vitro and in vivo differentiation of hEG-derived cells (LVEC line) toward mesenchymal tissues. The LVEC cells express many surface marker proteins characteristic of mesenchymal stem cells and differentiated into cartilage, bone, and fat. Homogenous hyaline cartilage was generated from cells after 63 population doublings. In vivo results demonstrate cell survival, differentiation, and tissue formation. The high proliferative capacity of hEG-derived cells and their ability to differentiate and form three-dimensional mesenchymal tissues without teratoma formation underscores their significant potential for regenerative medicine. The adopted coculture system also provides new insights into how a microenvironment comprised of extracellular and cellular components may be harnessed to generate hierarchically complex tissues from pluripotent cells.

  6. Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system

    PubMed Central

    Zhu, Huanqi; Scharnhorst, Kelsey S.; Stieg, Adam Z.; Gimzewski, James K.; Minami, Itsunari; Nakatsuji, Norio; Nakano, Haruko; Nakano, Atsushi

    2017-01-01

    Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals. PMID:28266620

  7. Chondrogenic differentiation of human embryonic germ cell derived cells in hydrogels.

    PubMed

    Varghese, Shyni; Theprungsirikul, Paranduangji; Ferran, Angela; Hwang, Nathaniel; Canver, Adam; Elisseeff, Jennifer

    2006-01-01

    Human embryonic germ (hEG) cells have the potential to self-renew over long periods of time and differentiate into various lineages. Cells derived from embryoid bodies of hEG cells express a broad spectrum of gene markers and have been induced towards cells of ecto-dermal and recently endo-dermal and mesenchymal lineages. LVEC cells express a number of surface marker proteins characteristic of mesenchymal stem cells (MSCs), indicating the potential of these cells to differentiate into mesenchymal tissues. Here we demonstrate the homogenous differentiation of LVEC cells into hyaline cartilage. Three dimensional tissue formation is achieved by encapsulating cells in synthetic hydrogels followed by incubation in chondrocyte-conditioned culture medium. Homogenous hyaline cartilage was produced, even after 63 population doublings (13 passages). The high proliferative capacity of these cells without teratoma formation, homogenous differentiation, and three-dimensional cartilage tissue formation suggests the significant potential of LVEC cells for cartilage tissue engineering applications.

  8. Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

    PubMed

    Antonucci, Ivana; Di Pietro, Roberta; Alfonsi, Melissa; Centurione, Maria Antonietta; Centurione, Lucia; Sancilio, Silvia; Pelagatti, Francesca; D'Amico, Maria Angela; Di Baldassarre, Angela; Piattelli, Adriano; Tetè, Stefano; Palka, Giandomenico; Borlongan, Cesar V; Stuppia, Liborio

    2014-01-01

    Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

  9. A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

    PubMed

    Moe, Morten C; Kolberg, Rebecca S; Sandberg, Cecilie; Vik-Mo, Einar; Olstorn, Havard; Varghese, Mercy; Langmoen, Iver A; Nicolaissen, Bjørn

    2009-01-01

    Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human

  10. [Wheat androgenic embryoids and calli: data of scanning electron microscopy].

    PubMed

    Kruglova, N N; Gorbunova, V Iu; Abramov, S N; Sel'dimirova, O A

    2001-01-01

    The surface of wheat androgenic embryoids and calli at different developmental stages was studied using SEM. The embryoids were already characterized by regular cell divisions at the early developmental stages, while the calli were represented by irregular cell conglomerates. This trend was preserved during further development of androgenic structures. SEM studies of the surface of so-called secondary embryoids confirmed these observations.

  11. Embryoids derived from isolated protoplasts of carrot.

    PubMed

    Kameya, T; Uchimiya, H

    1972-12-01

    Protoplasts isolated enzymatically from carrot root tissues developed into cell clusters in a liquid medium containing coconut milk and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Cells of the resulting calluses differentiated into embryoids on an agar medium containing coconut milk or kinetin.

  12. Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

    PubMed

    Dirja, Bayu Tirta; Yoshie, Susumu; Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Nomoto, Yukio; Wada, Ikuo; Hazama, Akihiro; Omori, Koichi

    2016-01-01

    Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

  13. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

    PubMed Central

    Matsumoto, Takuya; Fujimori, Koki; Andoh-Noda, Tomoko; Ando, Takayuki; Kuzumaki, Naoko; Toyoshima, Manabu; Tada, Hirobumi; Imaizumi, Kent; Ishikawa, Mitsuru; Yamaguchi, Ryo; Isoda, Miho; Zhou, Zhi; Sato, Shigeto; Kobayashi, Tetsuro; Ohtaka, Manami; Nishimura, Ken; Kurosawa, Hiroshi; Yoshikawa, Takeo; Takahashi, Takuya; Nakanishi, Mahito; Ohyama, Manabu; Hattori, Nobutaka; Akamatsu, Wado; Okano, Hideyuki

    2016-01-01

    Summary Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases. PMID:26905201

  14. Immature embryoid teratoma of the gall bladder: case of a primary primitive neoplasm.

    PubMed

    Naim, Mohammed

    2009-01-01

    This study presents diagnostic histopathological features of a primary embryoid teratomatous neoplasm in the gall bladder of a 60-year-old woman, and also discusses its pathogenesis. Sections revealed immature and typical embryoid bodies and dispersed syncytial trophoblasts along with mature and immature neuroectodermal and mesodermal elements. The residual endothelial lining of the gall bladder showed in situ and proliferative changes characteristic of an endodermal tumour. Ultrasonographic and magnetic resonance imaging findings of the rest of the abdominal and pelvic organs appeared normal. Results show that the mature adult gall bladder is susceptible to primary neoplasms of a primitive nature ranging from immature embryoma and teratoma to neuroectodermal tumour. Such primitive neoplasms in adult organs where benign neuroectodermal elements are present may evolve from a constituent cell of an adult organ acquired during embryogenesis from a morula that differentiated into trophoblastic (outer) and embryoblastic (inner) cells, and existing dormant cells at rest until reactivation during oncogenesis.

  15. Long-term culture of mouse embryonic stem cell-derived adherent neurospheres and functional neurons.

    PubMed

    Hayashi, Mirian A F; Guerreiro, Juliano R; Cassola, Antonio C; Lizier, Nelson F; Kerkis, Alexandre; Camargo, Antonio C M; Kerkis, Irina

    2010-12-01

    Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of

  16. Molecular Profiling of Human Induced Pluripotent Stem Cell-Derived Hypothalamic Neurones Provides Developmental Insights into Genetic Loci for Body Weight Regulation.

    PubMed

    Yao, L; Liu, Y; Qiu, Z; Kumar, S; Curran, J E; Blangero, J; Chen, Y; Lehman, D M

    2017-02-01

    Recent data suggest that common genetic risks for metabolic disorders such as obesity may be human-specific and exert effects via the central nervous system. To overcome the limitation of human tissue access for study, we have generated induced human pluripotent stem cell (hiPSC)-derived neuronal cultures that recapture many features of hypothalamic neurones within the arcuate nucleus. In the present study, we have comprehensively characterised this model across development, benchmarked these neurones to in vivo events, and demonstrate a link between obesity risk variants and hypothalamic development. The dynamic transcriptome across neuronal maturation was examined using microarray and RNA sequencing methods at nine time points. K-means clustering of the longitudinal data was conducted to identify co-regulation and microRNA control of biological processes. The transcriptomes were compared with those of 103 samples from 13 brain regions reported in the Genotype-Tissue Expression database (GTEx) using principal components analysis. Genes with proximity to body mass index (BMI)-associated genetic variants were mapped to the developmentally expressed genesets, and enrichment significance was assessed with Fisher's exact test. The human neuronal cultures have a transcriptional and physiological profile of neuropeptide Y/agouti-related peptide arcuate nucleus neurones. The neuronal transcriptomes were highly correlated with adult hypothalamus compared to any other brain region from the GTEx. Also, approximately 25% of the transcripts showed substantial changes in expression across neuronal development and potential co-regulation of biological processes that mirror neuronal development in vivo. These developmentally expressed genes were significantly enriched for genes in proximity to BMI-associated variants. We confirmed the utility of this in vitro human model for studying the development of key hypothalamic neurones involved in energy balance and show that genes at

  17. Efficient generation of human embryonic stem cell-derived cardiac progenitors based on tissue-specific enhanced green fluorescence protein expression.

    PubMed

    Szebényi, Kornélia; Péntek, Adrienn; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I; Sarkadi, Balázs; Apáti, Ágota

    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-EGFP(high) rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFP(high) rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFP(high) 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.

  18. Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

    PubMed

    Maya-Espinosa, Guadalupe; Collazo-Navarrete, Omar; Millán-Aldaco, Diana; Palomero-Rivero, Marcela; Guerrero-Flores, Gilda; Drucker-Colín, René; Covarrubias, Luis; Guerra-Crespo, Magdalena

    2015-02-01

    A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

  19. Clonal propagation of chemically uniform fennel plants through somatic embryoids.

    PubMed

    Miura, Y; Fukui, H; Tabata, M

    1987-02-01

    Somatic embryoids obtained from cell suspension cultures of fennel in Linsmaier-Skoog medium containing 2,4-D and kinetin readily developed into plantlets when plated on a hormone-free agar medium. These plants were transplanted to the field to be tested for the uniformity of the chemically as well as the morphologically important characteristics of fruits. The results of field trials conducted for two years have confirmed that the clonal plants derived from somatic embryoids are remarkably uniform in all the characteristics examined in comparison with the control plants propagated through seeds. It is suggested, therefore, that the quality control of fennel fruits used for spice or medicine could be achieved by means of clonal propagation through somatic embryoids.

  20. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    PubMed

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  1. Ultrasmall Magnetically Engineered Ag2Se Quantum Dots for Instant Efficient Labeling and Whole-Body High-Resolution Multimodal Real-Time Tracking of Cell-Derived Microvesicles.

    PubMed

    Zhao, Jing-Ya; Chen, Gang; Gu, Yi-Ping; Cui, Ran; Zhang, Zhi-Ling; Yu, Zi-Li; Tang, Bo; Zhao, Yi-Fang; Pang, Dai-Wen

    2016-02-17

    Cell-derived microvesicles (MVs) are natural carriers that can transport biological molecules between cells, which are expected to be promising delivery vehicles for therapeutic purposes. Strategies to label MVs are very important for investigation and application of MVs. Herein, ultrasmall Mn-magnetofunctionalized Ag2Se quantum dots (Ag2Se@Mn QDs) integrated with excellent near-infrared (NIR) fluorescence and magnetic resonance (MR) imaging capabilities have been developed for instant efficient labeling of MVs for their in vivo high-resolution dual-mode tracking. The Ag2Se@Mn QDs were fabricated by controlling the reaction of Mn(2+) with the Ag2Se nanocrystals having been pretreated in 80 °C NaOH solution, with an ultrasmall size of ca. 1.8 nm, water dispersibility, high NIR fluorescence quantum yield of 13.2%, and high longitudinal relaxivity of 12.87 mM(-1) s(-1) (almost four times that of the commercial contrast agent Gd-DTPA). The ultrasmall size of the Ag2Se@Mn QDs enables them to be directly and efficiently loaded into MVs by electroporation, instantly and reliably conferring both NIR fluorescence and MR traceability on MVs. Our method for labeling MVs of different origins is universal and free of unfavorable influence on intrinsic behaviors of MVs. The complementary imaging capabilities of the Ag2Se@Mn QDs have made the long-term noninvasive whole-body high-resolution dual-mode tracking of MVs in vivo realized, by which the dynamic biodistribution of MVs has been revealed in a real-time and in situ quantitative manner. This work not only opens a new window for labeling with QDs, but also facilitates greatly the investigation and application of MVs.

  2. Mouse embryonic stem cell-derived cardiac myocytes in a cell culture dish.

    PubMed

    Glass, Carley; Singla, Reetu; Arora, Anshu; Singla, Dinender K

    2015-01-01

    Embryonic stem (ES) cells are pluripotent stem cells capable of self-renewal and have broad differentiation potential yielding cell types from all three germ layers. In the absence of differentiation inhibitory factors, when cultured in suspension, ES cells spontaneously differentiate and form three-dimensional cell aggregates termed embryoid bodies (EBs). Although various methods exist for the generation of EBs, the hanging drop method offers reproducibility and homogeneity from a predetermined number of ES cells. Herein, we describe the in vitro differentiation of mouse embryonic stem cells into cardiac myocytes using the hanging drop method and immunocytochemistry to identify cardiomyogenic differentiation. In brief, ES cells, placed in droplets on the lid of culture dishes following a 2-day incubation, yield embryoid bodies, which are resuspended and plated. 1-2 weeks following plating of the EBs, spontaneous beating areas can be observed and staining for specific cardiac markers can be achieved.

  3. [Effects of lingfasu on embryoid and plantlet formation of Panax notoginseng in vitro].

    PubMed

    Xu, Hongyuan; Meng, Aidong; Li, Chunxia; Deng, Xiqing; Zhou, Qiwei; Yang, Meichun

    2004-10-01

    On the medium MS added the right amount of 2,4-D and LFS (Angustmycin) and cultured under dark condition, the callus from stem segments of Panax notoginseng could induce a lot of embryoids. In 2-3 months, the ratio of embryoid formation reached about 90%. Then transplanted on MS + 2,4-D 1.5 mg/L + LFS 2 mg/L and cultured under light 20001x, near 30% embryoids could grow and develop as robust plantlets.

  4. Embryoid Formation by High Temperature Treatment from Multiple Shoots of Panax ginseng1.

    PubMed

    Asaka, I; Li, I; Yoshikawa, T; Hirotani, M; Furuya, T

    1993-08-01

    We developed a new technology to induce embryoids by a moderate high temperature treatment from multiple shoots of PANAX GINSENG (Araliaceae). The number of formed embryoids was 10 times higher than that of untreated tissue. Normal plantlets were regenerated from the embryoids by transplanting them on a hormone-free medium. They contained ginsenosides Rb (1), Rg (1) and the other saponins as well as those of natural ginseng.

  5. A Systemized Approach to Investigate Ca2+ Synchronization in Clusters of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes

    PubMed Central

    Jones, Aled R.; Edwards, David H.; Cummins, Michael J.; Williams, Alan J.; George, Christopher H.

    2016-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM) are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB) vs. “on plate” culture on spontaneous activity and regional Ca2+ synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca2+ spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca2+ synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (>2 weeks). The maintenance of all spontaneously active IPS-CM clusters under “on plate” culture conditions promoted the progressive reduction in regional Ca2+ synchronization and the loss of spontaneous Ca2+ spiking. Raising the extracellular [Ca2+] surrounding these quiescent IPS-CM clusters from ~0.4 to 1.8 mM unmasked discrete behaviors typified by either (a) long-lasting Ca2+ elevation that returned to baseline or (b) persistent, large-amplitude Ca2+ oscillations around an increased cytoplasmic [Ca2+]. The different responses of IPS-CM to elevated extracellular [Ca2+] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation. PMID:26793710

  6. Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis.

    PubMed

    Simunovic, Mijo; Brivanlou, Ali H

    2017-03-15

    Cells have an intrinsic ability to self-assemble and self-organize into complex and functional tissues and organs. By taking advantage of this ability, embryoids, organoids and gastruloids have recently been generated in vitro, providing a unique opportunity to explore complex embryological events in a detailed and highly quantitative manner. Here, we examine how such approaches are being used to answer fundamental questions in embryology, such as how cells self-organize and assemble, how the embryo breaks symmetry, and what controls timing and size in development. We also highlight how further improvements to these exciting technologies, based on the development of quantitative platforms to precisely follow and measure subcellular and molecular events, are paving the way for a more complete understanding of the complex events that help build the human embryo.

  7. A new method for the purification of the different stages of carrot embryoids.

    PubMed

    Giuliano, G; Rosellini, D; Terzi, M

    1983-08-01

    An easy method is presented for the purification of the different stages of carrot embryoids. This is based on a synchronization of the regenerating culture and on a filtration through filters of various pore sizes. A differential sedimentation was used for removing undifferentiated cells. At the end of the process, the different stages: globular, heart- and torpedo-shaped were obtained with a degree of purity that always exceeded 90%. This method can be used for the separation of relatively large numbers of embryoids (from thousands to a million) of haploid and diploid carrot lines and is very gentle on embryoids in that it does not affect their viability or further development.

  8. 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.

  9. Mass Production of Ginseng (Panax ginseng) Embryoids on Media Containing High Concentrations of Sugar1.

    PubMed

    Asaka, I; Ii, I; Hirotani, M; Asada, Y; Yoshokawa, T; Furuya, T

    1994-04-01

    A lot of ginseng embryoids were produced by culturing on high concentrations of sugar media from the embryogenic tissues obtained by moderately high temperature treatment. When the sucrose concentration was 100 g/l, the number of embryoids produced were over 100 pieces per g of inoculum weight. It was about ten times of that produced by culturing on 30 g/l of sucrose. Glucose showed an effect similar to sucrose on the basis of weight percentage. However, mannitol did not show this effect. The embryoids obtained by these processes redifferentiated to normal plantlets on culturing on the medium containing 30 g/l of sucrose. The saponin components of the tissue containing embryoids showed a similar pattern to those of natural GINSENG by HPLC.

  10. Aescin Content in Embryogenic Callus and in Embryoids from Leaf Explants of Aesculus hippocastanum.

    PubMed

    Profumo, P; Caviglia, A M; Gastaldo, P; Dameri, R M

    1991-02-01

    HPLC determinations of the aescin contents in calli and embryoids from leaf explants of AESCULUS HIPPOCASTANUM L. were carried out in order to determine whether it was possible to obtain aescin-forming proliferations IN VITRO. The results indicate that embryogenic calli and embryoids derived from them produce an amount of active principle higher than that of horse-chestnut seeds. The data are discussed in terms of the relation between tissue differentiation and secondary metabolites biosynthesis.

  11. Identification of Embryoid-Abundant Genes That Are Temporally Expressed during Pollen Embryogenesis in Wheat Anther Cultures.

    PubMed

    Reynolds, T L; Kitto, S L

    1992-12-01

    Uninucleate microspores in anther cultures of bread wheat (Triticum aestivum cv Pavon) are capable of producing haploid pollen embryoids and plants. To gain an understanding of this alternate pathway of pollen development, we constructed a cDNA library to young pollen embryoids, isolated embryoid-specific genes, and analyzed their expression patterns during morphogenesis. Two embryoid-abundant clones, pEMB4 and 94, were expressed very early during culture, suggesting that these genes are associated with development and are not simply expressed as a consequence of differentiation. The accumulation patterns of five cloned mRNAs may indicate the activation of specific genes associated with the major morphological and physiological activities connected with the differentiation of embryoids in vitro. These results suggest that embryoid-abundant gene expression is causally related to this pathway because gene expression is spatially and temporally specific and is not observed when microspores are cultured under noninductive conditions.

  12. Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues.

    PubMed

    Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

    2016-01-01

    Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to

  13. Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues

    PubMed Central

    Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

    2016-01-01

    Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to

  14. The development of haploid embryoids from anther cultures of Atropa belladonna L.

    PubMed

    Rashid, A; Street, H E

    1973-09-01

    Development of haploid embryoids from the microspores of Atropa belladonna occurs with relatively high frequency when anthers are excised from buds in which the petals are shorter than the sepals (at this stage microspores are predominantly uninucleate) and cultured on a medium containing iron as the ferric salt of ethylenediamine-di-O-hydroxyphenylacetic acid (FeEDDHA). Additions of combinations of kinetin, auxin and casamino-acids to the culture medium induce callusing in both haploid and diploid tissues, lead to the origin of embryoids from somatic tissues of the anther and should be avoided. Simple techniques for the maintenance of haploid clones are described.Stages in early embryogenesis in the pollen grains have been observed and these indicate that embryogenesis is most frequently initiated by an equal division in the uninucleate spore. The frequency of grains showing embryoid formation is very low and it is estimated that plantlets are formed from up to 50% of these grains.

  15. Human embryonic stem cell derivation and directed differentiation.

    PubMed

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  16. Reduced Inhomogeneity of Angelica acutiloba Plants Propagated Clonally Through Somatic Embryoids.

    PubMed

    Miura, Y; Fukui, H; Tabata, M

    1988-02-01

    Clonal plants propagated from a single plant of a commercial variety of ANGELICA ACUTILOBA (Umbelliferae) through somatic embryoids induced in cell suspension cultures proved to be significantly more uniform with respect to the contents of medicinally important chemical constituents (ligustilide and choline) of the root when compared with seed-propagated plants.

  17. Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. Merr.).

    PubMed

    Kerns, H R; Barwale, U B; Meyer, M M; Widholm, J M

    1986-04-01

    Suspension cultures of soybean were initiated from hypocotyl or cotyledon callus tissue of several soybean genotypes. When these were grown on L2 medium with 0.4 mg/liter 2,4-D several genotypes produced numerous embryoids while others produced only a few such structures. Due to internal anatomy, no embryoid developed into a complete plant. A genotype's propensity to form normal appearing embryoids was correlated with the ability to proliferate shoots at the cotyledonary node on a medium with benzylaminopurine as determined in previous testing.

  18. Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells

    PubMed Central

    Kossack, Nina; Meneses, Juanito; Shefi, Shai; Nguyen, Ha Nam; Chavez, Shawn; Nicholas, Cory; Gromoll, Joerg; Turek, Paul J; Reijo-Pera, Renee A

    2009-01-01

    Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia-derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post-transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming. PMID:18927477

  19. Human primordial germ cell-derived progenitors give rise to neurons and glia in vivo

    SciTech Connect

    Teng, Yincheng; Chen, Bin; Tao, Minfang

    2009-12-18

    We derived a cell population from cultured human primordial germ cells from early human embryos. The derivates, termed embryoid body-derived (EBD) cells, displayed an extensive capacity for proliferation and expressed a panel of markers in all three germ layers. Interestingly, EBD cells were also positive for markers of neural stem/progenitor cells, such as nestin and glial fibrillary acidic protein. When these cells were transplanted into the brain cavities of fetal sheep and postnatal NOD-SCID mice or nerve-degenerated tibialis anterior muscles, they readily gave rise to neurons or glial cells. To our knowledge, our data are the first to demonstrate that EBD cells can undergo further neurogenesis under suitable environments in vivo. Hence, with the abilities of extensive expansion, self-renewal, and differentiation, EBD cells may provide a useful donor source for neural stem/progenitor cells to be used in cell-replacement therapies for diseases of the nervous system.

  20. Identification of Embryoid-Abundant Genes That Are Temporally Expressed during Pollen Embryogenesis in Wheat Anther Cultures 1

    PubMed Central

    Reynolds, Thomas L.; Kitto, Sherry L.

    1992-01-01

    Uninucleate microspores in anther cultures of bread wheat (Triticum aestivum cv Pavon) are capable of producing haploid pollen embryoids and plants. To gain an understanding of this alternate pathway of pollen development, we constructed a cDNA library to young pollen embryoids, isolated embryoid-specific genes, and analyzed their expression patterns during morphogenesis. Two embryoid-abundant clones, pEMB4 and 94, were expressed very early during culture, suggesting that these genes are associated with development and are not simply expressed as a consequence of differentiation. The accumulation patterns of five cloned mRNAs may indicate the activation of specific genes associated with the major morphological and physiological activities connected with the differentiation of embryoids in vitro. These results suggest that embryoid-abundant gene expression is causally related to this pathway because gene expression is spatially and temporally specific and is not observed when microspores are cultured under noninductive conditions. Images Figure 1 Figure 2 Figure 3 PMID:16653192

  1. Aescin formation in calli and embryoids from cotyledon and stem explants of Aesculus hippocastanum L.

    PubMed

    Profumo, P; Caviglia, A M; Gastaldo, P

    1994-11-01

    Aescin in calli and embryoids obtained from both cotyledon and stem explants of Aesculus hippocastanum were investigated by HPLC. Determinations were carried out on tissues cultured in agarized medium supplemented with growth substances (2,4-dichlorophenoxyacetic acid; kinetin; 1-naphthaleneacetic acid). The results indicate that aescin was produced in all the analysed samples. The amount of active principle present in some samples was higher than that found in horse-chestnut seeds.

  2. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    EPA Pesticide Factsheets

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  3. 3D high throughput screening and profiling of embryoid bodies in thermoformed microwell plates.

    PubMed

    Vrij, E J; Espinoza, S; Heilig, M; Kolew, A; Schneider, M; van Blitterswijk, C A; Truckenmüller, R K; Rivron, N C

    2016-02-21

    3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow high-throughput screens (HTS). Here, we introduce a microwell platform, integrated in standard culture plates, for functional HTS. Using micro-thermoforming, we form round-bottom microwell arrays from optically clear cyclic olefin polymer films, and assemble them with bottom-less 96-well plates. We show that embryonic stem cells aggregate faster and more reproducibly (centricity, circularity) as compared to a state-of-the-art microwell array. We then run a screen of a chemical library to direct differentiation into primitive endoderm (PrE) and, using on-chip high content imaging (HCI), we identify molecules, including regulators of the cAMP pathway, regulating tissue size, morphology and PrE gene activity. We propose that this platform will benefit to the systematic study of organogenesis in vitro.

  4. High frequency embryoid and plantlet formation from tissue cultures of the Finger millet-Eleusine coracana (L.) Gaertn.

    PubMed

    Sivadas, P; Kothari, S L; Chandra, N

    1990-07-01

    Compact nodulated embryogenic callus differentiated from cultured seeds of Eleusine coracana (Finger Millet) on Murashige and Skoog (1962) basal medium with 2,4-dichlorophenoxyacetic acid (1.0, 3.0 mg (l)). This embryogenic callus was maintained on a medium with a lower level of 2,4 - dichlorophenoxyacetic acid. At every subculture the embryogenic callus had some preexisting embryoids in it. With this method of subculture the callus has retained its morphogenic potential for four years. Following transfer to media with different levels of auxins and cytokinins, the callus showed varied patterns of growth and morphogenesis. Embryoids could be germinated in profusion to form plantlets which could be transferred to the field. Shoot buds also differentiated from the whole surface of the embryoid or from the flattened meristemoids.

  5. Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations

    PubMed Central

    Santana, Steven M.; Antonyak, Marc A.; Cerione, Richard A.

    2015-01-01

    Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer. PMID:25342569

  6. In Vitro Imaging of Angiogenesis Using Embryonic Stem Cell-Derived Endothelial Cells

    PubMed Central

    Stuhlmann, Heidi

    2012-01-01

    Angiogenesis is an important event during developmental processes, and it plays a key role in neovascularization. The development of an in vitro model that can be used for live imaging of vessel growth will facilitate the study of molecular and cellular mechanisms for the growth of blood vessels. Embryonic stem cells (ESCs) are considered to be a novel renewable source for the derivation of genetically manipulable endothelial cells (ECs). To derive green fluorescence protein (GFP)-expressing ECs, we used a transgenic ESC line in which a GFP reporter was driven by the endothelial-specific promoter fetal liver kinase 1. ESC-ECs were isolated from 11-day embryoid bodies by fluorescence-activated cell sorting. Embedding the aggregated ESC-ECs in a 3-dimensional collagen gel matrix resulted in ESC-EC migration out of the aggregates and coalescence into a capillary network. Time-lapse microscopy revealed EC migration, proliferation, lumen formation, and anastomosis to other capillary vessels during this process, which were reminiscent of angiogenic processes. Vascular endothelial growth factor plays major roles in the induction of ESC-EC angiogenesis in vitro. Blockage of the β1 integrin subunit severely impaired ESC-EC survival and migration. We demonstrate that our in vitro ESC-EC angiogenesis model represents a high-resolution dynamic video-image system for observing the cellular events underlying angiogenic cascades. We also consider this model as an image screening tool for the identification of pro-angiogenic and anti-angiogenic molecules. PMID:21385073

  7. Towards Personalized Regenerative Cell Therapy: Mesenchymal Stem Cells Derived from Human Induced Pluripotent Stem Cells.

    PubMed

    Lin, Lin; Bolund, Lars; Luo, Yonglun

    2016-01-01

    Mesenchymal stem cells (MSCs) are adult stem cells with the capacity of self-renewal and multilineage differentiation, and can be isolated from several adult tissues. However, isolating MSCs from adult tissues for cell therapy is hampered by the invasive procedure, the rarity of the cells and their attenuated proliferation capacity when cultivated and expanded in vitro. Human MSCs derived from induced pluripotent stem cells (iPSC-MSCs) have now evolved as a promising alternative cell source for MSCs and regenerative medicine. Several groups, including ours, have reported successful derivation of functional iPSC-MSCs and applied these cells in MSC-based therapeutic testing. Still, the current experience and understanding of iPSC-MSCs with respect to production methods, safety and efficacy are primitive. In this review, we highlight the methodological progress in iPSC-MSC research, describing the importance of choosing the right sources of iPSCs, iPSC reprogramming methods, iPSC culture systems, embryoid body intermediates, pathway inhibitors, basal medium, serum, growth factors and culture surface coating. We also highlight some progress in the application of iPSC-MSCs in direct cell therapy, tissue engineering and gene therapy.

  8. Changes in the Essential Oil Components during the Development of Fennel Plants from Somatic Embryoids.

    PubMed

    Miura, Y; Ogawa, K; Fukui, H; Tabata, M

    1987-02-01

    Quantitative and qualitative changes of essential oils during the development of clonal plants of fennel propagated through somatic embryogenesis were investigated. Although no essential oil could be detected either in cultured cells or in somatic embryoids, monoter-penes such as alpha-phellandrene and alpha-pinene were found in radical leaves of regenerated plantlets cultured on a hormone-free agar medium. The regenerated plants cultivated in the field for about one month accumulated phenylpropanoids such as estragole, anethole, and fenchone in addition to the two monoterpenes described above in radical leaves. Rich accumulations of phenylpropanoids and monoterpenes were observed in the fruits; especially the contents of estragole and anethole were much higher than in radical leaves.

  9. Structural Phenotyping of Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-01-01

    Summary 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. PMID:25733020

  10. Quality Metrics for Stem Cell-Derived Cardiac Myocytes

    PubMed Central

    Sheehy, Sean P.; Pasqualini, Francesco; Grosberg, Anna; Park, Sung Jin; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2014-01-01

    Summary Advances in stem cell manufacturing methods have made it possible to produce stem cell-derived cardiac myocytes at industrial scales for in vitro muscle physiology research purposes. Although FDA-mandated quality assurance metrics address safety issues in the manufacture of stem cell-based products, no standardized guidelines currently exist for the evaluation of stem cell-derived myocyte functionality. As a result, it is unclear whether the various stem cell-derived myocyte cell lines on the market perform similarly, or whether any of them accurately recapitulate the characteristics of native cardiac myocytes. We propose a multiparametric quality assessment rubric in which genetic, structural, electrophysiological, and contractile measurements are coupled with comparison against values for these measurements that are representative of the ventricular myocyte phenotype. We demonstrated this procedure using commercially available, mass-produced murine embryonic stem cell- and induced pluripotent stem cell-derived myocytes compared with a neonatal mouse ventricular myocyte target phenotype in coupled in vitro assays. PMID:24672752

  11. Stem Cell-Derived Extracellular Vesicles and Immune-Modulation

    PubMed Central

    Burrello, Jacopo; Monticone, Silvia; Gai, Chiara; Gomez, Yonathan; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system. PMID:27597941

  12. Stem Cell-Derived Extracellular Vesicles and Immune-Modulation.

    PubMed

    Burrello, Jacopo; Monticone, Silvia; Gai, Chiara; Gomez, Yonathan; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system.

  13. The effect of anther orientation on the production of microspore-derived embryoids and plants of Hordeum vulgare cv. Sabarlis.

    PubMed

    Hunter, C P

    1985-10-01

    The orientation of barley anthers on culture medium had a marked effect on their response. Embryoids developed only in the upper lobes of anthers cultured on edge, i.e. with a single lobe in contact with the culture medium. Anthers cultured flat, i.e. with both lobes in contact with the medium, did not respond. Careful orientation of anthers at the start of incubation resulted in a mean yield of 20 green plants per 100 anthers cultured.

  14. Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid

    PubMed Central

    Makoolati, Zohreh; Movahedin, Mansoureh; Forouzandeh-Moghadam, Mehdi

    2016-01-01

    An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0–5  μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs. PMID:27834666

  15. Influence of activin A supplementation during human embryonic stem cell derivation on germ cell differentiation potential.

    PubMed

    Duggal, Galbha; Heindryckx, Björn; Warrier, Sharat; O'Leary, Thomas; Van der Jeught, Margot; Lierman, Sylvie; Vossaert, Liesbeth; Deroo, Tom; Deforce, Dieter; Chuva de Sousa Lopes, Susana M; De Sutter, Petra

    2013-12-01

    Human embryonic stem cells (hESCs) are more similar to "primed" mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro.

  16. Early gene expression along the animal-vegetal axis in sea urchin embryoids and grafted embryos.

    PubMed

    Ghiglione, C; Emily-Fenouil, F; Chang, P; Gache, C

    1996-10-01

    The HE gene is the earliest strictly zygotic gene activated during sea urchin embryogenesis. It is transiently expressed in a radially symmetrical domain covering the animal-most two-thirds of the blastula. The border of this domain, which is orthogonal to the primordial animal-vegetal axis, is shifted towards the animal pole in Li+-treated embryos. Exogenous micromeres implanted at the animal pole of whole embryos, animal or vegetal halves do not modify the extent and localization of the HE expression domain. In grafted embryos or animal halves, the Li+ effect is not affected by the presence of ectopic micromeres at the animal pole. A Li+-induced shift of the border, similar to that seen in whole embryos, occurs in embryoids developing from animal halves isolated from 8-cell stage embryos or dissected from unfertilised eggs. Therefore, the spatial restriction of the HE gene is not controlled by the inductive cascade emanating from the micromeres and the patterning along the AV-axis revealed by Li+ does not require interactions between cells from the animal and vegetal halves. This suggests that maternal primary patterning in the sea urchin embryo is not limited to a small vegetal center but extends along the entire AV axis.

  17. Micropost arrays for measuring stem cell-derived cardiomyocyte contractility

    PubMed Central

    Beussman, Kevin M.; Rodriguez, Marita L.; Leonard, Andrea; Taparia, Nikita; Thompson, Curtis R.; Sniadecki, Nathan J.

    2015-01-01

    Stem cell-derived cardiomyocytes have the potential to be used to study heart disease and maturation, screen drug treatments, and restore heart function. Here, we discuss the procedures involved in using micropost arrays to measure the contractile forces generated by stem cell-derived cardiomyocytes. Cardiomyocyte contractility is needed for the heart to pump blood, so measuring the contractile forces of cardiomyocytes is a straightforward way to assess their function. Microfabrication and soft lithography techniques are utilized to create identical arrays of flexible, silicone microposts from a common master. Micropost arrays are functionalized with extracellular matrix protein to allow cardiomyocytes to adhere to the tips of the microposts. Live imaging is used to capture videos of the deflection of microposts caused by the contraction of the cardiomyocytes. Image analysis code provides an accurate means to quantify these deflections. The contractile forces produced by a beating cardiomyocyte are calculated by modeling the microposts as cantilever beams. We have used this assay to assess techniques for improving the maturation and contractile function of stem cell-derived cardiomyocytes. PMID:26344757

  18. Electrophysiological Properties of Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Risner-Janiczek, Jessica R.; Ungless, Mark A.; Li, Meng

    2011-01-01

    In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP+ neurons in culture display functional neuronal properties even at early stages of differentiation. PMID:21887381

  19. Functional Characterization of Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Kirsch, Authors Glenn E.; Obejero-Paz, Carlos A.; Bruening-Wright, Andrew

    2014-01-01

    Cardiac toxicity is a leading contributor to late-stage attrition in the drug discovery process and to withdrawal of approved from the market. In vitro assays that enable earlier and more accurate testing for cardiac risk provide early stage predictive indicators that aid in mitigating risk. Human cardiomyocytes, the most relevant subjects for early stage testing, are severely limited in supply. But human stem cell-derived cardiomyocytes (SC-hCM) are readily available from commercial sources and are increasingly used in academic research, drug discovery and safety pharmacology. As a result, SC-hCM electrophysiology has become a valuable tool to assess cardiac risk associated with drugs. This unit describes techniques for recording individual currents carried by sodium, calcium and potassium ions, as well as single cell action potentials, and impedance recordings from contracting syncytia of thousands of interconnected cells. PMID:25152802

  20. Balancing Ethical Pros and Cons of Stem Cell Derived Gametes.

    PubMed

    Segers, Seppe; Mertes, Heidi; de Wert, Guido; Dondorp, Wybo; Pennings, Guido

    2017-01-13

    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.

  1. Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids.

    PubMed

    Kawakami, Yutaka; Goto-Kazeto, Rie; Saito, Taiju; Fujimoto, Takafumi; Higaki, Shogo; Takahashi, Yoshiyuki; Arai, Katsutoshi; Yamaha, Etsuro

    2010-01-01

    Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. In our previous study, a single PGC transplanted into a host differentiated into fertile gametes and produced germ-line chimeras of cyprinid fish, including zebrafish. In this study, we aimed to induce germ-line chimeras by transplanting donor PGCs from various sources (normal embryos at different stages, dissociated blastomeres, embryoids, or embryoids cryopreserved by vitrification) into host blastulae, and compare the migration rates of the PGCs towards the gonadal ridge. Isolated, cultured blastomeres not subject to mesodermal induction were able to differentiate into PGCs that retained their motility. Moreover, these PGCs successfully migrated towards the gonadal ridge of the host and formed viable gametes. Motility depended on developmental stage and culture duration: PGCs obtained at earlier developmental stages and with shorter cultivation periods showed an increased rate of migration to the gonadal ridge. Offspring were obtained from natural spawning between normal females and chimeric males. These results provide the basis for new methods of gene preservation in zebrafish.

  2. The early ontogeny of embryoids and callus from pollen and subsequent organogenesis in anther cultures of Datura metel and rice.

    PubMed

    Iyer, R D; Raina, S K

    1972-06-01

    Haploidy induction through anther culture has been examined in Datura metel and rice with a view to tracing the precise sequence of development of the pollen, either directly or through an intervening callus, into an embryo and seedling. In D. metel, the vegetative cell of the young pollen grain assumes the major role in formation of embryos whereas the generative cell and its few derivatives degenerate. Embryos and seedlings arising directly from pollen without an intervening callus phase always proved to be haploids, whereas those differentiating from pollen-derived callus gave haploid, diploid and even triploid plants. Cytological analysis of callus tissue showed cells of various ploidy levels ranging from haploid to triploid, and in rare instances even with higher chromosome numbers.In rice anther cultures the embryoids arose from an initial callus phase. Of 15 different rice cultivars tried, only four produced a callus, and in only one, was there differentiation of plants, both haploid and diploid ones. Among other species tried, egg plant has also yielded plantlets through a callus phase whereas only callus production has been achieved in jute, tea and petunia. No response has been obtained in wheat, maize, cotton and coconut.Coconut milk (CM) appears to be the most important component of the medium for the initial induction of embryoids and callus in anther cultures of most of the species tried. However, further growth and differentiation of plants may require a simpler medium; in D. metel, continued culture on CM led to dedifferntiation.

  3. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  4. Cartilage repair using human embryonic stem cell-derived chondroprogenitors.

    PubMed

    Cheng, Aixin; Kapacee, Zoher; Peng, Jiang; Lu, Shibi; Lucas, Robert J; Hardingham, Timothy E; Kimber, Susan J

    2014-11-01

    In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions.

  5. Improved Flow Cytometric Assessment Reveals Distinct Microvesicle (Cell-Derived Microparticle) Signatures in Joint Diseases

    PubMed Central

    György, Bence; Szabó, Tamás G.; Turiák, Lilla; Wright, Matthew; Herczeg, Petra; Lédeczi, Zsigmond; Kittel, Ágnes; Polgár, Anna; Tóth, Kálmán; Dérfalvi, Beáta; Zelenák, Gergő; Böröcz, István; Carr, Bob; Nagy, György; Vékey, Károly; Gay, Steffen; Falus, András; Buzás, Edit I.

    2012-01-01

    Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures. PMID:23185418

  6. Cells derived from young bone marrow alleviate renal aging.

    PubMed

    Yang, Hai-Chun; Rossini, Michele; Ma, Li-Jun; Zuo, Yiqin; Ma, Ji; Fogo, Agnes B

    2011-11-01

    Bone marrow-derived stem cells may modulate renal injury, but the effects may depend on the age of the stem cells. Here we investigated whether bone marrow from young mice attenuates renal aging in old mice. We radiated female 12-mo-old 129SvJ mice and reconstituted them with bone marrow cells (BMC) from either 8-wk-old (young-to-old) or 12-mo-old (old-to-old) male mice. Transfer of young BMC resulted in markedly decreased deposition of collagen IV in the mesangium and less β-galactosidase staining, an indicator of cell senescence. These changes paralleled reduced expression of plasminogen activator inhibitor-1 (PAI-1), PDGF-B (PDGF-B), the transdifferentiation marker fibroblast-specific protein-1 (FSP-1), and senescence-associated p16 and p21. Tubulointerstitial and glomerular cells derived from the transplanted BMC did not show β-galactosidase activity, but after 6 mo, there were more FSP-1-expressing bone marrow-derived cells in old-to-old mice compared with young-to-old mice. Young-to-old mice also exhibited higher expression of the anti-aging gene Klotho and less phosphorylation of IGF-1 receptor β. Taken together, these data suggest that young bone marrow-derived cells can alleviate renal aging in old mice. Direct parenchymal reconstitution by stem cells, paracrine effects from adjacent cells, and circulating anti-aging molecules may mediate the aging of the kidney.

  7. Large-scale generation of cell-derived nanovesicles.

    PubMed

    Jo, W; Kim, J; Yoon, J; Jeong, D; Cho, S; Jeong, H; Yoon, Y J; Kim, S C; Gho, Y S; Park, J

    2014-10-21

    Exosomes are enclosed compartments that are released from cells and that can transport biological contents for the purpose of intercellular communications. Research into exosomes is hindered by their rarity. In this article, we introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles. The device has a simple polycarbonate structure to hold the filter, and operates in a common centrifuge. Nanovesicles are similar in size and membrane structure to exosomes. Nanovesicles contain intracellular RNAs ranging from microRNA to mRNA, intracellular proteins, and plasma membrane proteins. The quantity of nanovesicles produced using the device is 250 times the quantity of naturally secreted exosomes. Also, the quantity of intracellular contents in nanovesicles is twice that in exosomes. Nanovesicles generated from murine embryonic stem cells can transfer RNAs to target cells. Therefore, this novel device and the nanovesicles that it generates are expected to be used in exosome-related research, and can be applied in various applications such as drug delivery and cell-based therapy.

  8. Large-scale generation of cell-derived nanovesicles

    NASA Astrophysics Data System (ADS)

    Jo, W.; Kim, J.; Yoon, J.; Jeong, D.; Cho, S.; Jeong, H.; Yoon, Y. J.; Kim, S. C.; Gho, Y. S.; Park, J.

    2014-09-01

    Exosomes are enclosed compartments that are released from cells and that can transport biological contents for the purpose of intercellular communications. Research into exosomes is hindered by their rarity. In this article, we introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles. The device has a simple polycarbonate structure to hold the filter, and operates in a common centrifuge. Nanovesicles are similar in size and membrane structure to exosomes. Nanovesicles contain intracellular RNAs ranging from microRNA to mRNA, intracellular proteins, and plasma membrane proteins. The quantity of nanovesicles produced using the device is 250 times the quantity of naturally secreted exosomes. Also, the quantity of intracellular contents in nanovesicles is twice that in exosomes. Nanovesicles generated from murine embryonic stem cells can transfer RNAs to target cells. Therefore, this novel device and the nanovesicles that it generates are expected to be used in exosome-related research, and can be applied in various applications such as drug delivery and cell-based therapy.

  9. Enriched retinal ganglion cells derived from human embryonic stem cells

    PubMed Central

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  10. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes.

  11. Muse Cells Derived from Dermal Tissues Can Differentiate into Melanocytes.

    PubMed

    Tian, Ting; Zhang, Ru-Zhi; Yang, Yu-Hua; Liu, Qi; Li, Di; Pan, Xiao-Ru

    2017-02-07

    The objective of the authors has been to obtain multilineage-differentiating stress-enduring cells (Muse cells) from primary cultures of dermal fibroblasts, identify their pluripotency, and detect their ability to differentiate into melanocytes. The distribution of SSEA-3-positive cells in human scalp skin was assessed by immunohistochemistry, and the distribution of Oct4, Sox2, Nanog, and SSEA-3-positive cells was determined by immunofluorescence staining. The expression levels of Sox2, Oct4, hKlf4, and Nanog mRNAs and proteins in Muse cells were determined by reverse transcription polymerase chain reaction (RT-PCR) analyses and Western blots, respectively. These Muse cells differentiated into melanocytes in differentiation medium. The SSEA-3-positive cells were scattered in the basement membrane zone and the dermis, with comparatively more in the sebaceous glands, vascular and sweat glands, as well as the outer root sheath of hair follicles, the dermal papillae, and the hair bulbs. Muse cells, which have the ability to self-renew, were obtained from scalp dermal fibroblasts by flow cytometry sorting with an anti-SSEA-3 antibody. The results of RT-PCR, Western blot, and immunofluorescence staining showed that the expression levels of Oct4, Nanog, Sox2, and Klf4 mRNAs and proteins in Muse cells were significantly different from their parental dermal fibroblasts. Muse cells differentiated into melanocytes when cultured in melanocyte differentiation medium, and the Muse cell-derived melanocytes expressed the melanocyte-specific marker HMB45. Muse cells could be obtained by flow cytometry from primary cultures of scalp dermal fibroblasts, which possessed the ability of pluripotency and self-renewal, and could differentiate into melanocytes in vitro.

  12. Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning

    PubMed Central

    Doss, Michael Xavier; Chen, Shuhua; Winkler, Johannes; Hippler-Altenburg, Rita; Odenthal, Margareta; Wickenhauser, Claudia; Balaraman, Sridevi; Schulz, Herbert; Hummel, Oliver; Hübner, Norbert; Ghosh-Choudhury, Nandini; Sotiriadou, Isaia; Hescheler, Jürgen; Sachinidis, Agapios

    2007-01-01

    Background Bone morphogenetic protein (BMP)2 is a late mesodermal marker expressed during vertebrate development and plays a crucial role in early embryonic development. The nature of the BMP2-expressing cells during the early stages of embryonic development, their transcriptome and cell phenotypes developed from these cells have not yet been characterized. Results We generated a transgenic BMP2 embryonic stem (ES) cell lineage expressing both puromycin acetyltransferase and enhanced green fluorescent protein (EGFP) driven by the BMP2 promoter. Puromycin resistant and EGFP positive BMP2+ cells with a purity of over 93% were isolated. Complete transcriptome analysis of BMP2+ cells in comparison to the undifferentiated ES cells and the control population from seven-day-old embryoid bodies (EBs; intersection of genes differentially expressed between undifferentiated ES cells and BMP2+ EBs as well as differentially expressed between seven-day-old control EBs and BMP2+ EBs by t-test, p < 0.01, fold change >2) by microarray analysis led to identification of 479 specifically upregulated and 193 downregulated transcripts. Transcription factors, apoptosis promoting factors and other signaling molecules involved in early embryonic development are mainly upregulated in BMP2+ cells. Long-term differentiation of the BMP2+ cells resulted in neural crest stem cells (NCSCs), smooth muscle cells, epithelial-like cells, neuronal-like cells, osteoblasts and monocytes. Interestingly, development of cardiomyocytes from the BMP2+ cells requires secondary EB formation. Conclusion This is the first study to identify the complete transcriptome of BMP2+ cells and cell phenotypes from a mesodermal origin, thus offering an insight into the role of BMP2+ cells during embryonic developmental processes in vivo. PMID:17784959

  13. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

    NASA Astrophysics Data System (ADS)

    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  14. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    DTIC Science & Technology

    2012-10-01

    Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS PRINCIPAL INVESTIGATOR: Nicholas J. Maragakis, M.D...Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS 5b. GRANT NUMBER W81XWH-10-1-0520 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d...into astrocytes following transplantation. 15. SUBJECT TERMS Stem Cells , iPS cells , astrocytes, familial ALS 16. SECURITY CLASSIFICATION OF

  15. Cell-Derived Extracellular Matrix: Basic Characteristics and Current Applications in Orthopedic Tissue Engineering.

    PubMed

    Zhang, Weixiang; Zhu, Yun; Li, Jia; Guo, Quanyi; Peng, Jiang; Liu, Shichen; Yang, Jianhua; Wang, Yu

    2016-06-01

    The extracellular matrix (ECM) is a dynamic and intricate microenvironment with excellent biophysical, biomechanical, and biochemical properties, which can directly or indirectly regulate cell proliferation, adhesion, migration, and differentiation, as well as plays key roles in homeostasis and regeneration of tissues and organs. The ECM has attracted a great deal of attention with the rapid development of tissue engineering in the field of regenerative medicine. Tissue-derived ECM scaffolds (also referred to as decellularized tissues and whole organs) are considered a promising therapy for the repair of musculoskeletal defects, including those that are widely used in orthopedics, although there are a few shortcomings. Similar to tissue-derived ECM scaffolds, cell-derived ECM scaffolds also have highly advantageous biophysical and biochemical properties, in particular their ability to be produced in vitro from a number of different cell types. Furthermore, cell-derived ECM scaffolds more closely resemble native ECM microenvironments. The products of cell-derived ECM have a wide range of biomedical applications; these include reagents for cell culture substrates and biomaterials for scaffolds, hybrid scaffolds, and living cell sheet coculture systems. Although cell-derived ECM has only just begun to be investigated, it has great potential as a novel approach for cell-based tissue repair in orthopedic tissue engineering. This review summarizes and analyzes the various types of cell-derived ECM products applied in cartilage, bone, and nerve tissue engineering in vitro or in vivo and discusses future directions for investigation of cell-derived ECM.

  16. Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes.

    PubMed

    Kabeya, Tomoki; Matsumura, Wakana; Iwao, Takahiro; Hosokawa, Masakiyo; Matsunaga, Tamihide

    2017-04-22

    Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

  17. Leukocyte Cell-Derived Chemotaxin 2-Associated Amyloidosis: A Recently Recognized Disease with Distinct Clinicopathologic Characteristics.

    PubMed

    Nasr, Samih H; Dogan, Ahmet; Larsen, Christopher P

    2015-11-06

    Amyloidosis derived from leukocyte cell-derived chemotaxin 2 is a recently recognized form of amyloidosis, and it has already been established as a frequent form of systemic amyloidosis in the United States, with predominant involvement of kidney and liver. The disease has a strong ethnic bias, affecting mainly Hispanics (particularly Mexicans). Additional ethnic groups prone to develop amyloidosis derived from leukocyte cell-derived chemotaxin 2 include Punjabis, First Nations people in British Columbia, and Native Americans. Most patients are elderly who present with chronic renal insufficiency and bland urinary sediment. Proteinuria is variable, being absent altogether in about one third of patients. Liver involvement is frequently an incidental finding. Amyloidosis derived from leukocyte cell-derived chemotaxin 2 deposits shows a characteristic distribution: in the kidney, there is consistent involvement of cortical interstitium, whereas in the liver, there is a preferential involvement of periportal and pericentral vein regions. Concurrent renal disease is frequent, with diabetic nephropathy and IgA nephropathy being the most common. Patient survival is excellent, likely because of the rarity of cardiac involvement, whereas renal survival is guarded, with a median renal survival of 62 months in those without concurrent renal disease. There is currently no efficacious therapy for amyloidosis derived from leukocyte cell-derived chemotaxin 2 amyloidosis. Renal transplantation seems to be a reasonable treatment for patients with advanced renal failure, although the disease may recur in the allograft. The pathogenesis of amyloidosis derived from leukocyte cell-derived chemotaxin 2 amyloidosis has not yet been elucidated. It could be a result of leukocyte cell-derived chemotaxin 2 overexpression by hepatocytes either constitutively (controlled by yet-uncharacterized genetic defects) or secondary to hepatocellular damage. It is critical not to misdiagnose amyloidosis

  18. Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

    PubMed

    Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

    2012-05-01

    Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs.

  19. Maturation of Stem Cell-Derived Beta-cells Guided by the Expression of Urocortin 3

    PubMed Central

    van der Meulen, Talitha; Huising, Mark O.

    2014-01-01

    Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts. PMID:25148370

  20. Ethical aspects of the use of stem cell derived gametes for reproduction.

    PubMed

    Mertes, Heidi; Pennings, Guido

    2010-09-01

    A lot of interest has been generated by the possibility of deriving gametes from embryonic stem cells and bone marrow stem cells. These stem cell derived gametes may become useful for research and for the treatment of infertility. In this article we consider prospectively the ethical issues that will arise if stem cell derived gametes are used in the clinic, making a distinction between concerns that only apply to embryonic stem cell derived gametes and concerns that are also relevant for gametes derived from adult stem cells. At present, it appears preferable to use non-embryonic stem cells for the derivation of gametes. Adult stem cell derived gametes do not present any problems with regard to the moral status of the human embryo, bypass the safety risks linked to SCNT and do not present any ambiguity or novel problems with regard to informed consent, psychological consequences for the child or genetic parenthood. A remaining ethical concern, however, regards the safety of the procedure in terms of the welfare of the resulting children. This should spark a thorough reflection on how far one must go to accommodate a person's wish to have a genetically related child.

  1. 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

  2. Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

    PubMed

    Mamidi, Murali Krishna; Pal, Rajarshi; Mori, Nor Azah Binti; Arumugam, Greetha; Thrichelvam, Saratha Thevi; Noor, Puteri J; Abdullah, Hj Mohamad Farouk; Gupta, Pawan Kumar; Das, Anjan Kumar; Zakaria, Zubaidah; Bhonde, Ramesh

    2011-05-01

    Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

  3. Therapeutic Potential of Lung Epithelial Progenitor Cells Derived from Embryonic and Induced Pluripotent Stem Cells

    PubMed Central

    Wetsel, Rick A.; Wang, Dachun; Calame, Daniel G.

    2015-01-01

    Embryonic stem (ES) cells derived from preimplantation blastocysts and induced pluripotent stem (iPS) cells generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide a possible unlimited source of cells that could be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. Because of inherent difficulties in deriving endodermal cells from undifferentiated cell cultures, applications using lung epithelial cells derived from ES and iPS cells have lagged behind similar efforts devoted to other tissues, such as the heart and spinal cord. However, during the past several years, significant advances in culture, differentiation, and purification protocols, as well as in bioengineering methodologies, have fueled enthusiasm for the development of stem cell–based lung therapeutics. This article provides an overview of recent research achievements and discusses future technical challenges that must be met before the promise of stem cell applications for lung disease can be realized. PMID:21226612

  4. Pluripotent stem cell derived hepatocytes: using materials to define cellular differentiation and tissue engineering

    PubMed Central

    Lucendo-Villarin, B.; Rashidi, H.; Cameron, K.

    2016-01-01

    Pluripotent stem cell derived liver cells (hepatocytes) represent a promising alternative to primary tissue for biological and clinical applications. To date, most hepatocyte maintenance and differentiation systems have relied upon the use of animal derived components. This serves as a significant barrier to large scale production and application of stem cell derived hepatocytes. Recently, the use of defined biologics has overcome those limitations in two-dimensional monolayer culture. In order to improve the cell phenotype further, three-dimensional culture systems have been employed to better mimic the in vivo situation, drawing upon materials chemistry, engineering and biology. In this review we discuss efforts in the field, to differentiate pluripotent stem cells towards hepatocytes under defined conditions. PMID:27746914

  5. Pluripotent stem cell derived hepatocytes: using materials to define cellular differentiation and tissue engineering.

    PubMed

    Lucendo-Villarin, B; Rashidi, H; Cameron, K; Hay, D C

    2016-05-28

    Pluripotent stem cell derived liver cells (hepatocytes) represent a promising alternative to primary tissue for biological and clinical applications. To date, most hepatocyte maintenance and differentiation systems have relied upon the use of animal derived components. This serves as a significant barrier to large scale production and application of stem cell derived hepatocytes. Recently, the use of defined biologics has overcome those limitations in two-dimensional monolayer culture. In order to improve the cell phenotype further, three-dimensional culture systems have been employed to better mimic the in vivo situation, drawing upon materials chemistry, engineering and biology. In this review we discuss efforts in the field, to differentiate pluripotent stem cells towards hepatocytes under defined conditions.

  6. Stem cell derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model

    PubMed Central

    Donegan, Jennifer J.; Tyson, Jennifer A.; Branch, Sarah Y.; Beckstead, Michael J.; Anderson, Stewart A.; Lodge, Daniel J.

    2016-01-01

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol (MAM) rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity, and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia. PMID:27480492

  7. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes

    PubMed Central

    Eng, George; Lee, Benjamin W.; Protas, Lev; Gagliardi, Mark; Brown, Kristy; Kass, Robert S.; Keller, Gordon; Robinson, Richard B.; Vunjak-Novakovic, Gordana

    2016-01-01

    The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration. PMID:26785135

  8. Large scale production of a mammalian cell derived quadrivalent hepatitis C virus like particle vaccine.

    PubMed

    Earnest-Silveira, L; Christiansen, D; Herrmann, S; Ralph, S A; Das, S; Gowans, E J; Torresi, J

    2016-10-01

    A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus-like particles (HCV VLPs) is described. The HCV core E1 and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine.

  9. Glucose responsive insulin production from human embryonic germ (EG) cell derivatives

    SciTech Connect

    Clark, Gregory O.; Yochem, Robert L.; Axelman, Joyce; Sheets, Timothy P.; Kaczorowski, David J.; Shamblott, Michael J. . E-mail: mshambl1@jhmi.edu

    2007-05-11

    Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and {beta}-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.

  10. Xenotransplantation of embryonic stem cell-derived motor neurons into the developing chick spinal cord.

    PubMed

    Wichterle, Hynek; Peljto, Mirza; Nedelec, Stephane

    2009-01-01

    A growing number of specific cell types have been successfully derived from embryonic stem cells (ES cells), including a variety of neural cells. In vitro generated cells need to be extensively characterized to establish functional equivalency with their in vivo counterparts. The ultimate test for the ability of ES cell-derived neurons to functionally integrate into neural networks is transplantation into the developing central nervous system, a challenging technique limited by the poor accessibility of mammalian embryos. Here we describe xenotransplantation of mouse embryonic stem cell-derived motor neurons into the developing chick neural tube as an alternative for testing the ability of in vitro generated neurons to survive, integrate, extend axons, and form appropriate synaptic contacts with functionally relevant targets in vivo. Similar methods can be adapted to study functionality of other mammalian cells, including derivatives of human ES cells.

  11. PIEZO2 is required for mechanotransduction in human stem cell-derived touch receptors.

    PubMed

    Schrenk-Siemens, Katrin; Wende, Hagen; Prato, Vincenzo; Song, Kun; Rostock, Charlotte; Loewer, Alexander; Utikal, Jochen; Lewin, Gary R; Lechner, Stefan G; Siemens, Jan

    2015-01-01

    Human sensory neurons are inaccessible for functional examination, and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro, we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals, their most salient characteristic in vivo. Strikingly, mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors, which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity.

  12. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    DTIC Science & Technology

    2014-12-01

    transporter GLT1, connexin 43, aquaporin 4, the cell surface marker CD44 (a marker of astrocyte precursor identity), and the intermediate filament ... print ] 2. iPSC-Derived GRPs from FUS H517Q ALS do not significantly differ from control iPSC-derived GRP lines One question is whether...Cell-Derived Astrocyte Progenitors Following Spinal Cord Engraftment. Stem Cells Transl Med. 2014 Mar 6. [Epub ahead of print ] (see Appendix) Amanda

  13. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.

    PubMed

    Araki, Ryoko; Uda, Masahiro; Hoki, Yuko; Sunayama, Misato; Nakamura, Miki; Ando, Shunsuke; Sugiura, Mayumi; Ideno, Hisashi; Shimada, Akemi; Nifuji, Akira; Abe, Masumi

    2013-02-07

    The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues. However, partial reprogramming and genetic instabilities in iPSCs could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

  14. Learning-induced synaptic potentiation in implanted neural precursor cell-derived neurons

    PubMed Central

    Park, Kyungjoon; Heo, Hwon; Han, Ma Eum; Choi, Kyuhyun; Yi, Jee Hyun; Kang, Shin Jung; Kwon, Yunhee Kim; Shin, Ki Soon

    2015-01-01

    Neuronal loss caused by neurodegenerative diseases, traumatic brain injury and stroke results in cognitive dysfunctioning. Implantation of neural stem/precursor cells (NPCs) can improve the brain function by replacing lost neurons. Proper synaptic integration following neuronal differentiation of implanted cells is believed to be a prerequisite for the functional recovery. In the present study, we characterized the functional properties of immortalized neural progenitor HiB5 cells implanted into the rat hippocampus with chemically induced lesion. The implanted HiB5 cells migrated toward CA1 pyramidal layer and differentiated into vGluT1-positive glutamatergic neurons with morphological and electrophysiological properties of endogenous CA1 pyramidal cells. Functional synaptic integration of HiB5 cell-derived neurons was also evidenced by immunohistochemical and electrophysiological data. Lesion-caused memory deficit was significantly recovered after the implantation when assessed by inhibitory avoidance (IA) learning. Remarkably, IA learning preferentially produced long-term potentiation (LTP) at the synapses onto HiB5 cell-derived neurons, which occluded paring protocol-induced LTP ex vivo. We conclude that the implanted HiB5 cell-derived neurons actively participate in learning process through LTP formation, thereby counteracting lesion-mediated memory impairment. PMID:26634434

  15. 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.

  16. Methods for Assessing the Electromechanical Integration of Human Pluripotent Stem Cell-Derived Cardiomyocyte Grafts

    PubMed Central

    Zhu, Wei-Zhong; Filice, Dominic; Palpant, Nathan J.; Laflamme, Michael A.

    2014-01-01

    Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host–graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models. PMID:25070341

  17. Methods for assessing the electromechanical integration of human pluripotent stem cell-derived cardiomyocyte grafts.

    PubMed

    Zhu, Wei-Zhong; Filice, Dominic; Palpant, Nathan J; Laflamme, Michael A

    2014-01-01

    Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host-graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models.

  18. Angiogenic activity mediates bone repair from human pluripotent stem cell-derived osteogenic cells

    PubMed Central

    Zou, Li; Chen, Qingshan; Quanbeck, Zachary; Bechtold, Joan E.; Kaufman, Dan S.

    2016-01-01

    Human pluripotent stem cells provide a standardized resource for bone repair. However, criteria to determine which exogenous cells best heal orthopedic injuries remain poorly defined. We evaluated osteogenic progenitor cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Phenotypic and genotypic analyses demonstrated that these hESCs/hiPSCs are similar in their osteogenic differentiation efficiency and they generate osteogenic cells comparable to osteogenic cells derived from mesenchymal stromal cells (BM-MSCs). However, expression of angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor in these osteogenic progenitor cells are markedly different, suggesting distinct pro-angiogenic potential of these stem cell derivatives. Studies to repair a femur non-union fracture demonstrate only osteogenic progenitor cells with higher pro-angiogenic potential significantly enhance bone repair in vivo. Together, these studies highlight a key role of pro-angiogenic potential of transplanted osteogenic cells for effective cell-mediated bone repair. PMID:26980556

  19. Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization

    PubMed Central

    Han, Kyu-Yeon; Tran, Jennifer A.; Chang, Jin-Hong; Azar, Dimitri T.; Zieske, James D.

    2017-01-01

    Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions. PMID:28165027

  20. The influence and possible recombination of genotypes on the production of microspore embryoids in anther cultures of Solanum tuberosum and dihaploid hybrids.

    PubMed

    Jacobsen, E; Sopory, S K

    1978-05-01

    In addition to physical and chemical factors, genotype appears to be a very important factor influencing success in anther culture. Recombination by making crosses with selected responding clones has been introduced as a possible helpful method to positively influence the success and response type via the factor genotype. From the progeny of such a cross, one genotype could be selected, producing in 30 to 40 percent of the cultured anthers, fully developed embryoids and plantlets, which are a mixture of polyploids, dihaploids and monohaploids.Further, a pleiotropic marker 'embryo spot' visible as a 'nodal band' in the plant stage, has been used to confirm the microsporic origin of dihaploids and polyploids and to prove their homozygous nature. This marker also shows potential use in confirming the origin of calli from individual microspores.

  1. Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering

    PubMed Central

    Lowenthal, Justin; Gerecht, Sharon

    2016-01-01

    Proper blood vessel networks are necessary for constructing and re-constructing tissues, promoting wound healing, and delivering metabolic necessities throughout the body. Conversely, an understanding of vascular dysfunction has provided insight into the pathogenesis and progression of diseases both common and rare. Recent advances in stem cell-based regenerative medicine – including advances in stem cell technologies and related progress in bioscaffold design and complex tissue engineering – have allowed rapid advances in the field of vascular biology, leading in turn to more advanced modeling of vascular pathophysiology and improved engineering of vascularized tissue constructs. In this review we examine recent advances in the field of stem cell-derived vasculature, providing an overview of stem cell technologies as a source for vascular cell types and then focusing on their use in three primary areas: studies of vascular development and angiogenesis, improved disease modeling, and the engineering of vascularized constructs for tissue-level modeling and cell-based therapies. PMID:26427871

  2. Epithelial cell-derived micro RNA-146a generates interleukin-10-producing monocytes to inhibit nasal allergy.

    PubMed

    Luo, Xi; Han, Miaomiao; Liu, Jianqi; Wang, Yu; Luo, Xiangqian; Zheng, Jing; Wang, Shuai; Liu, Zhigang; Liu, Dabo; Yang, Ping-Chang; Li, Huabin

    2015-11-03

    The aberrant immunity plays an important role in the pathogenesis of allergic diseases. Micro RNAs (miR) are involved in regulating the immunity in the body. This study aims to test a hypothesis that miR-146a induces the expression of interleukin (IL)-10 in monocytes (Mos). In this study, the levels of miR-146a were determined by real time RT-PCR. The IL-10(+) Mos were evaluated by flow cytometry. The miR-146a-laden exosomes were generated with RPMI2650 cells (an airway epithelial cell line). An allergic rhinitis mouse model was developed. The results showed that nasal epithelial cells expressed miR-146a, which was markedly lower in the nasal epithelial cells of patients with nasal allergy than that in healthy controls. Exposure to T helper (Th)2 cytokines suppressed the levels of miR-146a in the nasal epithelial cells. The nasal epithelial cell-derived miR-146a up regulated the expression of IL-10 in Mos. The inducible IL-10(+) Mos showed an immune suppressor effect on the activities of CD4(+) effector T cells and the Th2 polarization in a mouse model of allergic rhinitis. In summary, nasal epithelial cells express miR-146a, the latter is capable of inducing IL-10 expression in Mos, which suppress allergic reactions in the mouse nasal mucosa.

  3. The effect of PVDF-TrFE scaffolds on stem cell derived cardiovascular cells.

    PubMed

    Hitscherich, Pamela; Wu, Siliang; Gordan, Richard; Xie, Lai-Hua; Arinzeh, Treena; Lee, Eun Jung

    2016-07-01

    Recently, electrospun polyvinylidene fluoride (PVDF) and polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) scaffolds have been developed for tissue engineering applications. These materials have piezoelectric activity, wherein they can generate electric charge with minute mechanical deformations. Since the myocardium is an electroactive tissue, the unique feature of a piezoelectric scaffold is attractive for cardiovascular tissue engineering applications. In this study, we examined the cytocompatibility and function of pluripotent stem cell derived cardiovascular cells including mouse embryonic stem cell-derived cardiomyocytes (mES-CM) and endothelial cells (mES-EC) on PVDF-TrFE scaffolds. MES-CM and mES-EC adhered well to PVDF-TrFE and became highly aligned along the fibers. When cultured on scaffolds, mES-CM spontaneously contracted, exhibited well-registered sarcomeres and expressed classic cardiac specific markers such as myosin heavy chain, cardiac troponin T, and connexin43. Moreover, mES-CM cultured on PVDF-TrFE scaffolds responded to exogenous electrical pacing and exhibited intracellular calcium handling behavior similar to that of mES-CM cultured in 2D. Similar to cardiomyocytes, mES-EC also demonstrated high viability and maintained a mature phenotype through uptake of low-density lipoprotein and expression of classic endothelial cell markers including platelet endothelial cell adhesion molecule, endothelial nitric oxide synthase, and the arterial specific marker, Notch-1. This study demonstrates the feasibility of PVDF-TrFE scaffold as a candidate material for developing engineered cardiovascular tissues utilizing stem cell-derived cells. Biotechnol. Bioeng. 2016;113: 1577-1585. © 2015 Wiley Periodicals, Inc.

  4. Magnetic Resonance Imaging of Iron Oxide-Labeled Human Embryonic Stem Cell-Derived Cardiac Progenitors

    PubMed Central

    Skelton, Rhys J.P.; Khoja, Suhail; Almeida, Shone; Rapacchi, Stanislas; Han, Fei; Engel, James; Zhao, Peng; Hu, Peng; Stanley, Edouard G.; Elefanty, Andrew G.; Kwon, Murray

    2016-01-01

    Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 μg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. Significance The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts

  5. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    NASA Astrophysics Data System (ADS)

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  6. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

    PubMed

    Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

    2017-02-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

  7. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice

    PubMed Central

    Boylan, Brendan T.; Moreira, Fernando R.; Carlson, Tim W.

    2017-01-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses. PMID:28187142

  8. Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

    PubMed

    Marton, Annamaria; Vizler, Csaba; Kusz, Erzsebet; Temesfoi, Viktoria; Szathmary, Zsuzsa; Nagy, Krisztina; Szegletes, Zsolt; Varo, Gyorgy; Siklos, Laszlo; Katona, Robert L; Tubak, Vilmos; Howard, O M Zack; Duda, Erno; Minarovits, Janos; Nagy, Katalin; Buzas, Krisztina

    2012-01-01

    To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

  9. Human Cytomegalovirus Carries a Cell-Derived Phospholipase A2 Required for Infectivity

    PubMed Central

    Allal, Cuider; Buisson-Brenac, Claire; Marion, Vincent; Claudel-Renard, Clotilde; Faraut, Thomas; Dal Monte, Paola; Streblow, Daniel; Record, Michel

    2004-01-01

    Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2α, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity. PMID:15220446

  10. Regenerating the injured kidney with human umbilical cord mesenchymal stem cell-derived exosomes.

    PubMed

    Dorronsoro, Akaitz; Robbins, Paul D

    2013-04-25

    Transplantation of adult stem cells is being used to facilitate repair or regeneration of damaged or diseased tissues. However, in many cases, the therapeutic effects of the injected stem cells are mediated by factors secreted by stem cells and not by differentiation of the transplanted stem cells. Recent reports have identified a class of microvesicles, termed exosomes, released by stem cells that are able to confer therapeutic effects on injured renal and cardiac tissue. In this issue of Stem Cell Research & Therapy, Zhou and colleagues demonstrate the ability of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs), but not non-stem cell-derived exosomes, to improve acute kidney injury induced by cisplatin in rats. The authors demonstrate that hucMSC exosomes can reduce cisplatin-mediated renal oxidative stress and apoptosis in vivo and increase renal epithelial cell proliferation in culture. These results suggest that stem cell-derived exosomes, which are easy to isolate and safer to use than the parental stem cells, could have significant clinical utility.

  11. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    SciTech Connect

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  12. Decellularized Tissue and Cell-Derived Extracellular Matrices as Scaffolds for Orthopaedic Tissue Engineering

    PubMed Central

    Cheng, Christina W.; Solorio, Loran D.; Alsberg, Eben

    2014-01-01

    The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic. PMID:24417915

  13. Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons.

    PubMed

    Chuang, Wenpo; Sharma, Arun; Shukla, Praveen; Li, Guang; Mall, Moritz; Rajarajan, Kuppusamy; Abilez, Oscar J; Hamaguchi, Ryoko; Wu, Joseph C; Wernig, Marius; Wu, Sean M

    2017-03-22

    Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

  14. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes.

    PubMed

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  15. Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons

    PubMed Central

    Chuang, Wenpo; Sharma, Arun; Shukla, Praveen; Li, Guang; Mall, Moritz; Rajarajan, Kuppusamy; Abilez, Oscar J.; Hamaguchi, Ryoko; Wu, Joseph C.; Wernig, Marius; Wu, Sean M.

    2017-01-01

    Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs. PMID:28327614

  16. Functional Properties of Human Stem Cell-Derived Neurons in Health and Disease

    PubMed Central

    Weick, Jason P.

    2016-01-01

    Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS) development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications. PMID:27274733

  17. Microcircuit formation following transplantation of mouse embryonic stem cell-derived neurons in peripheral nerve.

    PubMed

    Magown, Philippe; Rafuse, Victor F; Brownstone, Robert M

    2017-04-01

    Motoneurons derived from embryonic stem cells can be transplanted in the tibial nerve, where they extend axons to functionally innervate target muscle. Here, we studied spontaneous muscle contractions in these grafts 3 mo following transplantation. One-half of the transplanted grafts generated rhythmic muscle contractions of variable patterns, either spontaneously or in response to brief electrical stimulation. Activity generated by transplanted embryonic stem cell-derived neurons was driven by glutamate and was modulated by muscarinic and GABAergic/glycinergic transmission. Furthermore, rhythmicity was promoted by the same transmitter combination that evokes rhythmic locomotor activity in spinal cord circuits. These results demonstrate that there is a degree of self-assembly of microcircuits in these peripheral grafts involving embryonic stem cell-derived motoneurons and interneurons. Such spontaneous activity is reminiscent of embryonic circuit development in which spontaneous activity is essential for proper connectivity and function and may be necessary for the grafts to form functional connections with muscle.NEW & NOTEWORTHY This manuscript demonstrates that, following peripheral transplantation of neurons derived from embryonic stem cells, the grafts are spontaneously active. The activity is produced and modulated by a number of transmitter systems, indicating that there is a degree of self-assembly of circuits in the grafts.

  18. Expansion and Characterization of Human Embryonic Stem Cell-Derived Osteoblast-Like Cells

    PubMed Central

    Arpornmaeklong, Premjit; Wang, Zhuo; Pressler, Michael J.; Brown, Shelley E.

    2010-01-01

    Abstract Human embryonic stem cells (hESCs) have the potential to serve as a repository of cells for the replacement of damaged or diseased tissues and organs. However, to use hESCs in clinically relevant scenarios, a large number of cells are likely to be required. The aim of this study was to demonstrate an alternative cell culture method to increase the quantity of osteoblast-like cells directly derived from hESCs (hESCs-OS). Undifferentiated hESCs were directly cultivated and serially passaged in osteogenic medium (hESC-OS), and exhibited similar expression patterns of osteoblast-related genes to osteoblast-like cells derived from mesenchymal stem cells derived from hESCs (hESCs-MSCs-OS) and human bone marrow stromal cells (hBMSCs-OS). In comparison to hESCs-MSCs-OS, the hESCs-OS required a shorter expansion time to generate a homogenous population of osteoblast-like cells that did not contain contaminating undifferentiated hESCs. Identification of human specific nuclear antigen (HuNu) in the newly formed bone in calvarial defects verified the role of the transplanted hESCs-OS as active bone forming cells in vivo. Taken together, this study suggests that osteoblast-like cells directly derived from hESCs have the potential to serve as an alternative source of osteoprogenitors for bone tissue engineering strategies. PMID:20698777

  19. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  20. Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network.

    PubMed

    Tarczyluk, Marta A; Nagel, David A; O'Neil, John D; Parri, H Rheinallt; Tse, Erin H Y; Coleman, Michael D; Hill, Eric J

    2013-09-01

    The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture.

  1. In-vitro stem cell derived red blood cells for transfusion: are we there yet?

    PubMed

    Kim, Hyun Ok

    2014-03-01

    To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices.

  2. Stem cell-derived models to improve mechanistic understanding and prediction of human drug induced liver injury

    PubMed Central

    Goldring, Christopher; Antoine, Daniel J.; Bonner, Frank; Crozier, Jonathan; Denning, Chris; Fontana, Robert J.; Hanley, Neil A.; Hay, David C.; Ingelman-Sundberg, Magnus; Juhila, Satu; Kitteringham, Neil; Silva-Lima, Beatriz; Norris, Alan; Pridgeon, Chris; Ross, James A.; Sison Young, Rowena; Tagle, Danilo; Tornesi, Belen; van de Water, Bob; Weaver, Richard J.; Zhang, Fang; Park, B. Kevin

    2016-01-01

    Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalised toxicology to determine inter-individual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury (DILI) means that no current single cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human DILI. Nevertheless, a single cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment, and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally-differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. PMID:27775817

  3. Stem cell-derived models to improve mechanistic understanding and prediction of human drug-induced liver injury.

    PubMed

    Goldring, Christopher; Antoine, Daniel J; Bonner, Frank; Crozier, Jonathan; Denning, Chris; Fontana, Robert J; Hanley, Neil A; Hay, David C; Ingelman-Sundberg, Magnus; Juhila, Satu; Kitteringham, Neil; Silva-Lima, Beatriz; Norris, Alan; Pridgeon, Chris; Ross, James A; Young, Rowena Sison; Tagle, Danilo; Tornesi, Belen; van de Water, Bob; Weaver, Richard J; Zhang, Fang; Park, B Kevin

    2017-02-01

    Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).

  4. Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes.

    PubMed

    del Cacho, Emilio; Gallego, Margarita; Lee, Sung Hyen; Lillehoj, Hyun Soon; Quilez, Joaquin; Lillehoj, Erik P; Sánchez-Acedo, Caridad

    2012-05-01

    This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.

  5. Apoptosis induced by the Tibetan herbal remedy PADMA 28 in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2

    PubMed Central

    Jenny, Marcel; Schwaiger, Wolfgang; Bernhard, David; Wrulich, Oliver A; Cosaceanu, Daria; Fuchs, Dietmar; Ueberall, Florian

    2005-01-01

    The Tibetan herbal remedy PADMA 28 revealed promising results to support treatment of atherosclerosis, Charot syndrome (intermittent claudication), chronic active hepatitis and infection of the respiratory tract. The remedy was confirmed to be closely linked with anti- and pro-oxidative properties in vitro. In this study, apoptogenic and survival effects of PADMA 28 were investigated in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2. PADMA 28 led to a concentration-dependent inhibition of cell proliferation accompanied by the accumulation of CEM-C7H2 cells in subG1 phase, fragmentation of poly (ADP-ribose) polymerase (PARP) and nuclear body formation. Treatment with PADMA 28 rescued to some extent cells over-expressing Bcl-2 from apoptosis. This finding suggests that the mechanism of action of PADMA 28 may be via interference with Bcl-2 triggered survival pathways. PMID:16138918

  6. Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

    PubMed

    Muppidi, Jagan R; Schmitz, Roland; Green, Jesse A; Xiao, Wenming; Larsen, Adrien B; Braun, Sterling E; An, Jinping; Xu, Ying; Rosenwald, Andreas; Ott, German; Gascoyne, Randy D; Rimsza, Lisa M; Campo, Elias; Jaffe, Elaine S; Delabie, Jan; Smeland, Erlend B; Braziel, Rita M; Tubbs, Raymond R; Cook, J R; Weisenburger, Dennis D; Chan, Wing C; Vaidehi, Nagarajan; Staudt, Louis M; Cyster, Jason G

    2014-12-11

    Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

  7. Optical control of muscle function by transplantation of stem cell-derived motor neurons in mice.

    PubMed

    Bryson, J Barney; Machado, Carolina Barcellos; Crossley, Martin; Stevenson, Danielle; Bros-Facer, Virginie; Burrone, Juan; Greensmith, Linda; Lieberam, Ivo

    2014-04-04

    Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell-derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.

  8. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model?

    PubMed

    Knollmann, Björn C

    2013-03-15

    This article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize antiarrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders, such as long QT syndrome, Brugada Syndrome, or Catecholaminergic Polymorphic Ventricular Tachycardia.

  9. Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture

    PubMed Central

    Gieseck III, Richard L.; Hannan, Nicholas R. F.; Bort, Roque; Hanley, Neil A.; Drake, Rosemary A. L.; Cameron, Grant W. W.; Wynn, Thomas A.; Vallier, Ludovic

    2014-01-01

    Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard. PMID:24466060

  10. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    PubMed Central

    Bayzigitov, Daniel R.; Medvedev, Sergey P.; Dementyeva, Elena V.; Bayramova, Sevda A.; Pokushalov, Evgeny A.; Karaskov, Alexander M.; Zakian, Suren M.

    2016-01-01

    Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes. PMID:27110425

  11. Role of stromal cell-derived factor 1α pathway in bone metastatic prostate cancer

    PubMed Central

    Gupta, Nisha; Duda, Dan G.

    2016-01-01

    Abstract Metastatic prostate cancer is one of the leading causes of cancer-related death in men. The primary site of metastasis from prostate cancers is the bone. During the last decade, multiple studies have pointed to the role of the stromal cell-derived factor 1 alpha (SDF1α)/CXCR4 axis in the metastatic spread of the disease, but the mechanisms that underlie this effect are still incompletely understood. In this review, we summarize the current understanding of the role of the SDF1α/CXCR4 pathway in bone metastatic prostate cancer. We also discuss the therapeutic potential of disrupting the interaction between prostate tumor cells and bone environment with focus on the SDF1α pathway. PMID:27533927

  12. Myeloid and T Cell-Derived TNF Protects against Central Nervous System Tuberculosis

    PubMed Central

    Hsu, Nai-Jen; Francisco, Ngiambudulu M.; Keeton, Roanne; Allie, Nasiema; Quesniaux, Valérie F. J.; Ryffel, Bernhard; Jacobs, Muazzam

    2017-01-01

    Tuberculosis of the central nervous system (CNS-TB) is a devastating complication of tuberculosis, and tumor necrosis factor (TNF) is crucial for innate immunity and controlling the infection. TNF is produced by many cell types upon activation, in particularly the myeloid and T cells during neuroinflammation. Here we used mice with TNF ablation targeted to myeloid and T cell (MT-TNF−/−) to assess the contribution of myeloid and T cell-derived TNF in immune responses during CNS-TB. These mice exhibited impaired innate immunity and high susceptibility to cerebral Mycobacterium tuberculosis infection, a similar phenotype to complete TNF-deficient mice. Further, MT-TNF−/− mice were not able to control T cell responses and cytokine/chemokine production. Thus, our data suggested that collective TNF production by both myeloid and T cells are required to provide overall protective immunity against CNS-TB infection. PMID:28280495

  13. Bioengineering Approaches to Mature Human Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Sun, Xuetao; Nunes, Sara S

    2017-01-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) represent a potential unlimited cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, hPSC-CMs produced by current protocols are not representative of native adult human cardiomyocytes as they display immature gene expression profile, structure and function. In order to improve hPSC-CM maturity and function, various approaches have been developed, including genetic manipulations to induce gene expression, delivery of biochemical factors, such as triiodothyronine and alpha-adrenergic agonist phenylephrine, induction of cell alignment in 3D tissues, mechanical stress as a mimic of cardiac load and electrical stimulation/pacing or a combination of these. In this mini review, we discuss biomimetic strategies for the maturation for hPSC-CMs with a particular focus on electromechanical conditioning methods.

  14. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.

    PubMed

    Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei

    2014-02-21

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction.

  15. Bioengineering Approaches to Mature Human Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Sun, Xuetao; Nunes, Sara S.

    2017-01-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) represent a potential unlimited cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, hPSC-CMs produced by current protocols are not representative of native adult human cardiomyocytes as they display immature gene expression profile, structure and function. In order to improve hPSC-CM maturity and function, various approaches have been developed, including genetic manipulations to induce gene expression, delivery of biochemical factors, such as triiodothyronine and alpha-adrenergic agonist phenylephrine, induction of cell alignment in 3D tissues, mechanical stress as a mimic of cardiac load and electrical stimulation/pacing or a combination of these. In this mini review, we discuss biomimetic strategies for the maturation for hPSC-CMs with a particular focus on electromechanical conditioning methods. PMID:28337437

  16. Myeloid and T Cell-Derived TNF Protects against Central Nervous System Tuberculosis.

    PubMed

    Hsu, Nai-Jen; Francisco, Ngiambudulu M; Keeton, Roanne; Allie, Nasiema; Quesniaux, Valérie F J; Ryffel, Bernhard; Jacobs, Muazzam

    2017-01-01

    Tuberculosis of the central nervous system (CNS-TB) is a devastating complication of tuberculosis, and tumor necrosis factor (TNF) is crucial for innate immunity and controlling the infection. TNF is produced by many cell types upon activation, in particularly the myeloid and T cells during neuroinflammation. Here we used mice with TNF ablation targeted to myeloid and T cell (MT-TNF(-/-)) to assess the contribution of myeloid and T cell-derived TNF in immune responses during CNS-TB. These mice exhibited impaired innate immunity and high susceptibility to cerebral Mycobacterium tuberculosis infection, a similar phenotype to complete TNF-deficient mice. Further, MT-TNF(-/-) mice were not able to control T cell responses and cytokine/chemokine production. Thus, our data suggested that collective TNF production by both myeloid and T cells are required to provide overall protective immunity against CNS-TB infection.

  17. Replication of Human Noroviruses in Stem Cell-Derived Human Enteroids

    PubMed Central

    Ettayebi, Khalil; Crawford, Sue E.; Murakami, Kosuke; Broughman, James R.; Karandikar, Umesh; Tenge, Victoria R.; Neill, Frederick H.; Blutt, Sarah E.; Zeng, Xi-Lei; Qu, Lin; Kou, Baijun; Opekun, Antone R.; Burrin, Douglas; Graham, David Y.; Ramani, Sasirekha; Atmar, Robert L.

    2016-01-01

    The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections. PMID:27562956

  18. Application potential of mesenchymal stem cells derived from Wharton's jelly in liver tissue engineering.

    PubMed

    Zhang, Lei; Zhao, Yong-Hen; Guan, Zheng; Ye, Jun-Song; de Isla, Natalia; Stoltz, Jean-François

    2015-01-01

    The shortage of organ resource has been limiting the application of liver transplantation. Bioartificial liver construction is increasingly focused as a replacement treatment. To product a bioartificial liver, three elements must be considered: seeding cells, scaffold and bioreactor. Recent studies have shown that several methods can successfully differentiate MSC (mesenchymal stem cells) derived from Wharton's jelly into hepatocyte, such as stimulating MSC by cytokines and growth factors, direct and indirect co-culture MSC with hepatocytes, or promote MSC differentiation by 3-dimensional matrix. In some cases, differentiation of MSC into hepatocytes can also be an alternative approach for whole organ transplantation in treatment of acute and chronic liver diseases. In this review, the characterization of MSC from Wharton's jelly, their potential of application in liver tissue engineering on base of decellularized scaffold, their status of banking and their preclinical work performed will be discussed.

  19. A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

    PubMed

    Wiley, Luke A; Beebe, David C; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-05-12

    This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

  20. Cryopreservation of Human Pluripotent Stem Cell-derived Cardiomyocytes: Strategies, Challenges, and Future Directions

    PubMed Central

    Preininger, Marcela K.; Singh, Monalisa; Xu, Chunhui

    2017-01-01

    In recent years, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a vital cell source for in vitro modeling of genetic cardiovascular disorders, drug screening, and in vivo cardiac regeneration research. Looking forward, the ability to efficiently cryopreserve hPSC-CMs without compromising their normal biochemical and physiologic functions will dramatically facilitate their various biomedical applications. Although working protocols for freezing, storing, and thawing hPSC-CMs have been established, the question remains as to whether they are optimal. In this chapter, we discuss our current understanding of cryopreservation appertaining to hPSC-CMs, and proffer key questions regarding the mechanical, contractile, and regenerative properties of cryopreserved hPSC-CMs. PMID:27837559

  1. clickECM: Development of a cell-derived extracellular matrix with azide functionalities.

    PubMed

    Ruff, S M; Keller, S; Wieland, D E; Wittmann, V; Tovar, G E M; Bach, M; Kluger, P J

    2016-12-10

    In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this "clickECM" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM.

  2. Extracellular Matrix Mediated Maturation of Human Pluripotent Stem Cell Derived Cardiac Monolayer Structure and Electrophysiological Function

    PubMed Central

    Herron, Todd J.; Rocha, Andre Monteiro Da; Campbell, Katherine; Ponce-Balbuena, Daniela; Willis, B. Cicero; Guerrero-Serna, Guadalupe; Liu, Qinghua; Klos, Matt; Musa, Hassan; Zarzoso, Manuel; Bizy, Alexandra; Furness, Jamie; Anumonwo, Justus; Mironov, Sergey; Jalife, José

    2016-01-01

    Background Human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electrical impulse propagation velocity and immature action potential profiles. Methods and Results Here we have identified an optimal extracellular matrix (ECM) for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal ECM combination have impulse propagation velocities ~2X faster than previously reported (43.6±7.0 cm·s−1 n=9) and have mature cardiomyocyte action potential profiles including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s, N=5 monolayers). In addition the optimal ECM promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1 and Connexin43) and myofilament markers (cTroponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase (FAK) activation prevented structural maturation. Conclusions Maturation of human stem cell derived cardiomyocyte monolayers is achieved in a one week period by plating cardiomyocytes on PDMS coverslips rather than on conventional 2D cell culture formats such as glass coverslips or plastic dishes. Activation of integrin signaling and FAK are essential for significant maturation of human cardiac monolayers. PMID:27069088

  3. Stem Cells Derived from Tooth Periodontal Ligament Enhance Functional Angiogenesis by Endothelial Cells

    PubMed Central

    Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J.; Tarle, Susan A.

    2014-01-01

    In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential

  4. Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells.

    PubMed

    Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J; Tarle, Susan A; Kaigler, Darnell

    2014-04-01

    In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential

  5. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    PubMed

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-07

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

  6. A cloned toy poodle produced from somatic cells derived from an aged female dog.

    PubMed

    Jang, G; Hong, S G; Oh, H J; Kim, M K; Park, J E; Kim, H J; Kim, D Y; Lee, B C

    2008-03-15

    To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.

  7. Effects of tacrolimus on morphology, proliferation and differentiation of mesenchymal stem cells derived from gingiva tissue

    PubMed Central

    HA, DONG-HO; YONG, CHUL SOON; KIM, JONG OH; JEONG, JEE-HEON; PARK, JUN-BEOM

    2016-01-01

    Tacrolimus is a 23-membered macrolide lactone with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following kidney, heart and liver transplantation. Tacrolimus also exerts a variety of actions on bone metabolism. The aim of the present study was to evaluate the effects of different concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva. Gingival-derived stem cells were grown in the presence of tacrolimus at final concentrations ranging from 0.001 to 100 µg/ml. The morphology of the cells was viewed under an inverted microscope and the cell viability was analyzed using Cell Counting kit-8 (CCK-8) on days 1, 3, 5 and 7. Alizarin Red S staining was used to assess mineralization of treated cells. The control group showed spindle-shaped, fibroblast-like morphology and the shapes of the cells in 0.001, 0.01, 0.1, 1 and 10 µg/ml tacrolimus were similar to those of the control group. All groups except the 100 µg/ml group showed increased cell proliferation over time. Cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, 1 and 10 µg/ml were not identified to be significantly different compared with the control at days 1, 3 and 5 using the CCK-8 assays. Increased mineralized deposits were noted with increased incubation time. Treatment with tacrolimus from 0.001 to 1 µg/ml led to an increase in mineralization compared with the control group. Within the limits of this study, tacrolimus at the tested concentrations (ranging from 0.001 to 10 µg/ml) did not result in differences in the viability of stem cells derived from gingiva; however it did enhance osteogenic differentiation of the stem cells. PMID:27177273

  8. Reversible Commitment to Differentiation by Human Multipotent Stromal Cells (MSCs) in Single-Cell Derived Colonies

    PubMed Central

    Ylöstalo, Joni; Bazhanov, Nikolay; Prockop, Darwin J

    2008-01-01

    Objective Human multipotent stromal cells (MSCs) readily form single-cell derived colonies when plated at clonal densities. However, the colonies are heterogeneous since the cells from a colony form new colonies that vary in size and differentiation potential when re-plated at clonal densities. The experiments here tested the hypothesis that the cells in the inner regions of colonies are partially differentiated but the differentiation is reversible. Materials and Methods Cells were separately isolated from the dense inner regions (IN) and less dense outer regions (OUT) of single-cell derived colonies. The cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. Results The IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when re-plated at clonal densities and assayed for rates of propagation and clonogenicity. Also, the colonies formed by IN cells were similar to colonies formed by OUT cells in that they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30-75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. Conclusions The results demonstrated that the cells in the inner region of single-derived colonies are partially differentiated but the differentiation can be reversed by re-plating the cells at clonal densities. PMID:18619725

  9. Immunogenicity of single-dose Vero cell-derived Japanese encephalitis vaccine in Japanese adults.

    PubMed

    Takeshita, Nozomi; Lim, Chang-Kweng; Mizuno, Yasutaka; Shimbo, Takuro; Kotaki, Akira; Ujiie, Mugen; Hayakawa, Kayoko; Kato, Yasuyuki; Kanagawa, Shuzo; Kaku, Mitsuo; Takasaki, Tomohiko

    2014-04-01

    In Japan, intensive immunization against Japanese encephalitis (JE) was performed from 1967 to 1976, and regular JE immunization was performed thereafter. However, for Japanese adults facing JE risk, dates of vaccination with new inactivated Vero cell-derived JE vaccine are unavailable. This study investigated how a single dose of Vero cell-derived JE vaccine affects Japanese adults. Neutralizing antibodies were measured pre- and post-JE vaccination in 79 participants (age 40.7 ± 9.4 years), enrolled between October 2009 and March 2011, whose JE-vaccination data were gathered from vaccination records and history taking. Before vaccination, the participants' seroprotection rate (SPR) was 51.9%, whereas SPR after vaccination was 93.7%. The seroconversion rate (SCR), which measures seronegative cases that turn seropositive after vaccination, was 86.8%. The geometric mean titer (GMT) was 14.7 before vaccination and 70.1 after vaccination. Age was a significant difference between seroprotected (42.8 years) and non-seroprotected (38.7 years) groups before vaccination. Then the difference of age, SCR, pre-vaccination GMT, post-vaccination GMT and sex ratio were also significant in participants aged 25-39 years and ≥40 years, who represent generations born when Japan's JE-vaccination policy changed. SCR was 100% in participants aged 25-39 years with a vaccination recorded 55.6% in participants aged 25-39 without a vaccination record, and 96.0% in participants aged ≥40 years. Thus, more participants aged 25-39 years were seroprotected before vaccination, but SCR was higher in those aged ≥40 years. Most Japanese adults can be protected after one-dose vaccination, but this may be insufficient for people aged 25-39 years without recorded JE vaccination.

  10. Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells.

    PubMed

    Wu, Yuxin; Zhang, Jinghan; Ben, Xiaoming

    2013-08-05

    Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the "pour-off" method. Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts, and could be expanded by supplementation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from β-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice. At 8 weeks, cells positive for LacZ and β-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

  11. Direct comparison of progenitor cells derived from adipose, muscle, and bone marrow from wild-type or craniosynostotic rabbits

    PubMed Central

    GM, Cooper; EL, Lensie; JJ, Cray; MR, Bykowski; GE, DeCesare; MA, Smalley; MP, Mooney; PG, Campbell; JE, Losee

    2010-01-01

    Background Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell-types. Also, few have investigated the potential connection between a tissue-specific pathology and cells derived from seemingly unrelated tissues. Here, we compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures. Methods Cells were derived from bone marrow, adipose, and muscle of 10 day-old wild-type rabbits (WT; n=17) or from age-matched rabbits with familial nonsyndromic craniosynostosis (CS; n=18). Cells were stimulated with bone morphogenetic protein 4 (BMP4) and alkaline phosphatase expression and cell proliferation were assessed. Results In WT rabbits, cells derived from muscle had more alkaline phosphatase activity than cells derived from either adipose or bone marrow. The cells derived from CS rabbit bone marrow and muscle were significantly more osteogenic than WT. Adipose-derived cells demonstrated no significant differences. While muscle-derived cells were most osteogenic in WT rabbits, bone marrow-derived cells were most osteogenic in CS rabbits. Conclusions Results suggest that cells from different tissues have different potentials for differentiation. Furthermore, cells derived from rabbits with craniosynostosis were different from wild-type derived cells. Interestingly, cells derived from the craniosynostotic rabbits were not uniformly more responsive compared with wild-type cells, suggesting that specific tissue-derived cells may react differently in individuals with craniosynostosis. PMID:20871482

  12. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Datta, Rupsa; Heylman, Christopher; George, Steven C.; Gratton, Enrico

    2016-01-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  13. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Datta, Rupsa; Heylman, Christopher; George, Steven C; Gratton, Enrico

    2016-05-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy.

  14. Proinflammatory and prothrombotic effects on human vascular endothelial cells of Immune-cell-derived LIGHT

    PubMed Central

    2009-01-01

    Objective LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTßR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells. Methods and Results Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4+ or CD8+) significantly increased after stimulation with PMA (Phorbolester-12-Myristat-13-Acetat) + ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of ~60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-γ pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93 ± 9.41 vs.129.53 ± 49.14 and 172.13 ± 77.64; p < 0.0005). Conclusion These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma

  15. Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

    PubMed Central

    2013-01-01

    Background Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. Methods To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Results Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Conclusions Our findings suggest that chronic

  16. Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

    PubMed Central

    Klima, Stefanie; Karreman, Christiaan; Grinberg, Marianna; Meisig, Johannes; Henry, Margit; Rotshteyn, Tamara; Rahnenführer, Jörg; Blüthgen, Nils; Sachinidis, Agapios; Waldmann, Tanja; Leist, Marcel

    2016-01-01

    Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates. Significance The generation of human cells from pluripotent stem cells has aroused great hopes in biomedical research and safety sciences. Neurotoxicity testing is a particularly important application for stem cell-derived somatic cells, as

  17. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    SciTech Connect

    Cohen, J.D.; Babiarz, J.E.; Abrams, R.M.; Guo, L.; Kameoka, S.; Chiao, E.; Taunton, J.; Kolaja, K.L.

    2011-11-15

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC{sub 50} in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 {mu}M concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 {mu}M. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: Black-Right-Pointing-Pointer Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes Black

  18. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism

    PubMed Central

    James, S. Jill; Rose, Shannon; Melnyk, Stepan; Jernigan, Stefanie; Blossom, Sarah; Pavliv, Oleksandra; Gaylor, David W.

    2009-01-01

    Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.—James, S. J., Rose, S., Melnyk, S., Jernigan, S., Blossom, S., Pavliv, O., Gaylor, D. W. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism. PMID:19307255

  19. [Effects of radiation on capillary endothelial cells derived from Mongolian gerbil brain].

    PubMed

    Mori, S; Tanaka, R; Minakawa, T; Onda, K

    1990-09-01

    Confluent monolayers of capillary endothelial cells derived from Mongolian gerbil brain were irradiated with a single exposure of x-rays, and their radiosensitivity and sequential changes in morphology, staining intensity for factor VIII-related antigen (F VIII RAg), and capacity to produce prostacyclin (PGI2) were examined. The radiobiologic parameters that characterized the dose-response survival curve for these cells were found to be n = 1.9, Dq = 140 rad, and D0 = 190 rad. Morphologically, nuclear and cytoplasmic swelling, vacuolation of cytoplasm, and giant cell formation occurred in a dose dependent manner after 24 hours from irradiation. Decreased staining intensity for F VIII RAg was observed in morphologically affected cells. The capacity to synthesize PGI2 was significantly enhanced at 24 hours, but less significant at 72 hours after irradiation. The present data suggest that the radiosensitivity of brain capillary endothelial cells may be somewhat lower than that of endothelial cells originated from larger vessels, and that radiation induced morphological and functional changes in the brain capillary endothelial cells may be quantitatively similar to the changes in endothelial cells of larger vessels.

  20. Stromal cell-derived factor-1 potentiates bone morphogenetic protein-2 induced bone formation.

    PubMed

    Higashino, Kosaku; Viggeswarapu, Manjula; Bargouti, Maggie; Liu, Hui; Titus, Louisa; Boden, Scott D

    2011-02-01

    The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients.

  1. Electric impedance of human embryonic stem cell-derived retinal pigment epithelium.

    PubMed

    Onnela, Niina; Savolainen, Virpi; Juuti-Uusitalo, Kati; Vaajasaari, Hanna; Skottman, Heli; Hyttinen, Jari

    2012-02-01

    The barrier properties of epithelium are conventionally defined by transepithelial resistance (TER). TER provides information about the tightness of the epithelium. Electrical impedance spectroscopy (EIS) provides additional information regarding cell membrane properties, such as changes in electric capacitance and possible parallel or serial pathways that may correlate with the morphology of the cell layer. This study presents EIS of retinal pigment epithelial (RPE) cell model of the putative RPE differentiated from human embryonic stem cells (hESC-RPE). The generally utilized RPE cell model, ARPE-19, was used as immature control. The measured EIS was analyzed by fitting an equivalent electrical circuit model describing the resistive and capacitive properties of the RPE. Our results indicated that TER of hESC-RPE cells was close to the values of human RPE presented in the literature. This provides evidence that the stem cell-derived RPE in vitro can reach high-barrier function. Furthermore, hESC-RPE cells produced impedance spectra that can be modeled by the equivalent circuit of one time constant. ARPE-19 cells produced low-barrier properties, that is, an impedance spectra that suggested poor maturation of ARPE-19 cells. To conclude, EIS could give us means for non-invasively estimating the functionality and maturation of differentiated-RPE cells.

  2. Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons

    PubMed Central

    Bradford, Aaron B; McNutt, Patrick M

    2015-01-01

    Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments. PMID:26240679

  3. Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate Non-Human Primate Hearts

    PubMed Central

    Chong, James J.H.; Yang, Xiulan; Don, Creighton W.; Minami, Elina; Liu, Yen-Wen; Weyers, Jill J; Mahoney, William M.; Van Biber, Benjamin; Cook, Savannah M.; Palpant, Nathan J; Gantz, Jay; Fugate, James A.; Muskheli, Veronica; Gough, G. Michael; Vogel, Keith W.; Astley, Cliff A.; Hotchkiss, Charlotte E.; Baldessari, Audrey; Pabon, Lil; Reinecke, Hans; Gill, Edward A.; Nelson, Veronica; Kiem, Hans-Peter; Laflamme, Michael A.; Murry, Charles E.

    2014-01-01

    Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure 1 by providing human cardiomyocytes to support heart regeneration 2. Studies of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in small animal models have shown favorable effects of this treatment 3–7. It remains unknown, however, whether clinical scale hESC-CMs transplantation is feasible, safe or can provide large-scale myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (>1 billion cells/batch) and cryopreserved with good viability. Using a non-human primate (NHP) model of myocardial ischemia-reperfusion, we show that that cryopreservation and intra-myocardial delivery of 1 billion hESC-CMs generates significant remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a three-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small animal models 7, non-fatal ventricular arrhythmias were observed in hESC-CM engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome. PMID:24776797

  4. Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes

    PubMed Central

    Maillet, Agnes; Tan, Kim; Chai, Xiaoran; Sadananda, Singh N.; Mehta, Ashish; Ooi, Jolene; Hayden, Michael R.; Pouladi, Mahmoud A.; Ghosh, Sujoy; Shim, Winston; Brunham, Liam R.

    2016-01-01

    Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. PMID:27142468

  5. Isolation, Characterization, and Multipotent Differentiation of Mesenchymal Stem Cells Derived from Meniscal Debris

    PubMed Central

    Fu, Weili; Xie, Xing; Li, Qi; Chen, Gang; Zhang, Chenghao; Tang, Xin

    2016-01-01

    This study aimed to culture and characterize mesenchymal stem cells derived from meniscal debris. Cells in meniscal debris from patients with meniscal injury were isolated by enzymatic digestion, cultured in vitro to the third passage, and analyzed by light microscopy to observe morphology and growth. Third-passage cultures were also analyzed for immunophenotype and ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. After 4-5 days in culture, cells showed a long fusiform shape and adhered to the plastic walls. After 10–12 days, cell clusters and colonies were observed. Third-passage cells showed uniform morphology and good proliferation. They expressed CD44, CD90, and CD105 but were negative for CD34 and CD45. Cultures induced to differentiate via osteogenesis became positive for Alizarin Red staining as well as alkaline phosphatase activity. Cultures induced to undergo adipogenesis were positive for Oil Red O staining. Cultures induced to undergo chondrogenesis were positive for staining with Toluidine Blue, Alcian Blue, and type II collagen immunohistochemistry, indicating cartilage-specific matrix. These results indicate that the cells we cultured from meniscal debris are mesenchymal stem cells capable of differentiating along three lineages. These stem cells may be valuable source for meniscal regeneration. PMID:28044083

  6. Upscaling of hiPS Cell-Derived Neurons for High-Throughput Screening.

    PubMed

    Traub, Stefanie; Stahl, Heiko; Rosenbrock, Holger; Simon, Eric; Heilker, Ralf

    2017-03-01

    The advent of human-induced pluripotent stem (hiPS) cell-derived neurons promised to provide better model cells for drug discovery in the context of the central nervous system. This work demonstrates both the upscaling of cellular expansion and the acceleration of neuronal differentiation to accommodate the immense material needs of a high-throughput screening (HTS) approach. Using GRowth factor-driven expansion and INhibition of NotCH (GRINCH) during maturation, the derived cells are here referred to as GRINCH neurons. GRINCH cells displayed neuronal markers, and their functional activity could be demonstrated by electrophysiological recordings. In an application of GRINCH neurons, the brain-derived neurotrophic factor (BDNF)-mediated activation of tropomyosin receptor kinase (TrkB) was investigated as a promising drug target to treat synaptic dysfunctions. To assess the phosphorylation of endogenous TrkB in the GRINCH cells, the highly sensitive amplified luminescent proximity homogeneous assay LISA (AlphaLISA) format was established as a primary screen. A high-throughput reverse transcription (RT)-PCR format was employed as a secondary assay to analyze TrkB-mediated downstream target gene expression. In summary, an optimized differentiation protocol, highly efficient cell upscaling, and advanced assay miniaturization, combined with increased detection sensitivity, pave the way for a new generation of predictive cell-based drug discovery.

  7. Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche

    PubMed Central

    Luarte, Alejandro; Bátiz, Luis Federico; Wyneken, Ursula; Lafourcade, Carlos

    2016-01-01

    Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future. PMID:27195011

  8. Induced Pluripotent Stem Cell Derived Macrophages as a Cellular System to Study Salmonella and Other Pathogens

    PubMed Central

    Hale, Christine; Yeung, Amy; Goulding, David; Pickard, Derek; Alasoo, Kaur; Powrie, Fiona; Dougan, Gordon; Mukhopadhyay, Subhankar

    2015-01-01

    A number of pathogens, including several human-restricted organisms, persist and replicate within macrophages (Mφs) as a key step in pathogenesis. The mechanisms underpinning such host-restricted intracellular adaptations are poorly understood, in part, due to a lack of appropriate model systems. Here we explore the potential of human induced pluripotent stem cell derived macrophages (iPSDMs) to study such pathogen interactions. We show iPSDMs express a panel of established Mφ-specific markers, produce cytokines, and polarise into classical and alternative activation states in response to IFN-γ and IL-4 stimulation, respectively. iPSDMs also efficiently phagocytosed inactivated bacterial particles as well as live Salmonella Typhi and S. Typhimurium and were able to kill these pathogens. We conclude that iPSDMs can support productive Salmonella infection and propose this as a flexible system to study host/pathogen interactions. Furthermore, iPSDMs can provide a flexible and practical cellular platform for assessing host responses in multiple genetic backgrounds. PMID:25946027

  9. Multielectrode Array (MEA) Assay for Profiling Electrophysiological Drug Effects in Human Stem Cell-Derived Cardiomyocytes.

    PubMed

    Clements, Mike

    2016-05-04

    More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Human stem cell-derived cardiomyocytes (hSC-CMs) provide a potential model for the development of superior assays for preclinical drug safety screening. One such hSC-CM assay that has shown significant potential for enabling more predictive drug cardiac risk assessment is the MEA assay. The Multi-electrode Array (MEA) assay is an electrophysiology-based technique that uses microelectrodes embedded in the culture surface of each well to measure fluctuations in extracellular field potential (FP) generated from spontaneously beating hSC-CMs. Perturbations to the recorded FP waveform can be used as an unbiased method of predicting the identity of ion channel(s) impacted on drug exposure. Here, a higher throughput MEA assay using hSC-CMs in 48-well MEA plates is described for profiling compound-induced effects on cardiomyocyte electrophysiology. Techniques for preparing hSC-CM monolayers in MEA plates and methods to contextualize MEA assay experimental results are also covered. © 2016 by John Wiley & Sons, Inc.

  10. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation

    PubMed Central

    McCabe, Kathryn L.; Kunzevitzky, Noelia J.; Chiswell, Brian P.; Xia, Xin; Goldberg, Jeffrey L.; Lanza, Robert

    2015-01-01

    Aim To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. Materials and Methods Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression. Results hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. Conclusion hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium. PMID:26689688

  11. Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format

    PubMed Central

    Sherman, Sydney A.; Phillips, Jack K.; Costa, J. Tighe; Cho, Frances S.; Oungoulian, Sevan R.; Finan, John D.

    2016-01-01

    Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology. PMID:27671211

  12. Increasing doublecortin expression promotes migration of human embryonic stem cell-derived neurons.

    PubMed

    Filipovic, Radmila; Santhosh Kumar, Saranya; Fiondella, Chris; Loturco, Joseph

    2012-09-01

    Human embryonic stem cell-derived neuronal progenitors (hNPs) provide a potential source for cellular replacement following neurodegenerative diseases. One of the greatest challenges for future neuron replacement therapies will be to control extensive cell proliferation and stimulate cell migration of transplanted cells. The doublecortin (DCX) gene encodes the protein DCX, a microtubule-associated protein essential for the migration of neurons in the human brain. In this study, we tested whether increasing the expression of DCX in hNPs would favorably alter their proliferation and migration. Migration and proliferation of hNPs was compared between hNPs expressing a bicistronic DCX/IRES-GFP transgene and those expressing a green fluorescent protein (GFP) transgene introduced by piggyBac-mediated transposition. The DCX-transfected hNPs showed a significant decrease in their proliferation and migrated significantly further on two different substrates, Matrigel and brain slices. Additionally, a dense network of nestin-positive (+) and vimentin+ fibers were found to extend from neurospheres transplanted onto brain slices, and this fiber growth was increased from neurospheres containing DCX-transfected hNPs. In summary, our results show that increased DCX expression inhibits proliferation and promotes migration of hNPs.

  13. 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

  14. Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell.

    PubMed

    Ren, Yue; Dashtsoodol, Nyambayar; Watarai, Hiroshi; Koseki, Haruhiko; Quan, Chengshi; Taniguchi, Masaru

    2014-10-01

    NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vβ7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vβ loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in peripheral lymphoid organs. NKT-iPSC-derived mice also showed normal development of other immune cells except for the absence of γδT cells and disturbed development of conventional CD4 αβT cells. These results suggest that the NKT-iPSC-derived mice are a better model for NKT cell development and function study rather than transgenic mouse models reported previously and also that the presence of a pre-rearranged Vα14Jα18 in the natural chromosomal context favors the developmental fate of NKT cells.

  15. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

    PubMed

    Rezania, Alireza; Bruin, Jennifer E; Arora, Payal; Rubin, Allison; Batushansky, Irina; Asadi, Ali; O'Dwyer, Shannon; Quiskamp, Nina; Mojibian, Majid; Albrecht, Tobias; Yang, Yu Hsuan Carol; Johnson, James D; Kieffer, Timothy J

    2014-11-01

    Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

  16. Generating and characterizing the mechanical properties of cell-derived matrices using atomic force microscopy.

    PubMed

    Tello, Marta; Spenlé, Caroline; Hemmerlé, Joseph; Mercier, Luc; Fabre, Roxane; Allio, Guillaume; Simon-Assmann, Patricia; Goetz, Jacky G

    2016-02-01

    Mechanical interaction between cells and their surrounding extracellular matrix (ECM) controls key processes such as proliferation, differentiation and motility. For many years, two-dimensional (2D) models were used to better understand the interactions between cells and their surrounding ECM. More recently, variation of the mechanical properties of tissues has been reported to play a major role in physiological and pathological scenarios such as cancer progression. The 3D architecture of the ECM finely tunes cellular behavior to perform physiologically relevant tasks. Technical limitations prevented scientists from obtaining accurate assessment of the mechanical properties of physiologically realistic matrices. There is therefore a need for combining the production of high-quality cell-derived 3D matrices (CDMs) and the characterization of their topographical and mechanical properties. Here, we describe methods that allow to accurately measure the young modulus of matrices produced by various cellular types. In the first part, we will describe and review several protocols for generating CDMs matrices from endothelial, epithelial, fibroblastic, muscle and mesenchymal stem cells. We will discuss tools allowing the characterization of the topographical details as well as of the protein content of such CDMs. In a second part, we will report the methodologies that can be used, based on atomic force microscopy, to accurately evaluate the stiffness properties of the CDMs through the quantification of their young modulus. Altogether, such methodologies allow characterizing the stiffness and topography of matrices deposited by the cells, which is key for the understanding of cellular behavior in physiological conditions.

  17. Tomosyn Negatively Regulates Arginine Vasopressin Secretion in Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Takeuchi, Seiji; Iwama, Shintaro; Takagi, Hiroshi; Kiyota, Atsushi; Nakashima, Kohtaro; Izumida, Hisakazu; Fujisawa, Haruki; Iwata, Naoko; Suga, Hidetaka; Watanabe, Takashi; Kaibuchi, Kozo; Oiso, Yutaka; Arima, Hiroshi; Sugimura, Yoshihisa

    2016-01-01

    Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxin-binding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons. PMID:27732637

  18. Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors.

    PubMed

    Iyer, S; Xiao, E; Alsayegh, K; Eroshenko, N; Riggs, M J; Bennett, J P; Rao, R R

    2012-05-01

    Human pluripotent stem cell-derived neural progenitor (hNP) cells are an excellent resource for understanding early neural development and neurodegenerative disorders. Given that many neurodegenerative disorders can be correlated with defects in the mitochondrial genome, optimal utilization of hNP cells requires an ability to manipulate and monitor changes in the mitochondria. Here, we describe a novel approach that uses recombinant human mitochondrial transcription factor A (rhTFAM) protein to transfect and express a pathogenic mitochondrial genome (mtDNA) carrying the G11778A mutation associated with Leber's hereditary optic neuropathy (LHON) disease, into dideoxycytidine (ddC)-treated hNPs. Treatment with ddC reduced endogenous mtDNA and gene expression, without loss of hNP phenotypic markers. Entry of G11778A mtDNA complexed with the rhTFAM was observed in mitochondria of ddC-hNPs. Expression of the pathogenic RNA was confirmed by restriction enzyme analysis of the SfaN1-digested cDNA. On the basis of the expression of neuron-specific class III beta-tubulin, neuronal differentiation occurred. Our results show for the first time that pathogenic mtDNA can be introduced and expressed into hNPs without loss of phenotype or neuronal differentiation potential. This mitochondrial gene replacement technology allows for creation of in vitro stem cell-based models useful for understanding neuronal development and treatment of neurodegenerative disorders.

  19. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

    PubMed

    Chong, James J H; Yang, Xiulan; Don, Creighton W; Minami, Elina; Liu, Yen-Wen; Weyers, Jill J; Mahoney, William M; Van Biber, Benjamin; Cook, Savannah M; Palpant, Nathan J; Gantz, Jay A; Fugate, James A; Muskheli, Veronica; Gough, G Michael; Vogel, Keith W; Astley, Cliff A; Hotchkiss, Charlotte E; Baldessari, Audrey; Pabon, Lil; Reinecke, Hans; Gill, Edward A; Nelson, Veronica; Kiem, Hans-Peter; Laflamme, Michael A; Murry, Charles E

    2014-06-12

    Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

  20. Evaluation of MDCK Cell-Derived Influenza H7N9 Vaccine Candidates in Ferrets

    PubMed Central

    Tseng, Yu-Fen; Weng, Tsai-Chuan; Lai, Chia-Chun; Lin, Jun-Yang; Chen, Po-Ling; Wang, Ya-Fang; Chao, Sin-Ru; Chang, Jui-Yuan; Hwang, Yi-Shiuh; Yeh, Chia-Tsui; Yu, Cheng-Ping; Chen, Yee-Chun; Su, Ih-Jen; Lee, Min-Shi

    2015-01-01

    Avian-origin influenza A (H7N9) viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300μg aluminum hydroxide, 1.5μg HA, and 1.5μg HA plus 300μg aluminum hydroxide) were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA) inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only), 17 and 80 for the unadjuvanted (HA only) group, and 190 and 640 for the adjuvanted group (HA plus adjuvant), respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted. PMID:25799397

  1. Process Extension from Embryonic Stem Cell-Derived Motor Neurons through Synthetic Extracellular Matrix Mimics

    NASA Astrophysics Data System (ADS)

    McKinnon, Daniel Devaud

    This thesis focuses on studying the extension of motor axons through synthetic poly(ethylene glycol) PEG hydrogels that have been modified with biochemical functionalities to render them more biologically relevant. Specifically, the research strategy is to encapsulate embryonic stem cell-derived motor neurons (ESMNs) in synthetic PEG hydrogels crosslinked through three different chemistries providing three mechanisms for dynamically tuning material properties. First, a covalently crosslinked, enzymatically degradable hydrogel is developed and exploited to study the biophysical dynamics of axon extension and matrix remodeling. It is demonstrated that dispersed motor neurons require a battery of adhesive peptides and growth factors to maintain viability and extend axons while those in contact with supportive neuroglial cells do not. Additionally, cell-degradable crosslinker peptides and a soft modulus mimicking that of the spinal cord are requirements for axon extension. However, because local degradation of the hydrogel results in a cellular environment significantly different than that of the bulk, enzymatically degradable peptide crosslinkers were replaced with reversible covalent hydrazone bonds to study the effect of hydrogel modulus on axon extension. This material is characterized in detail and used to measure forces involved in axon extension. Finally, a hydrogel with photocleavable linkers incorporated into the network structure is exploited to explore motor axon response to physical channels. This system is used to direct the growth of motor axons towards co-cultured myotubes, resulting in the formation of an in vitro neural circuit.

  2. Mitochondrial Toxicity of Perfluorooctane Sulfonate in Mouse Embryonic Stem Cell-Derived Cardiomyocytes.

    PubMed

    Tang, Lei-Lei; Wang, Jia-Dan; Xu, Ting-Ting; Zhao, Zhe; Zheng, Jia-Jie; Ge, Ren-Shan; Zhu, Dan-Yan

    2017-03-10

    Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may cause cardiotoxicity in animals and humans. However, little is known about the underlying mechanism by which it affects the organelle toxicity in cardiomyocytes during the cardiogenesis. Our previous proteomic study showed that differences of protein expression mainly existed in mitochondria of cardiomyocytes differentiated from embryonic stem (ES) cells after exposure to PFOS. Here, we focused on mitochondrial toxicity of PFOS in ES cell-derived cardiomyocytes. The cardiomyogenesis from ES cells in vitro was inhibited, and the expression of L-type Ca(2+) channel (LTCC) was decreased to interrupt [Ca(2+)]c transient amplitude in cardiomyocytes after PFOS treatment. Transmission electron microscope revealed that swollen mitochondrion with vacuole in PFOS-treated cells. Meanwhile, mitochondrial transmembrane potential (ΔYm) was declined and ATP production was lowered. These changes were related to the increased EGFR phosphorylation, activated Rictor signaling, then mediated HK2 binding to mitochondrial membrane. Furthermore, PFOS reduced the interaction of IP3R-Grp75-VDAC and accumulated intracellular fatty acids by activating Rictor, thereby attenuating PGC-1a and Mfn2 expressions, then destroying mitochondria-associated endoplasmic reticulum membrane (MAM), which resulted in the decrease of [Ca(2+)]mito transient amplitude triggered by ATP. In conclusion, mitochondrial structure damages and abnormal Ca(2+) shuttle were the important aspects in PFOS-induced cardiomyocytes toxicity from ES cells by activating Rictor signaling pathway.

  3. Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.

    PubMed

    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D; Jordan, Maria C; Roos, Kenneth P; Yao, Yucheng; Boström, Kristina I

    2012-12-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

  4. Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts

    PubMed Central

    Oki, Yoshinao; Ono, Hiromasa; Motohashi, Takeharu; Sugiura, Nobuki; Nobusue, Hiroyuki; Kano, Koichiro

    2012-01-01

    Transdifferentiation is the conversion of cells from one differentiated cell type into another. How functionally differentiated cells already committed to a specific cell lineage can transdifferentiate into other cell types is a key question in cell biology and regenerative medicine. In the present study we show that porcine ovarian follicular GCs (granulosa cells) can transdifferentiate into osteoblasts in vitro and in vivo. Pure GCs isolated and cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS (fetal bovine serum) proliferated and dedifferentiated into fibroblast-like cells. We referred to these cells as DFOG (dedifferentiated follicular granulosa) cells. Microarray analysis showed that DFOG cells lost expression of GC-specific marker genes, but gained the expression of osteogenic marker genes during dedifferentiation. After osteogenic induction, DFOG cells underwent terminal osteoblast differentiation and matrix mineralization in vitro. Furthermore, when DFOG cells were transplanted subcutaneously into SCID mice, these cells formed ectopic osteoid tissue. These results indicate that DFOG cells derived from GCs can differentiate into osteoblasts in vitro and in vivo. We suggest that GCs provide a useful model for studying the mechanisms of transdifferentiation into other cell lineages in functionally differentiated cells. PMID:22839299

  5. Endothelial Differentiation in Multipotent Cells Derived from Mouse and Human White Mature Adipocytes

    PubMed Central

    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D.; Jordan, Maria C.; Roos, Kenneth P.; Yao, Yucheng; Boström, Kristina I.

    2012-01-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhance the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues. PMID:22999861

  6. In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development

    PubMed Central

    Tsai, Yu-Hwai; Nattiv, Roy; Dedhia, Priya H.; Nagy, Melinda S.; Chin, Alana M.; Thomson, Matthew; Klein, Ophir D.

    2017-01-01

    ABSTRACT The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research. PMID:27927684

  7. Revisiting tumor angiogenesis: vessel co-option, vessel remodeling, and cancer cell-derived vasculature formation.

    PubMed

    Qian, Chao-Nan; Tan, Min-Han; Yang, Jun-Ping; Cao, Yun

    2016-01-08

    Tumor growth and metastasis depend on the establishment of tumor vasculature to provide oxygen, nutrients, and other essential factors. The well-known vascular endothelial growth factor (VEGF) signaling is crucial for sprouting angiogenesis as well as recruitment of circulating progenitor endothelial cells to tumor vasculature, which has become therapeutic targets in clinical practice. However, the survival benefits gained from targeting VEGF signaling have been very limited, with the inevitable development of treatment resistance. In this article, we discuss the most recent findings and understanding on how solid tumors evade VEGF-targeted therapy, with a special focus on vessel co-option, vessel remodeling, and tumor cell-derived vasculature establishment. Vessel co-option may occur in tumors independently of sprouting angiogenesis, and sprouting angiogenesis is not always required for tumor growth. The differences between vessel-like structure and tubule-like structure formed by tumor cells are also introduced. The exploration of the underlying mechanisms of these alternative angiogenic approaches would not only widen our knowledge of tumor angiogenesis but also provide novel therapeutic targets for better controlling cancer growth and metastasis.

  8. Stromal Cell-Derived Factor-1 Alpha is Cardioprotective After Myocardial Infarction

    PubMed Central

    Saxena, Ankur; Fish, Jason E.; White, Michael D.; Yu, Sangho; Smyth, James WP; Shaw, Robin M.; DiMaio, J. Michael; Srivastava, Deepak

    2009-01-01

    Background Heart disease is a leading cause of mortality throughout the world. Tissue damage from vascular occlusive events results in the replacement of contractile myocardium by nonfunctional scar tissue. The potential of new technologies to regenerate damaged myocardium is significant, although cell-based therapies must overcome several technical barriers. One possible cell-independent alternative is the direct administration of small proteins to damaged myocardium. Methods and Results Here we show that the secreted signaling protein stromal cell-derived factor-1 alpha (SDF-1α), which activates the cell-survival factor protein kinase B (PKB/Akt) via the G-protein-coupled receptor CXCR4, protected tissue after an acute ischemic event in mice and activated Akt within endothelial cells and myocytes of the heart. Significantly better cardiac function than in control mice was evident as early as 24 hours post-infarction as well as at 3, 14 and 28 days post-infarction. Prolonged survival of hypoxic myocardium was followed by an increase in levels of vascular endothelial growth factor (VEGF) protein and neo-angiogenesis. Consistent with improved cardiac function, mice exposed to SDF-1α demonstrated significantly decreased scar formation than control mice. Conclusions These findings suggest that SDF-1α may serve a tissue-protective and regenerative role for solid organs suffering a hypoxic insult. PMID:18427137

  9. Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Rajamohan, Divya; Kalra, Spandan; Duc Hoang, Minh; George, Vinoj; Staniforth, Andrew; Russell, Hugh; Yang, Xuebin

    2016-01-01

    Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose–response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation. PMID:26906236

  10. Embryonic stem cell-derived M2-like macrophages delay cutaneous wound healing.

    PubMed

    Dreymueller, Daniela; Denecke, Bernd; Ludwig, Andreas; Jahnen-Dechent, Willi

    2013-01-01

    In adults, repair of deeply injured skin wounds results in the formation of scar tissue, whereas in embryos wounds heal almost scar-free. Macrophages are important mediators of wound healing and secrete cytokines and tissue remodeling enzymes. In contrast to host defense mediated by inflammatory M1 macrophages, wound healing and tissue repair involve regulatory M2/M2-like macrophages. Embryonic/fetal macrophages are M2-like, and this may promote scar-free wound healing. In the present study, we asked whether atopical application of ex vivo generated, embryonic stem cell-derived macrophages (ESDM) improve wound healing in mice. ESDM were tested side by side with bone marrow-derived macrophages (BMDM). Compared to BMDM, ESDM resembled a less inflammatory and more M2-like macrophage subtype as indicated by their reduced responsiveness to lipopolysaccharide, reduced expression of Toll-like receptors, and reduced bacterial phagocytosis. Despite this anti-inflammatory phenotype in cell culture, ESDM prolonged the healing of deep skin wounds even more than BMDM. Healed wounds had more scar formation compared to wounds receiving BMDM or cell-free treatment. Our data indicate that atopical application of ex vivo generated macrophages is not a suitable cell therapy of dermal wounds.

  11. Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

    PubMed

    Xing, Qi; Zhang, Lijun; Redman, Travis; Qi, Shaohai; Zhao, Feng

    2015-12-01

    During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

  12. Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy.

    PubMed

    Darabi, Radbod; Baik, June; Clee, Mark; Kyba, Michael; Tupler, Rossella; Perlingeiro, Rita C R

    2009-11-01

    Muscular dystrophies (MDs) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon; however, to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell-derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD.

  13. Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease

    PubMed Central

    Suzuki, Etsu; Fujita, Daishi; Takahashi, Masao; Oba, Shigeyoshi; Nishimatsu, Hiroaki

    2016-01-01

    Mesenchymal stem cells (MSCs) have been used to treat patients suffering from acute myocardial infarction (AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microRNAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease (CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD. PMID:27679686

  14. FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons

    PubMed Central

    Errichelli, Lorenzo; Dini Modigliani, Stefano; Laneve, Pietro; Colantoni, Alessio; Legnini, Ivano; Capauto, Davide; Rosa, Alessandro; De Santis, Riccardo; Scarfò, Rebecca; Peruzzi, Giovanna; Lu, Lei; Caffarelli, Elisa; Shneider, Neil A.; Morlando, Mariangela; Bozzoni, Irene

    2017-01-01

    The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro-derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUSP525L mutation associated with ALS. PMID:28358055

  15. Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy

    PubMed Central

    Darabi, Radbod; Baik, June; Clee, Mark; Kyba, Michael; Tupler, Rossella; Perlingeiro, Rita C.R.

    2009-01-01

    Muscular dystrophies (MD) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon, however to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell- derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with Facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD. PMID:19682990

  16. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.

  17. In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development.

    PubMed

    Tsai, Yu-Hwai; Nattiv, Roy; Dedhia, Priya H; Nagy, Melinda S; Chin, Alana M; Thomson, Matthew; Klein, Ophir D; Spence, Jason R

    2017-03-15

    The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research.

  18. Stromal cell derived factor-1 (SDF-1) targeting reperfusion reduces myocardial infarction in isolated rat hearts.

    PubMed

    Jang, Young-Ho; Kim, June-Hong; Ban, Changill; Ahn, Kyohan; Cheong, Jae-Hun; Kim, Hyung-Hoi; Kim, Jung-Soo; Park, Yong-Hyun; Kim, Jun; Chun, Kook-Jin; Lee, Gyeong-Ho; Kim, Miju; Kim, Cheolmin; Xu, Zhelong

    2012-10-01

    Recent studies have shown that stromal cell derived factor-1 (SDF-1), first known as a cytokine involved in recruiting stem cells into injured organs, confers myocardial protection in myocardial infarction, which is not dependent on stem cell recruitment but related with modulation of ischemia-reperfusion (I/R) injury. However, the effect of SDF has been studied only in a preischemic exposure model, which is not clinically relevant if SDF is to be used as a therapeutic agent. Our study was aimed at evaluating whether or not SDF-1 confers cardioprotection during the reperfusion period. Hearts from SD rats were isolated and perfused with the Langendorff system. Proximal left coronary artery ligation, reperfusion, and SDF perfusion in KH buffer was done according to study protocol. Area of necrosis (AN) relative to area at risk (AR) was the primary endpoint of the study. Significant reduction of AN/AR by SDF in an almost dose-dependent manner was noted during both the preischemic exposure and reperfusion periods. In particular, infusion of a high concentration of SDF (25 nM/L) resulted in a dramatic reduction of infarct size, which was greater than that achieved with ischemic pre- or postconditioning. SDF perfusion during reperfusion was associated with a similar significant reduction of infarct size as preischemic SDF exposure. Further studies are warranted to assess the potential of SDF as a therapeutic agent for reducing I/R injury in clinical practice.

  19. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity.

    PubMed

    Schwartz, Michael P; Hou, Zhonggang; Propson, Nicholas E; Zhang, Jue; Engstrom, Collin J; Santos Costa, Vitor; Jiang, Peng; Nguyen, Bao Kim; Bolin, Jennifer M; Daly, William; Wang, Yu; Stewart, Ron; Page, C David; Murphy, William L; Thomson, James A

    2015-10-06

    Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.

  20. FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons.

    PubMed

    Errichelli, Lorenzo; Dini Modigliani, Stefano; Laneve, Pietro; Colantoni, Alessio; Legnini, Ivano; Capauto, Davide; Rosa, Alessandro; De Santis, Riccardo; Scarfò, Rebecca; Peruzzi, Giovanna; Lu, Lei; Caffarelli, Elisa; Shneider, Neil A; Morlando, Mariangela; Bozzoni, Irene

    2017-03-30

    The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro-derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS(P525L) mutation associated with ALS.

  1. Excitation–contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Kane, Christopher; Couch, Liam; Terracciano, Cesare M. N.

    2015-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation–contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca2+ handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells. PMID:26484342

  2. Accurate prediction of drug-induced liver injury using stem cell-derived populations.

    PubMed

    Szkolnicka, Dagmara; Farnworth, Sarah L; Lucendo-Villarin, Baltasar; Storck, Christopher; Zhou, Wenli; Iredale, John P; Flint, Oliver; Hay, David C

    2014-02-01

    Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug-inducible, and predictive human hepatocyte populations. Most importantly, stem cell-derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum-free, scalable, and shippable cell-based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell-based therapies.

  3. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

    PubMed

    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  4. Genotoxicity of 3-nitrobenzanthrone and 3-aminobenzanthrone in MutaMouse and lung epithelial cells derived from MutaMouse.

    PubMed

    Arlt, Volker M; Gingerich, John; Schmeiser, Heinz H; Phillips, David H; Douglas, George R; White, Paul A

    2008-11-01

    FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.

  5. Visceral fat area is a strong predictor of leukocyte cell-derived chemotaxin 2, a potential biomarker of dyslipidemia

    PubMed Central

    Tanisawa, Kumpei; Taniguchi, Hirokazu; Sun, Xiaomin; Ito, Tomoko; Kawakami, Ryoko; Sakamoto, Shizuo; Higuchi, Mitsuru

    2017-01-01

    Background Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine linking obesity to skeletal muscle insulin resistance. Although previous studies reported that obesity was associated with high levels of circulating LECT2 in human, the associations of detailed body fat distribution with LECT2 levels have not been examined. Furthermore, although animal study suggested that exercise decreased circulating LECT2 levels, it remains unknown whether physical fitness is associated with LECT2 levels in human. We therefore examined the relationship of plasma LECT2 levels with various adiposity indices and cardiorespiratory fitness (CRF) in middle-aged and elderly Japanese men. Furthermore, we examined the relationship of LECT2 levels with the presence of metabolic syndrome, hypertension, insulin resistance and dyslipidemia to determine the clinical significance of measuring circulating LECT2. Materials and methods This was a cross-sectional study of 143 Japanese men (age: 30–79 years). Participants’ plasma LECT2 levels were measured by an enzyme-linked immunosorbent assay. To assess their abdominal fat distributions, visceral fat area (VFA) and subcutaneous fat area (SFA) were measured using magnetic resonance imaging. CRF was assessed by measuring peak oxygen uptake (V˙O2peak). Results All adiposity indices measured in this study were positively correlated with plasma LECT2 levels, while V˙O2peak was negatively correlated with LECT2 levels after adjustment for age. The correlations, except for VFA were no longer significant with further adjustment for VFA. Stepwise multiple linear regression analysis revealed that VFA was the strongest predictor of plasma LECT2 levels. Plasma LECT2 levels differed based on the presence of metabolic syndrome and dyslipidemia, but not hypertension and insulin resistance. Logistic regression analyses revealed that plasma LECT2 levels were significantly associated with dyslipidemia independently of VFA; VFA was not significantly

  6. Induced pluripotent stem cell-derived neuron as a human model for testing environmentally induced developmental neurotoxicity

    EPA Science Inventory

    Induced pluripotent stem cell-derived neurons as a human model for testing environmentally induced developmental neurotoxicity Ingrid L. Druwe1, Timothy J. Shafer2, Kathleen Wallace2, Pablo Valdivia3 ,and William R. Mundy2. 1University of North Carolina, Curriculum in Toxicology...

  7. Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras.

    PubMed

    Karagenç, Levent; Sandikci, Mustafa

    2010-01-01

    The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-mum intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm.

  8. Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons*

    PubMed Central

    Debaisieux, Solène; Encheva, Vesela; Chakravarty, Probir; Snijders, Ambrosius P.; Schiavo, Giampietro

    2016-01-01

    Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first

  9. Insulin - producing cells derived from stem cells: recent progress and future directions

    PubMed Central

    Santana, A; Enseñat - Waser, R; Arribas, Maria Isabel; Reig, J A; Roche, E

    2006-01-01

    Type 1 diabetes is characterized by the selective destruction of pancreatic β-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to β-cell loss caused by apoptotic programs, includes β-cell dedifferentiation and peripheric insulin resistance. β-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreaticderived insulin secretion exerts on the body’s glycemia. Restoration of damaged β-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including β-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic β-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the clinical

  10. Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, C. K.; Fox, P. L.

    1998-01-01

    Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.

  11. Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer.

    PubMed

    Hermanson, David L; Bendzick, Laura; Pribyl, Lee; McCullar, Valarie; Vogel, Rachel Isaksson; Miller, Jeff S; Geller, Melissa A; Kaufman, Dan S

    2016-01-01

    Natural killer (NK) cells can provide effective immunotherapy for ovarian cancer. Here, we evaluated the ability of NK cells isolated from peripheral blood (PB) and NK cells derived from induced pluripotent stem cell (iPSC) to mediate killing of ovarian cancer cells in a mouse xenograft model. A mouse xenograft model was used to evaluate the intraperitoneal delivery of three different NK cell populations: iPSC-derived NK cells, PB-NK cells that had been activated and expanded in long-term culture, and overnight activated PB-NK cells that were isolated through CD3/CD19 depletion of PB B and T cells. Bioluminescent imaging was used to monitor tumor burden of luciferase expressing tumor lines. Tumors were allowed to establish prior to administering NK cells via intraperitoneal injection. These studies demonstrate a single dose of any of the three NK cell populations significantly reduced tumor burden. When mice were given three doses of either iPSC-NK cells or expanded PB-NK cells, the median survival improved from 73 days in mice untreated to 98 and 97 days for treated mice, respectively. From these studies, we conclude iPSC-derived NK cells mediate antiovarian cancer killing at least as well as PB-NK cells, making these cells a viable resource for immunotherapy for ovarian cancer. Due to their ability to be easily differentiated into NK cells and their long-term expansion potential, iPSCs can be used to produce large numbers of well-defined NK cells that can be banked and used to treat a large number of patients including treatment with multiple doses if necessary.

  12. Stromal cell-derived factor-1 (SDF-1) as a target in liver diseases.

    PubMed

    Liepelt, Anke; Tacke, Frank

    2016-08-01

    The chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is constitutively expressed in healthy liver. However, its expression increases following acute or chronic liver injury. Liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC), and malignant hepatocytes are important sources of SDF-1/CXCL12 in liver diseases. CXCL12 is able to activate two chemokine receptors with different downstream signaling pathways, CXCR4 and CXCR7. CXCR7 expression is relevant on LSEC, while HSC, mesenchymal stem cells, and tumor cells mainly respond via CXCR4. Here, we summarize recent developments in the field of liver diseases involving this chemokine and its receptors. SDF-1-dependent signaling contributes to modulating acute liver injury and subsequent tissue regeneration. By activating HSC and recruiting mesenchymal cells from bone marrow, CXCL12 can promote liver fibrosis progression, while CXCL12-CXCR7 interactions endorse proregenerative responses in chronic injury. Moreover, the SDF-1 pathway is linked to development of hepatocellular carcinoma (HCC) by promoting tumor growth, angiogenesis, and HCC metastasis. High hepatic CXCR4 expression has been suggested as a biomarker indicating poor prognosis of HCC patients. Tumor-infiltrating myeloid-derived suppressor cells (MDSC) also express CXCR4 and migrate toward CXCL12. Thus CXCL12 inhibition might not only directly block HCC growth but also modulate the tumor microenvironment (angiogenesis, MDSC), thereby sensitizing HCC patients to conventional or emerging novel cancer therapies (e.g., sorafenib, regorafenib, nivolumab, pembrolizumab). We herein summarize the current knowledge on the complex interplay between CXCL12 and CXCR4/CXCR7 in liver diseases and discuss approaches on the therapeutic targeting of these axes in hepatitis, fibrosis, and liver cancer.

  13. Knockout of Endothelial Cell-Derived Endothelin-1 Attenuates Skin Fibrosis but Accelerates Cutaneous Wound Healing

    PubMed Central

    Makino, Katsunari; Jinnin, Masatoshi; Aoi, Jun; Kajihara, Ikko; Makino, Takamitsu; Fukushima, Satoshi; Sakai, Keisuke; Nakayama, Kazuhiko; Emoto, Noriaki; Yanagisawa, Masashi; Ihn, Hironobu

    2014-01-01

    Endothelin (ET)-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF)-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF)-α and connective tissue growth factor (CTGF) were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach. PMID:24853267

  14. A novel broadband impedance method for detection of cell-derived microparticles

    PubMed Central

    Lvovich, Vadim; Srikanthan, Sowmya; Silverstein, Roy L.

    2010-01-01

    A novel label-free method is presented to detect and quantify cell-derived microparticles (MPs) by the electrochemical potential-modulated electrochemical impedance spectroscopy (EIS). MPs are present in elevated concentrations during pathological conditions and play a major role in the establishment and pathogenesis of many diseases. Considering this, accurate detection and quantification of MPs is very important in clinical diagnostics and therapeutics. A combination of bulk solution electrokinetic sorting and interfacial impedance responses allows achieving detection limits as low as several MPs per µL. By fitting resulting EIS spectra with an equivalent electrical circuit, the bulk solution electrokinetic and interfacial impedance responses were characterized. In the bulk solution two major relaxations were prominent - β-relaxation in low MHz region due to the MP capacitive membrane bridging, and α-relaxation at ∼ 10 kHz due to counter ions diffusion. At low frequencies (10-0.1 Hz) at electrochemical potentials exceeding −100 mV, a facile interfacial Faradaic process of oxidation in MPs coupled with diffusion and non Faradaic double layer charging dominate, probably due to oxidation of phospholipids and/or proteins on the MP surface and MP lysis. Buffer influence on the MP detection demonstrated that that a relatively low conductivity Tyrode’s buffer background solution is preferential for the MP electrokinetic separation and characterization. This study also demonstrated that standard laboratory methods such as flow cytometry underestimate MP concentrations, especially those with smaller average sizes, by as much as a factor of 2 to 40. PMID:20729061

  15. Cell-Derived Nanoparticles are Endogenous Modulators of Sepsis with Therapeutic Potential.

    PubMed

    Kunz, Natalia; Xia, Brent T; Kalies, Kai-Uwe; Klinger, Matthias; Gemoll, Timo; Habermann, Jens K; Whitacre, Brynne E; Seitz, Aaron P; Kalies, Kathrin; Caldwell, Charles C

    2017-02-22

    Cell-derived nanoparticles (CDNPs) containing cytosolic proteins and RNAs/DNAs can be isolated from stressed eukaryotic cells. Previously, CDNPs isolated from cultured cells exerted immunomodulatory activities in different infections. Here, we sought to elucidate the role of CDNPs using a murine model of cecal ligation and puncture (CLP). We hypothesized that CDNPs influence the immune response at the site of infection, where severe cellular stress occurs. We observed early CDNP accumulation in the peritoneum after 4 h and continued CDNP presence 24 h after CLP. To determine whether CDNPs influence the host response to sepsis, we isolated CDNPs from a murine fibroblast cell line stressed by nutrient-deprivation, and injected them into septic mice. CDNP-treated mice demonstrated decreased peritoneal interleukin 6 levels and an approximately 2-log lower bacterial load compared with control mice 24 h after CLP. Additionally, a 20% CFU reduction was observed when incubating CDNPs with Pseudomona aeroginosa, indicating that CDNPs are bactericidal. To identify CDNP-responsive cells, CFSE-labeled CDNPs were injected into mice at the time of CLP. We observed that CDNPs were preferentially ingested by F4/80 macrophages, and to a lesser degree, associated with inflammatory monocytes and neutrophils. Strikingly, CDNP-ingesting cells demonstrated elevated CD11b and MHCII expression compared with control cells. Altogether, our data indicate that CDNPs enhance the immune response at the site of infection and promote bacterial clearance, by direct bacterial killing and increasing phagocyte activation. Thus, CDNPs represent a novel, unexplored endogenous sepsis modulator with therapeutic potential.

  16. Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

    PubMed

    Linta, Leonhard; Boeckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2013-07-01

    The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

  17. Radiation response of mesenchymal stem cells derived from bone marrow and human pluripotent stem cells.

    PubMed

    Islam, Mohammad S; Stemig, Melissa E; Takahashi, Yutaka; Hui, Susanta K

    2015-03-01

    Mesenchymal stem cells (MSCs) isolated from human pluripotent stem cells are comparable with bone marrow-derived MSCs in their function and immunophenotype. The purpose of this exploratory study was comparative evaluation of the radiation responses of mesenchymal stem cells derived from bone marrow- (BMMSCs) and from human embryonic stem cells (hESMSCs). BMMSCs and hESMSCs were irradiated at 0 Gy (control) to 16 Gy using a linear accelerator commonly used for cancer treatment. Cells were harvested immediately after irradiation, and at 1 and 5 days after irradiation. Cell cycle analysis, colony forming ability (CFU-F), differentiation ability, and expression of osteogenic-specific runt-related transcription factor 2 (RUNX2), adipogenic peroxisome proliferator-activated receptor gamma (PPARγ), oxidative stress-specific dismutase-1 (SOD1) and Glutathione peroxidase (GPX1) were analyzed. Irradiation arrested cell cycle progression in BMMSCs and hESMSCs. Colony formation ability of irradiated MSCs decreased in a dose-dependent manner. Irradiated hESMSCs showed higher adipogenic differentiation compared with BMMSCs, together with an increase in the adipogenic PPARγ expression. PPARγ expression was upregulated as early as 4 h after irradiation, along with the expression of SOD1. More than 70% downregulation was found in Wnt3A, Wnt4, Wnt 7A, Wnt10A and Wnt11 in BMMSCs, but not in hESMSCs. hESMSCs are highly proliferative but radiosensitive compared with BMMSCs. Increased PPARγ expression relative to RUNX2 and downregulation of Wnt ligands in irradiated MSCs suggest Wnt mediated the fate determination of irradiated MSCs.

  18. Enhanced Human-Induced Pluripotent Stem Cell Derived Cardiomyocyte Maturation Using a Dual Microgradient Substrate

    PubMed Central

    2016-01-01

    Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) raise many possibilities for cardiac research but they exhibit an immature phenotype, which influences experimental outcomes. The aim of our research is to investigate the effects of a topographical gradient substrate on the morphology and function of commercially available hiPSC-CM. The lateral dimensions the microgrooves on the substrate varied from 8 to 100 μm space between the 8 μm grooves on one axis and from ∼5 nm to ∼1 μm in depth on the other axis. Cells were seeded homogeneously across the substrate and according to the manufacturers protocols. At days 4 and 10, measures of eccentricity, elongation, orientation, sarcomere length (SL), and contractility of the hiPSC-CM were taken. Only the deepest and widest region (8–30 μm wide and 0.85–1 μm deep) showed a significantly higher percentage of hiPSC-CM with an increased eccentricity (31.3 ± 6.4%), elongation (10.4 ± 4.3%), and orientation (<10°) (32.1 ± 2.7%) when compared with the control (flat substrate) (15.8 ± 5.0%, 3.4 ± 2.7%, and 10.6 ± 1.1%, respectively). Additionally, during stimulus-induced contraction, the relaxation phase of the twitch was prolonged (400 ms) compared to nonelongated cells (200 ms). These findings support the potential use of dual microgradient substrates to investigate substrate topographies that stimulate migration and/or maturation of hiPSC-CM. PMID:27990488

  19. Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing.

    PubMed

    Kim, In Gul; Hwang, Mintai P; Du, Ping; Ko, Jaehoon; Ha, Chul-won; Do, Sun Hee; Park, Kwideok

    2015-05-01

    Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications.

  20. ROCK Inhibition Extends Passage of Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium.

    PubMed

    Croze, Roxanne H; Buchholz, David E; Radeke, Monte J; Thi, William J; Hu, Qirui; Coffey, Peter J; Clegg, Dennis O

    2014-09-01

    Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-β pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture.

  1. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    SciTech Connect

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  2. Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene

    SciTech Connect

    Shirozu, Michio; Takano, Toru; Tada, Hideaki; Honjo, Tasuku

    1995-08-10

    Stromal cell-derived factors 1{alpha} and 1{beta} are small cytokines belonging to the intercrine CXC subfamily and originally isolated from a murine bone-marrow stroma cell line by the signal sequence trap method. cDNA and genomic clones of human SDF1{alpha} and SDF1{beta} (SDF1A and SDF1B) were isolated and characterized. cDNAs of SDF1{alpha} and SDF1{beta} encode proteins of 89 and 93 amino acids, respectively. SDF1{alpha} and SDF1{beta} sequences are more than 92% identical to those of the human counterparts. The genomic structure of the SDF1 gene revealed that human SDF1{alpha} and SDF1{beta} are encoded by a single gene and arise by alternative splicing. SDF1{alpha} and SDF1{beta} are encoded by 3 and 4 exons, respectively. Ubiquitous expression of the SDF1 gene, except in blood cells, was consistent with the presence of the GC-rich sequence in the 5{prime}-flanking region of the SDF1 gene, as is often the case in the {open_quotes}housekeeping{close_quotes} genes. Although genes encoding other members of the intercrine family are localized on chromosome 4q or 17q, the human SDF1 gene was mapped to chromosome 10q by fluorescence in situ hybridization. Strong evolutionary conservation and unique chromosomal localization of the SDF1 gene suggest that SDF1{alpha} and SDF1{beta} may have important functions distinct from those of other members of the intercrine family. 37 refs., 5 figs.

  3. Continuous delivery of stromal cell-derived factor-1 from alginate scaffolds accelerates wound healing.

    PubMed

    Rabbany, Sina Y; Pastore, Joseph; Yamamoto, Masaya; Miller, Tim; Rafii, Shahin; Aras, Rahul; Penn, Marc

    2010-01-01

    Proper wound diagnosis and management is an increasingly important clinical challenge and is a large and growing unmet need. Pressure ulcers, hard-to-heal wounds, and problematic surgical incisions are emerging at increasing frequencies. At present, the wound-healing industry is experiencing a paradigm shift towards innovative treatments that exploit nanotechnology, biomaterials, and biologics. Our study utilized an alginate hydrogel patch to deliver stromal cell-derived factor-1 (SDF-1), a naturally occurring chemokine that is rapidly overexpressed in response to tissue injury, to assess the potential effects SDF-1 therapy on wound closure rates and scar formation. Alginate patches were loaded with either purified recombinant human SDF-1 protein or plasmid expressing SDF-1 and the kinetics of SDF-1 release were measured both in vitro and in vivo in mice. Our studies demonstrate that although SDF-1 plasmid- and protein-loaded patches were able to release therapeutic product over hours to days, SDF-1 protein was released faster (in vivo K(d) 0.55 days) than SDF-1 plasmid (in vivo K(d) 3.67 days). We hypothesized that chronic SDF-1 delivery would be more effective in accelerating the rate of dermal wound closure in Yorkshire pigs with acute surgical wounds, a model that closely mimics human wound healing. Wounds treated with SDF-1 protein (n = 10) and plasmid (n = 6) loaded patches healed faster than sham (n = 4) or control (n = 4). At day 9, SDF-1-treated wounds significantly accelerated wound closure (55.0 +/- 14.3% healed) compared to nontreated controls (8.2 +/- 6.0%, p < 0.05). Furthermore, 38% of SDF-1-treated wounds were fully healed at day 9 (vs. none in controls) with very little evidence of scarring. These data suggest that patch-mediated SDF-1 delivery may ultimately provide a novel therapy for accelerating healing and reducing scarring in clinical wounds.

  4. The nature of activatory and tolerogenic dendritic cell-derived signal II

    PubMed Central

    Bakdash, Ghaith; Sittig, Simone P.; van Dijk, Tjeerd; Figdor, Carl G.; de Vries, I. Jolanda M.

    2013-01-01

    Dendritic cells (DCs) are central in maintaining the intricate balance between immunity and tolerance by orchestrating adaptive immune responses. Being the most potent antigen presenting cells, DCs are capable of educating naïve T cells into a wide variety of effector cells ranging from immunogenic CD4+ T helper cells and cytotoxic CD8+ T cells to tolerogenic regulatory T cells. This education is based on three fundamental signals. Signal I, which is mediated by antigen/major histocompatibility complexes binding to antigen-specific T cell receptors, guarantees antigen specificity. The co-stimulatory signal II, mediated by B7 family molecules, is crucial for the expansion of the antigen-specific T cells. The final step is T cell polarization by signal III, which is conveyed by DC-derived cytokines and determines the effector functions of the emerging T cell. Although co-stimulation is widely recognized to result from the engagement of T cell-derived CD28 with DC-expressed B7 molecules (CD80/CD86), other co-stimulatory pathways have been identified. These pathways can be divided into two groups based on their impact on primed T cells. Whereas pathways delivering activatory signals to T cells are termed co-stimulatory pathways, pathways delivering tolerogenic signals to T cells are termed co-inhibitory pathways. In this review, we discuss how the nature of DC-derived signal II determines the quality of ensuing T cell responses and eventually promoting either immunity or tolerance. A thorough understanding of this process is instrumental in determining the underlying mechanism of disorders demonstrating distorted immunity/tolerance balance, and would help innovating new therapeutic approaches for such disorders. PMID:23450201

  5. Body lice

    MedlinePlus

    ... Body lice are tiny insects (scientific name is Pediculus humanus corporis ) that are spread through close contact ... disease Images Body louse Lice, body with stool (Pediculus humanus) Body louse, female and larvae Head louse ...

  6. G-protein Coupled Receptor Signaling in Pluripotent Stem Cell-derived Cardiovascular Cells: Implications for Disease Modeling

    PubMed Central

    Dolatshad, Nazanin F.; Hellen, Nicola; Jabbour, Richard J.; Harding, Sian E.; Földes, Gabor

    2015-01-01

    Human pluripotent stem cell derivatives show promise as an in vitro platform to study a range of human cardiovascular diseases. A better understanding of the biology of stem cells and their cardiovascular derivatives will help to understand the strengths and limitations of this new model system. G-protein coupled receptors (GPCRs) are key regulators of stem cell maintenance and differentiation and have an important role in cardiovascular cell signaling. In this review, we will therefore describe the state of knowledge concerning the regulatory role of GPCRs in both the generation and function of pluripotent stem cell derived-cardiomyocytes, -endothelial, and -vascular smooth muscle cells. We will consider how far the in vitro disease models recapitulate authentic GPCR signaling and provide a useful basis for discovery of disease mechanisms or design of therapeutic strategies. PMID:26697426

  7. Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate.

    PubMed

    Rios, Natalia; Piacenza, Lucía; Trujillo, Madia; Martínez, Alejandra; Demicheli, Verónica; Prolo, Carolina; Álvarez, María Noel; López, Gloria V; Radi, Rafael

    2016-12-01

    more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.

  8. Producing fully ES cell-derived mice from eight-cell stage embryo injections.

    PubMed

    DeChiara, Thomas M; Poueymirou, William T; Auerbach, Wojtek; Frendewey, David; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    In conventional methods for the generation of genetically modified mice, gene-targeted embryonic stem (ES) cells are injected into blastocyst-stage embryos or are aggregated with morula-stage embryos, which are then transferred to the uterus of a surrogate mother. F0 generation mice born from the embryos are chimeras composed of genetic contributions from both the modified ES cells and the recipient embryos. Obtaining a mouse strain that carries the gene-targeted mutation requires breeding the chimeras to transmit the ES cell genetic component through the germ line to the next (F1) generation (germ line transmission, GLT). To skip the chimera stage, we developed the VelociMouse method, in which injection of genetically modified ES cells into eight-cell embryos followed by maturation to the blastocyst stage and transfer to a surrogate mother produces F0 generation mice that are fully derived from the injected ES cells and exhibit a 100% GLT efficiency. The method is simple and flexible. Both male and female ES cells can be introduced into the eight-cell embryo by any method of injection or aggregation and using all ES cell and host embryo combinations from inbred, hybrid, and outbred genetic backgrounds. The VelociMouse method provides several unique opportunities for shortening project timelines and reducing mouse husbandry costs. First, as VelociMice exhibit 100% GLT, there is no need to test cross chimeras to establish GLT. Second, because the VelociMouse method permits efficient production of ES cell-derived mice from female ES cells, XO ES cell subclones, identified by screening for spontaneous loss of the Y chromosome, can be used to generate F0 females that can be bred with isogenic F0 males derived from the original targeted ES cell clone to obtain homozygous mutant mice in the F1 generation. Third, as VelociMice are genetically identical to the ES cells from which they were derived, the VelociMouse method opens up myriad possibilities for creating mice with

  9. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions.

    PubMed

    Yang, Hao; Gao, Li-Na; An, Ying; Hu, Cheng-Hu; Jin, Fang; Zhou, Jun; Jin, Yan; Chen, Fa-Ming

    2013-09-01

    Gingival tissue-derived mesenchymal stem cells (MSCs) were recently identified and characterized as having multipotential differentiation and immunomodulatory properties in vitro and in vivo, and they represent new postnatal stem cell types for cytotherapy and regenerative medicine. However, the utility of gingival MSCs (GMSCs) as alternatives to periodontal ligament stem cells (PDLSCs), which have been demonstrated to be effective but with limited cell availability and reduced clinical feasibility, for periodontal regeneration in a previously diseased/inflamed environment remains obscure. In this study, patient-matched human GMSCs and PDLSCs were evaluated in terms of their colony-forming ability, proliferative capacity, cell surface epitopes, multi-lineage differentiation potentials, and related gene expression when incubated in different designed culture conditions, with or without the presence of inflammatory cytokines. An in vivo ectopic transplantation model using transplants from inflammatory cytokine-treated or untreated cells was applied to assess bone formation. We found that cells derived from both tissues expressed MSC markers, including CD146, CD105, CD90, CD29, and STRO-1. Both cells successfully differentiated under osteogenic, adipogenic, and chondrogenic microenvironments; PDLSCs displayed a more effective differentiation potential in all of the incubation conditions compared to GMSCs (P < 0.01). Although inflammatory cytokine-treated GMSCs and PDLSCs are inferior to normally cultured, patient and tissue-matched cells in terms of their osteogenic capacity and regenerative potential (P < 0.05), they retain the capacity for osteoblastic and adipose differentiation, as well as ectopic bone formation, similar to what has been demonstrated for other MSCs. Interestingly, GMSCs exhibited fewer inflammation-related changes in terms of osteogenic potential in vitro and bone formation in vivo compared to PDLSCs (P < 0.01). These results suggest

  10. Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment

    PubMed Central

    McClung, Joseph M.; Reinardy, Jessica L.; Mueller, Sarah B.; McCord, Timothy J.; Kontos, Christopher D.; Brown, David A.; Hussain, Sabah N. A.; Schmidt, Cameron A.; Ryan, Terence E.; Green, Tom D.

    2015-01-01

    Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy. PMID

  11. Vascular morphogenesis of human umbilical vein endothelial cells on cell-derived macromolecular matrix microenvironment.

    PubMed

    Du, Ping; Subbiah, Ramesh; Park, Jung-Hwan; Park, Kwideok

    2014-09-01

    Extracellular matrix (ECM) is a highly organized network of proteins and other macromolecules that plays a critical role in cell adhesion, migration, and differentiation. In this study, we hypothesize that ECM derived from in-vitro-cultured cells possesses unique surface texture, topography, and mechanical property, and consequently carries some distinct cues for vascular morphogenesis of human umbilical vein endothelial cells (ECs). Cell-derived matrix (CDM) was obtained by culturing fibroblasts, preosteoblasts, and chondrocytes, respectively, on coverslips and then by decellularizing them using detergents and enzymes. These matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). Immunofluorescence of each CDM shows that some of the matrix components are fibronectin (FN), type I collagen, and laminin. Atomic force microscopy analysis presented that average fiber diameter ranged from 2 to 7 μm and FDM holds much larger fibers. The matrix elasticity measurements revealed that average Young's modulus of CHDM (17.7 ± 4.2 kPa) was much greater than that of PDM (10.5 ± 1.1 kPa) or FDM (5.7 ± 0.5 kPa). During 5-day culture, EC morphologies were dramatically changed on PDM and FDM, but those on CHDM and gelatin were rather stable, regardless of time lapse. Cell migration assay discovered quicker repopulation of the scratched areas on PDM and FDM than on gelatin and CHDM. A capillary-like structure (CLS) assembly was also notable only in the PDM and FDM, as compared with CHDM, gelatin, or FN that were very poor in CLS formation. Quantitative analysis of mean CLS branch points and branch lengths demonstrated much better angiogenic activity of ECs on PDM and FDM. Interestingly, CLS formation was closely associated with matrix remodeling by ECs and the matrix clearance on PDM with time was sharply contrasted with that on CHDM that majority of the matrix FN was reserved. It was notable that membrane

  12. Peripheral antinociceptive effects of exogenous and immune cell-derived endomorphins in prolonged inflammatory pain.

    PubMed

    Labuz, Dominika; Berger, Stephan; Mousa, Shaaban A; Zöllner, Christian; Rittner, Heike L; Shaqura, Mohammed A; Segovia-Silvestre, Toni; Przewlocka, Barbara; Stein, Christoph; Machelska, Halina

    2006-04-19

    Endomorphins (EMs) are endogenous selective mu-opioid receptor agonists. Their role in inflammatory pain has not been fully elucidated. Here we examine peripheral antinociception elicited by exogenously applied EM-1 and EM-2 and the contribution of EM-containing leukocytes to stress- and corticotropin-releasing factor (CRF)-induced antinociception. To this end, we applied behavioral (paw pressure) testing, radioligand binding, immunohistochemistry, and flow cytometry in rats with unilateral hindpaw inflammation induced with Freund's adjuvant. EMs injected directly into both hindpaws produced antinociception exclusively in inflamed paws. This was blocked by locally applied mu-receptor-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) but not kappa-receptor-selective (nor-binaltorphimine) antagonists. Delta-receptor antagonists (naltrindole and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu) did not influence EM-1-induced but dose-dependently decreased EM-2-induced antinociception. Antibodies against beta-endorphin, methionine-enkephalin, or leucine-enkephalin did not significantly change EM-2-induced antinociception. Both EMs displaced binding of [3H]-[D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to mu-receptors in dorsal root ganglia (DRG). Using [3H]-naltrindole or [(125)I]-[D-Pen2,5]-enkephalin, no detectable delta-binding was found in DRG of inflamed hindlimbs. Numerous beta-endorphin-containing and fewer EM-1- and EM-2-containing leukocytes were detected in subcutaneous tissue of inflamed paws. Leukocyte-depleting serum decreased the number of immigrating opioid-containing immune cells and attenuated swim stress- and CRF-induced antinociception in inflamed paws. Both forms of antinociception were strongly attenuated by anti-beta-endorphin and to a lesser degree by anti-EM-1 and anti-EM-2 antibodies injected into inflamed paws. Together, exogenously applied and immune cell-derived EMs alleviate prolonged inflammatory pain through selective activation of peripheral opioid receptors

  13. Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury

    PubMed Central

    Li, Ke; Javed, Elham; Scura, Daniel; Hala, Tamara J.; Seetharam, Suneil; Falnikar, Aditi; Richard, Jean-Philippe; Chorath, Ashley; Maragakis, Nicholas J.; Wright, Megan C.; Lepore, Angelo C.

    2015-01-01

    Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor

  14. Transplantation of Embryonic and Induced Pluripotent Stem Cell-Derived 3D Retinal Sheets into Retinal Degenerative Mice

    PubMed Central

    Assawachananont, Juthaporn; Mandai, Michiko; Okamoto, Satoshi; Yamada, Chikako; Eiraku, Mototsugu; Yonemura, Shigenobu; Sasai, Yoshiki; Takahashi, Masayo

    2014-01-01

    Summary In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL) with complete inner and outer segments even in an advanced retinal degeneration model (rd1) that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a “proof of concept” for retinal sheet transplantation therapy for advanced retinal degenerative diseases. PMID:24936453

  15. Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

    DTIC Science & Technology

    2015-02-05

    derived 5a. CONTRACT NUMBER central nervous system neurons 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Hubbard, K, Beske, PH...numbers CBM.THRTOX.01.10.RC.023 and CBM.THRTOX.01.RC.014). 14. ABSTRACT See reprint. 15. SUBJECT TERMS embryonic stem cells, stem cell-derived neurons ...botulinum neurotoxin detection, electrophysiology, synapse, neuronal networks, glutamatergic synapse, GABAergic synapse 16. SECURITY CLASSIFICATION

  16. Growth and turning properties of adult glial cell-derived neurotrophic factor coreceptor α1 nonpeptidergic sensory neurons.

    PubMed

    Guo, GuiFang; Singh, Vandana; Zochodne, Douglas W

    2014-09-01

    An overlapping population of adult primary sensory neurons that innervate the skin express the glial cell-derived neurotrophic factor coreceptor α1 (GFRα1), the lectin IB4, and the "regenerative brake" phosphatase and tensin homolog deleted on chromosome 10. Using an adapted turning and growth assay, we analyzed the growth cone behavior of adult immunoselected GFRα1 sensory neurons. These neurons had less robust baseline growth and reluctant responsiveness to individual growth factors but responded to synergistic types of input from glial cell-derived neurotrophic factor, hepatocyte growth factor, a phosphatase and tensin homolog deleted on chromosome 10 inhibitor, or a downstream Rho kinase inhibitor. Hepatocyte growth factor and the phosphatase and tensin homolog deleted on chromosome 10 inhibitor were associated with growth cone turning. A gradient of protein extracted from skin samples, a primary target of GFRα1 axons, replicated the impact of synergistic support. Within the skin, glial cell-derived neurotrophic factor was expressed within epidermal axons, indicating an autocrine role accompanying local hepatocyte growth factor synthesis. Taken together, our findings identify unique growth properties and plasticity of a distinct population of epidermal axons that are relevant to neurologic repair and skin reinnervation.

  17. Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy

    PubMed Central

    OSADA, Hironari; OGAWA, Misato; HASEGAWA, Ayana; NAGAI, Makoto; SHIRAI, Junsuke; SASAKI, Kazuaki; SHIMODA, Minoru; ITOH, Hiroshi; KONDO, Hirotaka; OHMORI, Keitaro

    2016-01-01

    It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE. PMID:28049868

  18. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

    PubMed Central

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  19. Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy.

    PubMed

    Osada, Hironari; Ogawa, Misato; Hasegawa, Ayana; Nagai, Makoto; Shirai, Junsuke; Sasaki, Kazuaki; Shimoda, Minoru; Itoh, Hiroshi; Kondo, Hirotaka; Ohmori, Keitaro

    2017-02-28

    It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE.

  20. Stromal Cell-Derived Factor 1 Polymorphism in Retinal Vein Occlusion

    PubMed Central

    Szigeti, Andrea; Ecsedy, Mónika; Schneider, Miklós; Lénárt, Lilla; Lesch, Balázs; Nagy, Zoltán Zsolt

    2016-01-01

    Background Stromal cell-derived factor 1 (SDF1) has crucial role in the regulation of angiogenesis and ocular neovascularisation (NV). The purpose of this study was to evaluate the association between SDF1-3’G(801)A polymorphism and NV complications of retinal vein occlusion (RVO). Methods 130 patients with RVO (median age: 69.0, range 35–93 years; male/female– 58/72; 55 patients had central RVO, 75 patients had branch RVO) were enrolled in this study. In the RVO group, 40 (30.8%) patients were diagnosed with NV complications of RVO and 90 (69.2%) patients without NVs. The median follow up period was 40.3 months (range: 18–57 months). The SDF1-3’G(801)A polymorphism was detected by PCR-RFLP. Allelic prevalence was related to reference values obtained in the control group consisted of 125 randomly selected, age and gender matched, unrelated volunteers (median age: 68.0, range 36–95 years; male/female– 53/72). Statistical analysis of the allele and genotype differences between groups (RVO patients vs controls; RVO patients with NV vs RVO patients without NV) was determined by chi-squared test. P value of <0.05 was considered statistically significant. Results Hardy-Weinberg criteria was fulfilled in all groups. The SDF1-3’G(801)A allele and genotype frequencies of RVO patients were similar to controls (SDF1-3’A allele: 22.3% vs 20.8%; SDF1-3’(801)AA: 5.4% vs 4.8%, SDF1-3’(801)GG: 60.8% vs 63.2%). The frequency of SDF1-3’(801)AA and SDF1-3’(801)GA genotypes, as well as the SDF1-3’(801)A allele frequency were higher in RVO patients with NV versus in patients without NV complication (SDF1-3’(801)AA+AG genotypes: 57.5% vs 31.1%, p = 0.008; SDF1-3’(801)A allele: 35.0% vs 16.7%, p = 0.002) or versus controls (SDF1-3’(801)AA+AG genotypes 57.5% vs 36.8%, p = 0.021; SDF1-3’(801)A allele: 35.0% vs 20.8% p = 0.01). Carrying of SDF1-3’(801)A allele increased the risk of neovascularisation complications of RVO by 2.69 (OR, 95% CI = 1.47–4

  1. 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.

  2. Calcium regulation of vascular smooth muscle cell-derived matrix vesicles.

    PubMed

    Kapustin, Alexander N; Shanahan, Catherine M

    2012-07-01

    Vascular calcification is a pathological process common in patients with disorders of mineral metabolism and mediated by vascular smooth muscle cells (VSMCs). A key event in the initiation of VSMC calcification is the release of mineralization-competent matrix vesicles (MVs), small membrane-bound bodies with structural features enabling them to efficiently nucleate hydroxyapatite. These bodies are similar to MVs secreted by chondrocytes during bone development and their properties include the absence of calcification inhibitors, formation of nucleation sites, and accumulation of matrix metalloproteinases such as MMP-2. The mechanisms of MV biogenesis and loading remain poorly understood; however, emerging data have demonstrated that alterations in cytosolic calcium homeostasis can trigger multiple changes in MV composition that promote their mineralization.

  3. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts.

    PubMed

    Wen, Duancheng; Saiz, Nestor; Rosenwaks, Zev; Hadjantonakis, Anna-Katerina; Rafii, Shahin

    2014-01-01

    Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  4. Resolving the "egg supply problem" in human embryonic stem cell derivation through technical means--a legal and ethical analysis.

    PubMed

    Hammond, Natasha; Holm, Søren

    2008-03-01

    This paper seeks to briefly discuss the legal and ethical problems connected to scientific developments in the field of human embryonic stem cell derivation aimed at solving the "egg supply problem" in stem cell research. The legal situation is discussed in respect of the UK's current regulatory regime, proposed reform and the Oviedo Convention. The scientific developments which are examined are chimeric embryos, in vitro maturation of oocytes, derivation of stem cell lines in connection with pre-implantation genetic diagnosis and the derivation of oocytes from existing stem cell lines.

  5. Body Composition.

    ERIC Educational Resources Information Center

    Mayhew, Jerry L.

    1981-01-01

    Body composition refers to the types and amounts of tissues which make up the body. The most acceptable method for assessing body composition is underwater weighing. A subcutaneous skinfold provides a quantitative measurement of fat below the skin. The skinfold technique permits a valid estimate of the body's total fat content. (JN)

  6. Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Angiogenesis: Potencial Clinical Application

    PubMed Central

    Merino-González, Consuelo; Zuñiga, Felipe A.; Escudero, Carlos; Ormazabal, Valeska; Reyes, Camila; Nova-Lamperti, Estefanía; Salomón, Carlos; Aguayo, Claudio

    2016-01-01

    Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are able to differentiate into multiple specialized cell types including osteocytes, adipocytes, and chondrocytes. MSCs exert different functions in the body and have recently been predicted to have a major clinical/therapeutic potential. However, the mechanisms of self-renewal and tissue regeneration are not completely understood. It has been shown that the biological effect depends mainly on its paracrine action. Furthermore, it has been reported that the secretion of soluble factors and the release of extracellular vesicles, such as exosomes, could mediate the cellular communication to induce cell-differentiation/self-renewal. This review provides an overview of MSC-derived exosomes in promoting angiogenicity and of the clinical relevance in a therapeutic approach. PMID:26903875

  7. Application of a Persistent Heparin Treatment Inhibits the Malignant Potential of Oral Squamous Carcinoma Cells Induced by Tumor Cell-Derived Exosomes.

    PubMed

    Sento, Shinya; Sasabe, Eri; Yamamoto, Tetsuya

    2016-01-01

    Exosomes are 30-100 nm-sized membranous vesicles, secreted from a variety of cell types into their surrounding extracellular space. Various exosome components including lipids, proteins, and nucleic acids are transferred to recipient cells and affect their function and activity. Numerous studies have showed that tumor cell-derived exosomes play important roles in tumor growth and progression. However, the effect of exosomes released from oral squamous cell carcinoma (OSCC) into the tumor microenvironment remains unclear. In the present study, we isolated exosomes from OSCC cells and investigated the influence of OSCC cell-derived exosomes on the tumor cell behavior associated with tumor development. We demonstrated that OSCC cell-derived exosomes were taken up by OSCC cells themselves and significantly promoted proliferation, migration, and invasion through the activation of the PI3K/Akt, MAPK/ERK, and JNK-1/2 pathways in vitro. These effects of OSCC cell-derived exosomes were obviously attenuated by treatment with PI3K, ERK-1/2, and JNK-1/2 pharmacological inhibitors. Furthermore, the growth rate of tumor xenografts implanted into nude mice was promoted by treatment with OSCC cell-derived exosomes. The uptake of exosomes by OSCC cells and subsequent tumor progression was abrogated in the presence of heparin. Taken together, these data suggest that OSCC cell-derived exosomes might be a novel therapeutic target and the use of heparin to inhibit the uptake of OSCC-derived exosomes by OSCC cells may be useful for treatment.

  8. Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues

    PubMed Central

    Ravenscroft, Stephanie M.; Pointon, Amy; Williams, Awel W.; Cross, Michael J.; Sidaway, James E.

    2016-01-01

    The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity. PMID:27125969

  9. Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy

    PubMed Central

    Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L.; Dai, Long-Jun; Luo, Jie

    2015-01-01

    Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable. PMID:26496034

  10. Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

    PubMed

    Tang, Xiang-Jun; Sun, Xu-Yong; Huang, Kuan-Ming; Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L; Dai, Long-Jun; Luo, Jie

    2015-12-29

    Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

  11. Comparative gene expression profiling in human-induced pluripotent stem cell--derived cardiocytes and human and cynomolgus heart tissue.

    PubMed

    Puppala, Dinesh; Collis, Leon P; Sun, Sunny Z; Bonato, Vinicius; Chen, Xian; Anson, Blake; Pletcher, Mathew; Fermini, Bernard; Engle, Sandra J

    2013-01-01

    Cardiotoxicity is one of the leading causes of drug attrition. Current in vitro models insufficiently predict cardiotoxicity, and there is a need for alternative physiologically relevant models. Here we describe the gene expression profile of human-induced pluripotent stem cell-derived cardiocytes (iCC) postthaw over a period of 42 days in culture and compare this profile to human fetal and adult as well as adult cynomolgus nonhuman primate (NHP, Macaca fascicularis) heart tissue. Our results indicate that iCC express relevant cardiac markers such as ion channels (SCN5A, KCNJ2, CACNA1C, KCNQ1, and KCNH2), tissue-specific structural markers (MYH6, MYLPF, MYBPC3, DES, TNNT2, and TNNI3), and transcription factors (NKX2.5, GATA4, and GATA6) and lack the expression of stem cell markers (FOXD3, GBX2, NANOG, POU5F1, SOX2, and ZFP42). Furthermore, we performed a functional evaluation of contractility of the iCC and showed functional and pharmacological correlations with myocytes isolated from adult NHP hearts. These results suggest that stem cell-derived cardiocytes may represent a novel in vitro model to study human cardiac toxicity with potential ex vivo and in vivo translation.

  12. Tumor cell-derived exosome-targeted dendritic cells stimulate stronger CD8+ CTL responses and antitumor immunities.

    PubMed

    Yao, Ye; Chen, Linjun; Wei, Wei; Deng, Xiaohui; Ma, Liyuan; Hao, Siguo

    2013-06-21

    Tumor cell-derived exosomes (TEX) have been widely used to induce antitumor immune responses in animal models and clinical trials. However, the efficiency of the antitumor immunity that is induced by TEX is still relatively weak. In this study, we compared the antitumor immunities between EG7 tumor cell-derived exosomes (EXO(EG7)) and EXO(EG7)-targeted dendritic cells (DC(EXO)). We found that EXO(EG7) harbored OVA and peptide major histocompatibility complex I (pMHC-I), which were expressed on its parental EG7 tmor cells, and they could transfer OVA and pMHC-I to dendritic cells (DCs) in vitro. DC(EXO) could more efficiently induce antitumor immunity than EXO(EG7). In addition, we showed that the immune stimulatory effects of EXO(EG7) were dependent on the host DCs and, whereas those of DC(EXO) were not, indicating the important role of the host DCs in TEX vaccines. Taken together, TEX-targeted DCs may be more effective for EXO-based vaccines for the induction of antitumor immunity.

  13. Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived HGF and fibronectin in angiogenesis

    PubMed Central

    Newman, Andrew C.; Chou, Wayne; Welch-Reardon, Katrina M.; Fong, Ashley H.; Popson, Stephanie A.; Phan, Duc Thien; Sandoval, Daniel R.; Nguyen, Dananh P.; Gershon, Paul D.; Hughes, Christopher C. W.

    2013-01-01

    Objective Angiogenesis requires tightly coordinated cross-talk between endothelial cells and stromal cells such as fibroblasts and smooth muscle cells. The specific molecular mechanisms moderating this process are still poorly understood. Method and Results Stromal cell-derived factors are essential for endothelial cell sprouting and lumen formation. We therefore compared the abilities of two primary fibroblast isolates and a primary smooth muscle cell isolate to promote in vitro angiogenesis and analyzed their secretomes using a combination of nanoLC-MS/MS, qPCR and ELISA. Each isolate exhibited a different level of angiogenic ability. Using quantitative MS, we then compared the secretomes of a fibroblast isolate exhibiting low angiogenic activity, a fibroblast isolate exhibiting high angiogenic activity and human umbilical vein endothelial cells. High angiogenic fibroblast supernatants exhibited an over-abundance of proteins associated with extracellular matrix constituents compared to low angiogenic fibroblasts or endothelial cells. Finally, siRNA technology and purified protein were used to confirm a role for stromal cell-derived hepatocyte growth factor and fibronectin in inducing endothelial cell sprouting. Conclusion Differences in stromal cell ability to induce angiogenesis are due to differences in the secreted proteomes of both extracellular matrix proteins and pro-angiogenic growth factors. PMID:23288153

  14. Stem cell-derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model.

    PubMed

    Donegan, J J; Tyson, J A; Branch, S Y; Beckstead, M J; Anderson, S A; Lodge, D J

    2016-08-02

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that the stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia.Molecular Psychiatry advance online publication, 2 August 2016; doi:10.1038/mp.2016.121.

  15. Induction of the MCP chemokine cluster cascade in the periphery by cancer cell-derived Ccl3.

    PubMed

    Farmaki, Elena; Kaza, Vimala; Papavassiliou, Athanasios G; Chatzistamou, Ioulia; Kiaris, Hippokratis

    2017-03-28

    The induction of localized pro-inflammatory niches in the periphery is instrumental in metastasis. In order to better understand how tumors engage distal sites and activate a pro-inflammatory response we utilized syngeneic breast cancers as a model and showed that soluble factors from the neoplastic epithelium activate the expression of the monocyte chemoattractive protein (MCP) chemokines of the mouse 11C cluster that include Ccl1, Ccl2, Ccl7, Ccl8, Ccl11 and Ccl12. Tissues such as the lungs and the brain, that are more prone to colonization by breast cancer cells, were more sensitive to MCP cluster chemokine induction than others such as the liver. Subsequent analyses involving chemokine arrays in breast cancer cells and media followed by functional validation assays in in vitro and in vivo identified the cytokine Ccl3 as the principle mediator of the communication between the neoplastic epithelium and the peripheral tissues in terms of MCP cluster chemokine induction. Our results show that MCP chemokines are activated in peripheral tissues of breast cancer-bearing mice, by a mechanism that involves breast cancer cell-derived Ccl3. Interference with the expression of cancer cell-derived Ccl3 may find application in the management of breast cancer metastases.

  16. Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

    PubMed

    Shah, Sandeep N; Gelderman, Monique P; Lewis, Emily M A; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I; Vostal, Jaroslav G

    2016-01-01

    Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

  17. Pulmonary Epithelial Cell-Derived Cytokine TGF-β1 Is a Critical Cofactor for Enhanced Innate Lymphoid Cell Function

    PubMed Central

    Denney, Laura; Byrne, Adam J.; Shea, Thomas J.; Buckley, James S.; Pease, James E.; Herledan, Gaelle M.F.; Walker, Simone A.; Gregory, Lisa G.; Lloyd, Clare M.

    2015-01-01

    Summary Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-β had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-β1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-β by expressing the receptor TGF-βRII and ILC chemoactivity was enhanced by TGF-β. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions. PMID:26588780

  18. Prominent role for T cell-derived Tumour Necrosis Factor for sustained control of Mycobacterium tuberculosis infection

    PubMed Central

    Allie, Nasiema; Grivennikov, Sergei I.; Keeton, Roanne; Hsu, Nai-Jen; Bourigault, Marie-Laure; Court, Nathalie; Fremond, Cecile; Yeremeev, Vladimir; Shebzukhov, Yuriy; Ryffel, Bernhard; Nedospasov, Sergei A.; Quesniaux, Valerie F. J.; Jacobs, Muazzam

    2013-01-01

    Tumour Necrosis Factor (TNF) is critical for host control of M. tuberculosis, but the relative contribution of TNF from innate and adaptive immune responses during tuberculosis infection is unclear. Myeloid versus T-cell-derived TNF function in tuberculosis was investigated using cell type-specific TNF deletion. Mice deficient for TNF expression in macrophages/neutrophils displayed early, transient susceptibility to M. tuberculosis but recruited activated, TNF-producing CD4+ and CD8+ T-cells and controlled chronic infection. Strikingly, deficient TNF expression in T-cells resulted in early control but susceptibility and eventual mortality during chronic infection with increased pulmonary pathology. TNF inactivation in both myeloid and T-cells rendered mice critically susceptible to infection with a phenotype resembling complete TNF deficient mice, indicating that myeloid and T-cells are the primary TNF sources collaborating for host control of tuberculosis. Thus, while TNF from myeloid cells mediates early immune function, T-cell derived TNF is essential to sustain protection during chronic tuberculosis infection. PMID:23657146

  19. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-06-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

  20. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    PubMed Central

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-01-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis. PMID:27292795

  1. Optimization of surface-immobilized extracellular matrices for the proliferation of neural progenitor cells derived from induced pluripotent stem cells.

    PubMed

    Komura, Takashi; Kato, Koichi; Konagaya, Shuhei; Nakaji-Hirabayashi, Tadashi; Iwata, Hiroo

    2015-11-01

    Neural progenitor cells derived from induced pluripotent stem cells have been considered as a potential source for cell-transplantation therapy of central nervous disorders. However, efficient methods to expand neural progenitor cells are further required for their clinical applications. In this study, a protein array was fabricated with nine extracellular matrices and used to screen substrates suitable for the expansion of neural progenitor cells derived from mouse induced pluripotent stem cells. The results showed that neural progenitor cells efficiently proliferated on substrates with immobilized laminin-1, laminin-5, or Matrigel. Based on this result, further attempts were made to develop clinically compliant substrates with immobilized polypeptides that mimic laminin-1, one of the most effective extracellular matrices as identified in the array-based screening. We used here recombinant DNA technology to prepare polypeptide containing the globular domain 3 of laminin-1 and immobilized it onto glass-based substrates. Our results showed that neural progenitor cells selectively proliferated on substrate with the immobilized polypeptide while maintaining their differentiated state.

  2. Teratocarcinomas Arising from Allogeneic Induced Pluripotent Stem Cell-Derived Cardiac Tissue Constructs Provoked Host Immune Rejection in Mice

    PubMed Central

    Kawamura, Ai; Miyagawa, Shigeru; Fukushima, Satsuki; Kawamura, Takuji; Kashiyama, Noriyuki; Ito, Emiko; Watabe, Tadashi; Masuda, Shigeo; Toda, Koichi; Hatazawa, Jun; Morii, Eiichi; Sawa, Yoshiki

    2016-01-01

    Transplantation of induced pluripotent stem cell-derived cardiac tissue constructs is a promising regenerative treatment for cardiac failure: however, its tumourigenic potential is concerning. We hypothesised that the tumourigenic potential may be eliminated by the host immune response after allogeneic cell transplantation. Scaffold-free iPSC-derived cardaic tissue sheets of C57BL/6 mouse origin were transplanted into the cardiac surface of syngeneic C57BL/6 mice and allogeneic BALB/c mice with or without tacrolimus injection. Syngeneic mice and tacrolimus-injected immunosuppressed allogeneic mice formed teratocarcinomas with identical phenotypes, characteristic, and time courses, as assessed by imaging tools including 18F-fluorodeoxyglucose-positron emission tomography. In contrast, temporarily immunosuppressed allogeneic mice, following cessation of tacrolimus injection displayed diminished progression of the teratocarcinoma, accompanied by an accumulation of CD4/CD8-positive T cells, and finally achieved complete elimination of the teratocarcinoma. Our results indicated that malignant teratocarcinomas arising from induced pluripotent stem cell-derived cardiac tissue constructs provoked T cell-related host immune rejection to arrest tumour growth in murine allogeneic transplantation models. PMID:26763872

  3. Region-Specific Integration of Embryonic Stem Cell-Derived Neuronal Precursors into a Pre-Existing Neuronal Circuit

    PubMed Central

    Neuser, Franziska; Polack, Martin; Annaheim, Christine; Tucker, Kerry L.; Korte, Martin

    2013-01-01

    Enduring reorganization is accepted as a fundamental process of adult neural plasticity. The most dramatic example of this reorganization is the birth and continuously occurring incorporation of new neurons into the pre-existing network of the adult mammalian hippocampus. Based on this phenomenon we transplanted murine embryonic stem (ES)-cell derived neuronal precursors (ESNPs) into murine organotypic hippocampal slice cultures (OHC) and examined their integration. Using a precise quantitative morphological analysis combined with a detailed electrophysiology, we show a region-specific morphological integration of transplanted ESNPs into different subfields of the hippocampal tissue, resulting in pyramidal neuron-like embryonic stem cell-derived neurons (ESNs) in the Cornu Ammonis (CA1 and CA3) and granule neuron-like ESNs in the dentate gyrus (DG), respectively. Subregion specific structural maturation was accompanied by the development of dendritic spines and the generation of excitatory postsynaptic currents (EPSCs). This cell type specific development does not depend upon NMDA-receptor-dependent synaptic transmission. The presented integration approach was further used to determine the cell-autonomous function of the pan-neurotrophin receptor p75 (P75NTR), as a possible negative regulator of ESN integration. By this means we used p75NTR-deficient ESNPs to study their integration into a WT organotypic environment. We show here that p75NTR is not necessary for integration per se but plays a suppressing role in dendritic development. PMID:23840491

  4. Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

    PubMed Central

    1992-01-01

    We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types. PMID:1572895

  5. Pluripotent stem cell derivation and differentiation toward cardiac muscle: novel techniques and advances in patent literature.

    PubMed

    Quattrocelli, Mattia; Thorrez, Lieven; Sampaolesi, Maurilio

    2013-04-01

    Pluripotent stem cells hold unprecedented potential for regenerative medicine, disease modeling and drug screening. Embryonic stem cells (ESCs), standard model for pluripotency studies, have been recently flanked by induced pluripotent stem cells (iPSCs). iPSCs are obtained from somatic cells via epigenetic and transcriptional reprogramming, overcoming ESC-related ethical issues and enabling the possibility of donor-matching pluripotent cell lines. Since the European Court of Justice banned patents involving embryo disaggregation to generate human ESCs, iPSCs can now fuel the willingness of European companies to invest in treatments based on stem cells. Moreover, iPSCs share many unique features of ESCs, such as unlimited self-renewal potential and broad differentiation capability, even though iPSCs seem more susceptible to genomic instability and display epigenetic biases as compared to ESCs. Both ESCs and iPSCs have been intensely investigated for cardiomyocyte production and cardiac muscle regeneration, both in human and animal models. In vitro and in vivo studies are continuously expanding and refining this field via genetic manipulation and cell conditioning, trying to achieve standard and reproducible products, eligible for clinical and biopharmaceutical scopes. This review focuses on the recently growing body of patents, concerning technical advances in production, expansion and cardiac differentiation of ESCs and iPSCs.

  6. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    PubMed

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs.

  7. Pluripotency of adult stem cells derived from human and rat pancreas

    NASA Astrophysics Data System (ADS)

    Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

    Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

  8. Stem cell-derived tissue-associated regulatory T cells ameliorate the development of autoimmunity

    PubMed Central

    Haque, Mohammad; Song, Jianyong; Fino, Kristin; Sandhu, Praneet; Song, Xinmeng; Lei, Fengyang; Zheng, Songguo; Ni, Bing; Fang, Deyu; Song, Jianxun

    2016-01-01

    Pluripotent stem cells (PSCs) have the potential to produce almost all of the cells in the body, including regulatory T cells (Tregs). However, the exact conditions required for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) are not well delineated. Ag-specific PSC-Tregs can be tissue/organ-associated and migrate to local inflamed tissues/organs to suppress the autoimmune response after adoptive transfer, thereby avoiding potential overall immunosuppression from non-specific Tregs. In this study, we developed a new approach to generate functional Ag-specific Tregs from induced PSCs (iPSCs), i.e., iPSC-Tregs, which had the ability to generate an Ag-specific immunosuppressive response in a murine model of arthritis. We retrovirally transduced murine iPSCs with a construct containing genes of Ag-specific T cell receptor (TCR) and the transcriptional factor FoxP3. We differentiated the iPSCs into Ag-specific iPSC-Tregs using in vitro or in vivo Notch signaling, and demonstrated that adoptive transfer of such Tregs dramatically suppressed autoimmunity in a well-established Ag-induced arthritis model, including the inflammation, joint destruction, cartilage prostaglandin depletion, osteoclast activity, and Th17 production. Our results indicate that PSCs can be used to develop Ag-specific Tregs, which have a therapeutic potential for Treg-based therapies of autoimmune disorders. PMID:26846186

  9. Body Measurement.

    ERIC Educational Resources Information Center

    Neufeld, K. Allen

    1989-01-01

    Described are activities for measuring the human body. The activities include measurements and calculations, calculating volume and density, problems related to body measurement, and using a nomogram. Several charts, illustrations, and a nomogram are provided. (YP)

  10. The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages

    PubMed Central

    Xie, Jingwei; Willerth, Stephanie M.; Li, Xiaoran; Macewan, Matthew R.; Rader, Allison; Sakiyama-Elbert, Shelly E.; Xia, Younan

    2008-01-01

    Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair. PMID:18930315

  11. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons

    PubMed Central

    Machado, Carolina Barcellos; Kanning, Kevin C.; Kreis, Patricia; Stevenson, Danielle; Crossley, Martin; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David; Blanc, Eric; Lieberam, Ivo

    2014-01-01

    Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations. PMID:24496616

  12. Exogenous Nitric Oxide Protects Human Embryonic Stem Cell-Derived Cardiomyocytes against Ischemia/Reperfusion Injury

    PubMed Central

    Pálóczi, János; Varga, Zoltán V.; Szebényi, Kornélia; Sarkadi, Balázs; Madonna, Rosalinda; De Caterina, Raffaele; Csont, Tamás; Eschenhagen, Thomas; Ferdinandy, Péter; Görbe, Anikó

    2016-01-01

    Background and Aims. Human embryonic stem cell- (hESC-) derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP) in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days) and then on more differentiated cardiomyocytes (6 + 24 days), both derived from hESCs. Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (10−7, 10−6, and 10−5 M), BNP (10−9, 10−8, and 10−7 M), and the nonspecific NO synthase inhibitor Nω-nitro-L-arginine (L-NNA, 10−5 M). Results. SNAP (10−6, 10−5 M) significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10−6 M) also protected hESC-derived cardiomyocytes. Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms. PMID:27403231

  13. Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets

    PubMed Central

    KIM, JAEHYUP; BREUNIG, MELISSA J.; ESCALANTE, LEAH E.; BHATIA, NEEHAR; DENU, RYAN A.; DOLLAR, BRIDGET A.; STEIN, ANDREW P.; HANSON, SUMMER E.; NADERI, NADIA; RADEK, JAMES; HAUGHY, DERMOT; BLOOM, DEBRA D.; ASSADI-PORTER, FARIBA M.; HEMATTI, PEIMAN

    2012-01-01

    Background aims Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. Methods We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. Results Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. Conclusions Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts. PMID:22571381

  14. Mesenchymal stem cell-derived exosomes from different sources selectively promote neuritic outgrowth.

    PubMed

    Lopez-Verrilli, M A; Caviedes, A; Cabrera, A; Sandoval, S; Wyneken, U; Khoury, M

    2016-04-21

    Mesenchymal stem cells (MSCs) obtained from bone marrow (BM) have been shown to promote neuronal growth and survival. However, the comparative effects of MSCs of different sources, including menstrual MSCs (MenSCs), BM, umbilical cord and chorion stem cells on neurite outgrowth have not yet been explored. Moreover, the modulatory effects of MSCs may be mediated by paracrine mechanisms, i.e. by molecules contained in the MSC secretome that includes soluble factors and extracellular vesicles such as microvesicles and/or exosomes. The biogenesis of microvesicles, characterized by a vesicle diameter of 50 to 1000 nm, involves membrane shedding while exosomes, of 30 to 100 nm in diameter, originate in the multivesicular bodies within cells. Both vesicle types, which can be harvested from the conditioned media of cell cultures by differential centrifugation steps, regulate the function of target cells due to their molecular content of microRNA, mRNA, proteins and lipids. Here, we compared the effect of human menstrual MSCs (MenSCs) mediated by cell-cell contact, by their total secretome or by secretome-derived extracellular vesicles on neuritic outgrowth in primary neuronal cultures. The contact of MenSCs with cortical neurons inhibited neurite outgrowth while their total secretome enhanced it. The extracellular vesicle fractions showed a distinctive effect: while the exosome-enriched fraction enhanced neurite outgrowth, the microvesicle-enriched fraction displayed an inhibitory effect. When we compared exosome fractions of different human MSC sources, MenSC exosomes showed superior effects on the growth of the longest neurite in cortical neurons and had a comparable effect to BM-SC exosomes on neurite outgrowth in dorsal root ganglia neurons. Thus, the growth-stimulating effects of exosomes derived from MenSCs as well as the opposing effects of both extracellular vesicle fractions provide important information regarding the potential use of MenSCs as therapeutic

  15. The effect of inflammatory cell-derived MCP-1 loss on neuronal survival during chronic neuroinflammation

    PubMed Central

    Sawyer, Andrew J.; Tian, Weiming; Saucier-Sawyer, Jennifer K.; Rizk, Paul J.; Saltzman, W. Mark; Bellamkonda, Ravi; Kyriakides, Themis R.

    2014-01-01

    Intracranial implants elicit neurodegeneration via the foreign body response (FBR) that includes BBB leakage, macrophage/microglia accumulation, and reactive astrogliosis, in addition to neuronal degradation that limit their useful lifespan. Previously, monocyte chemoattractant protein 1 (MCP-1, also CCL2), which plays an important role in monocyte recruitment and propagation of inflammation, was shown to be critical for various aspects of the FBR in a tissue-specific manner. However, participation of MCP-1 in the brain FBR has not been evaluated. Here we examined the FBR to intracortical silicon implants in MCP-1 KO mice at 1, 2, and 8 weeks after implantation. MCP-1 KO mice had a diminished FBR compared to WT mice, characterized by reductions in BBB leakage, macrophage/microglia accumulation, and astrogliosis, and an increased neuronal density. Moreover, pharmacological inhibition of MCP-1 in implant-bearing WT mice maintained the increased neuronal density. To elucidate the relative contribution of microglia and macrophages, bone marrow chimeras were generated between MCP-1 KO and WT mice. Increased neuronal density was observed only in MCP-1 knockout mice transplanted with MCP-1 knockout marrow, which indicates that resident cells in the brain are major contributors. We hypothesized that these improvements are the result of a phenotypic switch of the macrophages/microglia polarization state, which we confirmed using PCR for common activation markers. Our observations suggest that MCP-1 influences neuronal loss, which is integral to the progression of neurological disorders like Alzheimer’s and Parkinson disease, via BBB leakage and macrophage polarization. PMID:24881026

  16. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    PubMed

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

  17. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

    PubMed

    Machado, Carolina Barcellos; Kanning, Kevin C; Kreis, Patricia; Stevenson, Danielle; Crossley, Martin; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David; Blanc, Eric; Lieberam, Ivo

    2014-02-01

    Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

  18. Human embryonic stem cell-derived mesoderm-like epithelium transitions to mesenchymal progenitor cells.

    PubMed

    Boyd, Nolan L; Robbins, Kelly R; Dhara, Sujoy K; West, Franklin D; Stice, Steven L

    2009-08-01

    Human embryonic stem cells (hESC) have the potential to produce all of the cells in the body. They are able to self-renew indefinitely, potentially making them a source for large-scale production of therapeutic cell lines. Here, we developed a monolayer differentiation culture that induces hESC (WA09 and BG01) to form epithelial sheets with mesodermal gene expression patterns (BMP4, RUNX1, and GATA4). These E-cadherin+ CD90low cells then undergo apparent epithelial-mesenchymal transition for the derivation of mesenchymal progenitor cells (hESC-derived mesenchymal cells [hES-MC]) that by flow cytometry are negative for hematopoietic (CD34, CD45, and CD133) and endothelial (CD31 and CD146) markers, but positive for markers associated with mesenchymal stem cells (CD73, CD90, CD105, and CD166). To determine their functionality, we tested their capacity to produce the three lineages associated with mesenchymal stem cells and found they could form osteogenic and chondrogenic, but not adipogenic lineages. The derived hES-MC were able to remodel and contract collagen I lattice constructs to an equivalent degree as keloid fibroblasts and were induced to express alpha-smooth muscle actin when exposed to transforming growth factor (TGF)-beta1, but not platelet derived growth factor-B (PDGF-B). These data suggest that the derived hES-MC are multipotent cells with potential uses in tissue engineering and regenerative medicine and for providing a highly reproducible cell source for adult-like progenitor cells.

  19. Effects of Asiasari radix on the morphology and viability of mesenchymal stem cells derived from the gingiva.

    PubMed

    Jeong, Su-Hyeon; Lee, Ji-Eun; Jin, Seong-Ho; Ko, Youngkyung; Park, Jun-Beom

    2014-12-01

    Medicinal herbs used in traditional Oriental medicine, which have been in use clinically for thousands of years, are attractive sources of novel therapeutics or preventatives. Asiasari radix (A. radix) has been suggested for use in the treatment of dental diseases, including toothache and aphthous stomatitis. The aim of this study was to evaluate the effects of A. radix extracts on the morphology and viability of human stem cells derived from the gingiva. An Asiasarum heterotropoides extract was centrifuged and freeze-dried in a lyophilizer. Stem cells derived from the gingiva were grown in the presence of A. radix at concentrations ranging between 0.1 µg/ml and 1 mg/ml (0, 0.1, 1, 10, 100 and 1,000 µg/ml). Cell morphology was evaluated with an optical microscope and the viability of the cells was quantitatively analyzed with a cell counting kit-8 (CCK-8) assay for up to seven days. The untreated control group exhibited normal fibroblast morphology. The shapes of the cells following 0.1, 1, 10 and 100 µg/ml A. radix treatments were similar to those of the control group. However, a significant change was noted in the 1,000 µg/ml group on day 1, when compared with the untreated group. Furthermore, on day 7, the shapes of the cells following 100 and 1,000 µg/ml A. radix treatments were rounder and fewer cells were present, when compared with those of the control group. The cultures that grew in the presence of A. radix did not exhibit any changes in the CCK‑8 assay on day 2; however, significant reductions in cell viability were noticed following 100 and 1,000 µg/ml A. radix treatment on days 5 and 7. Within the limits of this study, A. radix influenced the viability of the stem cells derived from the gingiva. Thus, the direct application of A. radix to oral tissues may produce adverse effects at high doses. Therefore, the concentration and application time of A. radix requires meticulous control to obtain optimal results. These effects require consideration

  20. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes.

    PubMed

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J

    2015-12-08

    The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening.

  1. Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

    PubMed Central

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-01-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells. PMID:24938227

  2. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

    PubMed

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein

    2014-05-01

    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  3. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    PubMed

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  4. Stromal cell-derived factor-1 promotes migration of cells from the upper rhombic lip in cerebellar development.

    PubMed

    Yu, Tao; Huang, Hai; Li, Hui-Fang

    2010-10-01

    During cerebellar development, the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha) has been shown to play an important role in recruiting cells from the upper rhombic lip (URL) and external granule cell layer (EGL). However, its function in cerebellar development is still poorly understood. Our results have demonstrated that SDF-1 is necessary for EGL development, and URL cells stream to the SDF-1 source in vitro. Results of embryonic URL explant assays and transwell assays indicated that SDF-1 induces neural cell migration from the URL region in chemotactic and chemokinetic responses. The time-lapse results showed that the migration speed of granule cell progenitors out of the URL was accelerated by the addition of recombinant SDF-1 alpha. Collectively, our study shows that SDF-1 increases the motility of URL cells in the absence of a gradient and promotes the migration of granule cell progenitors during cerebellar development.

  5. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J.

    2015-01-01

    Summary The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening. PMID:26626178

  6. Establishment of an ES cell-derived murine megakaryocytic cell line, MKD1, with features of primary megakaryocyte progenitors.

    PubMed

    Chagraoui, Hedia; Porcher, Catherine

    2012-01-01

    Because of the scarcity of megakaryocytes in hematopoietic tissues, studying megakaryopoiesis heavily relies on the availability of appropriate cellular models. Here, we report the establishment of a new mouse embryonic stem (ES) cell-derived megakaryocytic cell line, MKD1. The cells are factor-dependent, their cell surface immunophenotype and gene expression profile closely resemble that of primary megakaryocyte progenitors (MkPs) and they further differentiate along the megakaryocyte lineage upon valproic acid treatment. At a functional level, we show that ablation of SCL expression, a transcription factor critical for MkP maturation, leads to gene expression alterations similar to that observed in primary, Scl-excised MkPs. Moreover, the cell line is amenable to biochemical and transcriptional analyses, as we report for GpVI, a direct target of SCL. Thus, the MKD1 cell line offers a pertinent experimental model to study the cellular and molecular mechanisms underlying MkP biology and more broadly megakaryopoiesis.

  7. The Role of Tumor Cell-Derived Connective Tissue Growth Factor (CTGF/CCN2) in Pancreatic Tumor Growth

    PubMed Central

    Bennewith, Kevin L.; Huang, Xin; Ham, Christine M.; Graves, Edward E.; Erler, Janine T.; Kambham, Neeraja; Feazell, Jonathan; Yang, George P.; Koong, Albert

    2009-01-01

    Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted subcutaneously. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by PET imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed co-localization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer. PMID:19179545

  8. Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model

    PubMed Central

    Lewis, Emily M. A.; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I.; Vostal, Jaroslav G.

    2016-01-01

    Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as ‘the storage lesion’. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product. PMID:27959920

  9. Human Stem Cell-Derived Endothelial-Hepatic Platform for Efficacy Testing of Vascular-Protective Metabolites from Nutraceuticals.

    PubMed

    Narmada, Balakrishnan Chakrapani; Goh, Yeek Teck; Li, Huan; Sinha, Sanjay; Yu, Hanry; Cheung, Christine

    2017-03-01

    Atherosclerosis underlies many cardiovascular and cerebrovascular diseases. Nutraceuticals are emerging as a therapeutic moiety for restoring vascular health. Unlike small-molecule drugs, the complexity of ingredients in nutraceuticals often confounds evaluation of their efficacy in preclinical evaluation. It is recognized that the liver is a vital organ in processing complex compounds into bioactive metabolites. In this work, we developed a coculture system of human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and human pluripotent stem cell-derived hepatocytes (hPSC-HEPs) for predicting vascular-protective effects of nutraceuticals. To validate our model, two compounds (quercetin and genistein), known to have anti-inflammatory effects on vasculatures, were selected. We found that both quercetin and genistein were ineffective at suppressing inflammatory activation by interleukin-1β owing to limited metabolic activity of hPSC-ECs. Conversely, hPSC-HEPs demonstrated metabolic capacity to break down both nutraceuticals into primary and secondary metabolites. When hPSC-HEPs were cocultured with hPSC-ECs to permit paracrine interactions, the continuous turnover of metabolites mitigated interleukin-1β stimulation on hPSC-ECs. We observed significant reductions in inflammatory gene expressions, nuclear translocation of nuclear factor κB, and interleukin-8 production. Thus, integration of hPSC-HEPs could accurately reproduce systemic effects involved in drug metabolism in vivo to unravel beneficial constituents in nutraceuticals. This physiologically relevant endothelial-hepatic platform would be a great resource in predicting the efficacy of complex nutraceuticals and mechanistic interrogation of vascular-targeting candidate compounds. Stem Cells Translational Medicine 2017;6:851-863.

  10. Spatiotemporal recapitulation of central nervous system development by murine embryonic stem cell-derived neural stem/progenitor cells.

    PubMed

    Okada, Yohei; Matsumoto, Arifumi; Shimazaki, Takuya; Enoki, Ryosuke; Koizumi, Amane; Ishii, Seiji; Itoyama, Yasuto; Sobue, Gen; Okano, Hideyuki

    2008-12-01

    Neural stem/progenitor cells (NS/PCs) can generate a wide variety of neural cells. However, their fates are generally restricted, depending on the time and location of NS/PC origin. Here we demonstrate that we can recapitulate the spatiotemporal regulation of central nervous system (CNS) development in vitro by using a neurosphere-based culture system of embryonic stem (ES) cell-derived NS/PCs. This ES cell-derived neurosphere system enables the efficient derivation of highly neurogenic fibroblast growth factor-responsive NS/PCs with early temporal identities and high cell-fate plasticity. Over repeated passages, these NS/PCs exhibit temporal progression, becoming epidermal growth factor-responsive gliogenic NS/PCs with late temporal identities; this change is accompanied by an alteration in the epigenetic status of the glial fibrillary acidic protein promoter, similar to that observed in the developing brain. Moreover, the rostrocaudal and dorsoventral spatial identities of the NS/PCs can be successfully regulated by sequential administration of several morphogens. These NS/PCs can differentiate into early-born projection neurons, including cholinergic, catecholaminergic, serotonergic, and motor neurons, that exhibit action potentials in vitro. Finally, these NS/PCs differentiate into neurons that form synaptic contacts with host neurons after their transplantation into wild-type and disease model animals. Thus, this culture system can be used to obtain specific neurons from ES cells, is a simple and powerful tool for investigating the underlying mechanisms of CNS development, and is applicable to regenerative treatment for neurological disorders.

  11. Phenotypic and functional characterization of cytokine-induced killer cells derived from preterm and term infant cord blood.

    PubMed

    Zhang, Qian; Wang, Lili; Luo, Chenghan; Shi, Zanyang; Cheng, Xinru; Zhang, Zhen; Yang, Yi; Zhang, Yi

    2014-11-01

    Cord blood has gradually become an important source for hematopoietic stem cell transplantation (HSCT) in the human, particularly in pediatric patients. Adoptive cellular immunotherapy of patients with hematologic malignancies after umbilical cord blood transplant is crucial. Cytokine‑induced killer (CIK) cells derived from cord blood are a new type of antitumor immune effector cells in tumor prevention and treatment and have increasingly attracted the attention of researchers. On the other hand, it has been suggested that preterm infant cord blood retains an early differentiation phenotype suitable for immunotherapy. Therefore, we determined the phenotypic and functional characterization of CIK cells derived from preterm infant cord blood (PCB-CIK) compared with CIK cells from term infant cord blood (TCB-CIK). Twenty cord blood samples were collected and classified into two groups based on gestational age. Cord blood mononuclear cells (CBMCs) were isolated, cultured and induced to CIK cells in vitro. We used flow cytometry to detect cell surface markers, FlowJo software to analyze the proliferation profile and intracellular staining to test the secretion of cytokines. Finally, we evaluated the antitumor activity of CIK cells against K562 in vitro. Compared with TCB-CIK, PCB-CIK cells demonstrated faster proliferation and higher expression of activated cell surface markers. The secretion of IL-10 was lower in PCB-CIK cells while the expression of perforin and CD107a had no significant difference between the two cell groups. PCB-CIK cells exhibited a high proliferation rate while the cytotoxic activity had no difference between the PCB-CIK and TCB-CIK cells. Hence preterm infant cord blood may be a potential source for immunotherapy.

  12. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitors Aid in Functional Recovery of Sensory Pathways following Contusive Spinal Cord Injury

    PubMed Central

    All, Angelo H.; Bazley, Faith A.; Gupta, Siddharth; Pashai, Nikta; Hu, Charles; Pourmorteza, Amir; Kerr, Candace

    2012-01-01

    Background Transplantations of human stem cell derivatives have been widely investigated in rodent models for the potential restoration of function of neural pathways after spinal cord injury (SCI). Studies have already demonstrated cells survival following transplantation in SCI. We sought to evaluate survival and potential therapeutic effects of transplanted human embryonic stem (hES) cell-derived oligodendrocyte progenitor cells (OPCs) in a contusive injury in rats. Bioluminescence imaging was utilized to verify survivability of cells up to 4 weeks, and somatosensory evoked potential (SSEPs) were recorded at the cortex to monitor function of sensory pathways throughout the 6-week recovery period. Principal Findings hES cells were transduced with the firefly luciferase gene and differentiated into OPCs. OPCs were transplanted into the lesion epicenter of rat spinal cords 2 hours after inducing a moderate contusive SCI. The hES-treatment group showed improved SSEPs, including increased amplitude and decreased latencies, compared to the control group. The bioluminescence of transplanted OPCs decreased by 97% in the injured spinal cord compared to only 80% when injected into an uninjured spinal cord. Bioluminescence increased in both experimental groups such that by week 3, no statistical difference was detected, signifying that the cells survived and proliferated independent of injury. Post-mortem histology of the spinal cords showed integration of human cells expressing mature oligodendrocyte markers and myelin basic protein without the expression of markers for astrocytes (GFAP) or pluripotent cells (OCT4). Conclusions hES-derived OPCs transplanted 2 hours after contusive SCI survive and differentiate into OLs that produce MBP. Treated rats demonstrated functional improvements in SSEP amplitudes and latencies compared to controls as early as 1 week post-injury. Finally, the hostile injury microenvironment at 2 hours post-injury initially caused increased cell

  13. Generation of Neural Crest-Like Cells From Human Periodontal Ligament Cell-Derived Induced Pluripotent Stem Cells.

    PubMed

    Tomokiyo, Atsushi; Hynes, Kim; Ng, Jia; Menicanin, Danijela; Camp, Esther; Arthur, Agnes; Gronthos, Stan; Mark Bartold, Peter

    2017-02-01

    Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue-derived periodontal ligament (PDL) iPSC to differentiate into neural crest-like cells (NCLC) relative to iPSC generated from a non-neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC- and FF iPSC-derived NCLC, which highly expressed HNK1. A high proportion of the HNK1-positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1-positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell-associated markers than the PDL and FF HNK1-negative populations. Interestingly, the HNK1-positive cells derived from the PDL-iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1-positive cells derived from the FF-iPSC. Our work suggests that HNK1-enriched NCLC from neural crest tissue-derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1-low population and non-neural crest iPSC-derived NCLC. J. Cell. Physiol. 232: 402-416, 2017. © 2016 Wiley Periodicals, Inc.

  14. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-05-01

    Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic

  15. Isolation and Assessment of Mesenchymal Stem Cells Derived From Bone Marrow: Histologic and Histomorphometric Study in a Canine Periodontal Defect.

    PubMed

    Paknejad, Mojgan; Eslaminejad, Mohamadreza Baghaban; Ghaedi, Baharak; Rokn, Amir-Reza; Khorsand, Afshin; Etemad-Moghadam, Shahroo; Alaeddini, Mojgan; Dehghan, Mohammad Mehdi; Moslemi, Neda; Nowzari, Hessam

    2015-06-01

    The aim of the present study was to investigate an isolation procedure to culture mesenchymal stem cells derived from bone marrow and evaluate their potential in periodontal regeneration. Potential stem cells from bone marrow, aspirated from the iliac crest of nine mongrel canines 1 to 2 years of age, were cultivated. After the examination of surface epitopes of the isolated cells, the total RNA from osteogenic, adipogenic, and chondrogenic cell cultures were analyzed by reverse transcription polymerase chain reaction (RT-PCR) to confirm stem cell gene expressions. 2 × 10(7) mL of the stem cells were loaded on 0.2 mL of anorganic bovine bone mineral (ABBM) granules. In each animal, bilateral acute/chronic intrabony periodontal defects were created surgically and by placement of ligatures around the cervical aspect of the teeth. At week 5, after flap debridement, the bilateral defects were randomly assigned to 2 treatment groups: the control group received ABBM, and the test group received BMSCs-loaded ABBM. Eight weeks after transplantation, regenerative parameters were analyzed histologically and histometrically. The RNA expressions confirmed the cultivation of mesenchymal stem cell. More new cementum and periodontal ligament (PDL) were measured in the test group (cementum: 3.33 ± 0.94 vs 2.03 ± 1.30, P = 0.027; PDL: 2.69 ± 0.73 vs 1.53 ± 1.21, P = 0.026). New bone formation was similar in both groups (2.70 ± 0.86 vs 1.99 ± 1.31; P = 0.193). Mesenchymal stem cells derived from bone marrow should be considered a promising technique for use in patients with periodontal attachment loss and merits further investigations.

  16. Cancer cell-derived IL-1α induces CCL22 and the recruitment of regulatory T cells.

    PubMed

    Wiedemann, Gabriela Maria; Knott, Max Martin Ludwig; Vetter, Viola Katharina; Rapp, Moritz; Haubner, Sascha; Fesseler, Julia; Kühnemuth, Benjamin; Layritz, Patrick; Thaler, Raffael; Kruger, Stephan; Ormanns, Steffen; Mayr, Doris; Endres, Stefan; Anz, David

    2016-01-01

    In cancer patients, immunosuppression through regulatory T cells (Treg) is a crucial component of tumor immune evasion and contributes to disease progression. Tumor-infiltrating Treg in particular suppress local effector T cell responses and are associated with poor prognosis in tumors such as human pancreatic cancer or hepatocellular carcinoma (HCC). The chemokine CCL22 is known to recruit Treg into the tumor tissue and many types of human tumors are known to express high levels of CCL22. The mechanisms leading to intratumoral secretion of CCL22 are so far unknown. We demonstrate here that intratumoral CCL22 is induced in tumor-infiltrating immune cells through cancer cell-derived interleukin-1 (IL-1α). In pancreatic cancer and HCC, CCL22 is produced by intratumoral dendritic cells, while the cancer cells themselves do not secrete CCL22 in vitro and in vivo. Incubation of human peripheral blood mononuclear cells (PBMC) or murine splenocytes with tumor cells or tumor cell supernatants strongly induced CCL22 secretion in vitro. Tumor cell supernatants contained IL-1 and CCL22 induction in PBMC could be specifically prevented by the IL-1 receptor antagonist anakinra or by transfection of tumor cell lines with IL-1 siRNA, leading to a suppression of Treg migration. In conclusion, we identify here tumor cell-derived IL-1α as a major inducer of the Treg attracting chemokine CCL22 in human cancer cells. Therapeutic blockade of the IL-1 pathway could represent a promising strategy to inhibit tumor-induced immunosuppression.

  17. Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations.

    PubMed

    Iovino, Mariangela; Agathou, Sylvia; González-Rueda, Ana; Del Castillo Velasco-Herrera, Martin; Borroni, Barbara; Alberici, Antonella; Lynch, Timothy; O'Dowd, Sean; Geti, Imbisaat; Gaffney, Daniel; Vallier, Ludovic; Paulsen, Ole; Káradóttir, Ragnhildur Thóra; Spillantini, Maria Grazia

    2015-11-01

    Tauopathies, such as Alzheimer's disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development.

  18. Fas signal promotes lung cancer growth by recruiting myeloid-derived suppressor cells via cancer cell-derived PGE2.

    PubMed

    Zhang, Yongliang; Liu, Qiuyan; Zhang, Minggang; Yu, Yizhi; Liu, Xia; Cao, Xuetao

    2009-03-15

    Fas/FasL system has been extensively investigated with respect to its capacity to induce cellular apoptosis. However, accumulated evidences show that Fas signaling also exhibits nonapoptotic functions, such as induction of cell proliferation and differentiation. Lung cancer is one of cancer's refractory to the immunotherapy, however, the underlying mechanisms remain to be fully understood. In this study, we show that Fas overexpression does not affect in vitro growth of 3LL cells, but promotes lung cancer growth in vivo. However, such tumor-promoting effect is not observed in FasL-deficient (gld) mice, and also not observed in the immune competent mice once inoculation with domain-negative Fas-overexpressing 3LL cells, suggesting the critical role of Fas signal in the promotion of lung cancer growth in vivo. More accumulation of myeloid-derived suppressor cells (MDSC) and Foxp3(+) regulatory T cells is found in tumors formed by inoculation with Fas-overexpressing 3LL cells, but not domain-negative Fas-overexpressing 3LL cells. Accordingly, Fas-ligated 3LL lung cancer cells can chemoattract more MDSC but not regulatory T cells in vitro. Furthermore, Fas ligation induces 3LL lung cancer cells to produce proinflammatory factor PGE(2) by activating p38 pathway, and in turn, 3LL cells-derived PGE(2) contribute to the Fas ligation-induced MDSC chemoattraction. Furthermore, in vivo administration of cyclooxygenase-2 inhibitor can significantly reduce MDSC accumulation in the Fas-overexpressing tumor. Therefore, our results demonstrate that Fas signal can promote lung cancer growth by recruiting MDSC via cancer cell-derived PGE(2), thus providing new mechanistic explanation for the role of inflammation in cancer progression and immune escape.

  19. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-02-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.

  20. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components

    PubMed Central

    Li, Dameng; Wang, Jifeng; Hou, Dongxia; Jiang, Xiaohong; Zhang, Junfeng; Wang, Jin; Zen, Ke; Yang, Fuquan; Zhang, Chen-Yu

    2016-01-01

    Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects. PMID:27649079

  1. Cancer Cell-Derived Extracellular Vesicles Are Associated with Coagulopathy Causing Ischemic Stroke via Tissue Factor-Independent Way: The OASIS-CANCER Study

    PubMed Central

    Bang, Oh Young; Chung, Jong-Won; Lee, Mi Ji; Kim, Suk Jae; Cho, Yeon Hee; Kim, Gyeong-Moon; Chung, Chin-Sang; Lee, Kwang Ho; Ahn, Myung-Ju; Moon, Gyeong Joon

    2016-01-01

    Background Cancer and stroke, which are known to be associated with one another, are the most common causes of death in the elderly. However, the pathomechanisms that lead to stroke in cancer patients are not well known. Circulating extracellular vesicles (EVs) play a role in cancer-associated thrombosis and tumor progression. Therefore, we hypothesized that cancer cell-derived EVs cause cancer-related coagulopathy resulting in ischemic stroke. Methods Serum levels of D-dimer and EVs expressing markers for cancer cells (epithelial cell adhesion molecule [CD326]), tissue factor (TF [CD142]), endothelial cells (CD31+CD42b-), and platelets (CD62P) were measured using flow cytometry in (a) 155 patients with ischemic stroke and active cancer (116 − cancer-related, 39 − conventional stroke mechanisms), (b) 25 patients with ischemic stroke without cancer, (c) 32 cancer patients without stroke, and (d) 101 healthy subjects. Results The levels of cancer cell-derived EVs correlated with the levels of D-dimer and TF+ EVs. The levels of cancer cell-derived EVs (CD326+ and CD326+CD142+) were higher in cancer-related stroke than in other groups (P<0.05 in all the cases). Path analysis showed that cancer cell-derived EVs are related to stroke via coagulopathy as measured by D-dimer levels. Poor correlation was observed between TF+ EV and D-dimer, and path analysis demonstrated that cancer cell-derived EVs may cause cancer-related coagulopathy independent of the levels of TF+ EVs. Conclusions Our findings suggest that cancer cell-derived EVs mediate coagulopathy resulting in ischemic stroke via TF-independent mechanisms. PMID:27427978

  2. Body Basics

    MedlinePlus

    ... more about how the body works, what basic human anatomy is, and what happens when parts of the body don't function properly. Blood Bones, Muscles, and Joints Brain and Nervous System Digestive System Endocrine System Eyes Female Reproductive System ...

  3. Upregulation of CD4+T-Cell Derived MiR-223 in The Relapsing Phase of Multiple Sclerosis Patients

    PubMed Central

    Hosseini, Aref; Ghaedi, Kamran; Tanhaei, Somayeh; Ganjalikhani-Hakemi, Mazdak; Teimuri, Shohreh; Etemadifar, Masoud; Nasr Esfahani, Mohammad Hossein

    2016-01-01

    Objective MicroRNAs (miRNA) are a class of non-coding RNAs which play key roles in post-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis (MS). Th17 and regulatory T (Treg) cells are two subsets of CD4+T-cells which have critical functions in the onset and progression of MS. The current study seeks to distinguish fluctuations in expression of CD4+T-cell derived miR-223 during the relapsing-remitting (RR) phase of MS (RR-MS), as well as the expressions of Th17 and Treg cell markers. Materials and Methods This experimental study used real-time quantitative polymerase chain reaction (qRT-PCR) to evaluate CD4+ T cell derived miR-223 expression patterns in patients that experienced either of the RR-MS phases (n=40) compared to healthy controls (n=12), along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 (FOXP3) and RAR-related orphan receptor γt (RORγt) in CD4+T-cells. Putative and validated targets of miR-223 were investigated in the miRWalk and miRTarBase databases, respectively. Results miR-223 significantly upregulated in CD4+T-cells during the relapsing phase of RR-MS compared to the remitting phase (P=0.000) and healthy individuals (P=0.036). Expression of RORγt, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potential targets of miR-223, STAT1, FORKHEAD BOX O (FOXO1) and FOXO3 were predicted by in silico studies. Conclusion miR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS. PMID:27602319

  4. Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration

    PubMed Central

    Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J.; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y. Eugene; Ma, Peter X.

    2015-01-01

    Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue. PMID:26398309

  5. 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

  6. A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

    PubMed Central

    Hellewell, Andrew L.; Rosini, Silvia; Adams, Josephine C.

    2017-01-01

    The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation. Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs. PMID:28117783

  7. Recommended Ethical Safeguards on Fertilization of Human Germ Cells Derived from Pluripotent Stem Cells Solely for Research Purposes.

    PubMed

    Mizuno, Hiroshi

    2016-08-01

    Production of human fertilized embryos by using germ cells derived from pluripotent stem cells (PSCs) entails ethical issues that differ fundamentally depending on the aim. If the aim is solely to conduct research, then embryo generation, utilization and destruction must respect for the human embryo as having the innate potential to develop into a human being. If the aim is human reproduction, this technology must never be used to manipulate human life, confuse social order, or negatively affect future generations. Researchers should distinguish the aims and then accordingly establish a consensus on the safeguards needed to proceed with scientifically significant and socially accepted research, or otherwise set a moratorium. Currently, in Japan, germ cell production from human PSCs is permitted, whereas fertilization of these germ cells is not. The Japanese Expert Panel on Bioethics in the Cabinet Office has proposed that all of the following conditions must be met to approve fertilization for research purposes: (1) the research is significant for the life sciences and medicine; (2) the benefits or anticipated benefits are socially accepted; (3) human safety is assured; and (4) safeguards are put in place. If fertilization is ethically approved, I recommend the following safeguards: limitation of the purpose to improving conventional ART as an initial step; permitted culture of human embryos until the appearance of the primitive streak; restriction of the number of embryos produced to the minimum necessary; prohibition of transplantation into a human or animal uterus; and provision of human-derived ova that are not required for ART treatment.

  8. Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function

    PubMed Central

    Yeung, A. T. Y.; Hale, C.; Xia, J.; Tate, P. H.; Goulding, D.; Keane, J. A.; Mukhopadhyay, S.; Forrester, L.; Billker, O.; Skarnes, W. C.; Hancock, R. E. W.; Dougan, G.

    2015-01-01

    The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection. PMID:25752829

  9. Economic 3D-printing approach for transplantation of human stem cell-derived β-like cells.

    PubMed

    Song, Jiwon; Millman, Jeffrey R

    2016-12-01

    Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing β cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived β (SC-β) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-β cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-β cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.

  10. Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions.

    PubMed

    Klimchenko, Olena; Di Stefano, Antonio; Geoerger, Birgit; Hamidi, Sofiane; Opolon, Paule; Robert, Thomas; Routhier, Mélanie; El-Benna, Jamel; Delezoide, Anne-Lise; Boukour, Siham; Lescure, Bernadette; Solary, Eric; Vainchenker, William; Norol, Françoise

    2011-03-17

    The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

  11. BMS-708163 and Nilotinib restore synaptic dysfunction in human embryonic stem cell-derived Alzheimer’s disease models

    PubMed Central

    Nishioka, Hisae; Tooi, Norie; Isobe, Takehisa; Nakatsuji, Norio; Aiba, Kazuhiro

    2016-01-01

    Alzheimer’s disease (AD) is the most common form of dementia. Cellular AD models derived from human pluripotent stem cells are promising tools in AD research. We recently developed human embryonic stem cell-derived AD models which overexpress mutant Presenilin1 genes, and which exhibit AD phenotypes, including synaptic dysfunction. In this study, we found that our AD models showed reduced levels of RAB3A and SV2B proteins in the pre-synapses, which is a possible cause of electrophysiological abnormalities. Through the screening of chemical compounds using our AD models, we have identified Aβ peptide inhibitors which decrease the concentration of Aβ in culture supernatant. Among these, BMS-708163 and Nilotinib were found to improve the expression levels of RAB3A and SV2B proteins and to recover the electrophysiological function in our AD models. These results suggest that the AD models we developed are promising materials for the discovery of AD drugs that target the expression of pre-synaptic proteins and synaptic function. PMID:27641902

  12. Roles of epithelial cell-derived periostin in TGF-beta activation, collagen production, and collagen gel elasticity in asthma.

    PubMed

    Sidhu, Sukhvinder S; Yuan, Shaopeng; Innes, Anh L; Kerr, Sheena; Woodruff, Prescott G; Hou, Lydia; Muller, Susan J; Fahy, John V

    2010-08-10

    Periostin is considered to be a matricellular protein with expression typically confined to cells of mesenchymal origin. Here, by using in situ hybridization, we show that periostin is specifically up-regulated in bronchial epithelial cells of asthmatic subjects, and in vitro, we show that periostin protein is basally secreted by airway epithelial cells in response to IL-13 to influence epithelial cell function, epithelial-mesenchymal interactions, and extracellular matrix organization. In primary human bronchial epithelial cells stimulated with periostin and epithelial cells overexpressing periostin, we reveal a function for periostin in stimulating the TGF-beta signaling pathway in a mechanism involving matrix metalloproteinases 2 and 9. Furthermore, conditioned medium from the epithelial cells overexpressing periostin caused TGF-beta-dependent secretion of type 1 collagen by airway fibroblasts. In addition, mixing recombinant periostin with type 1 collagen in solution caused a dramatic increase in the elastic modulus of the collagen gel, indicating that periostin alters collagen fibrillogenesis or cross-linking and leads to stiffening of the matrix. Epithelial cell-derived periostin in asthma has roles in TGF-beta activation and collagen gel elasticity in asthma.

  13. Multipotent stromal cells derived from common marmoset Callithrix jacchus within alginate 3D environment: Effect of cryopreservation procedures.

    PubMed

    Gryshkov, Oleksandr; Hofmann, Nicola; Lauterboeck, Lothar; Pogozhykh, Denys; Mueller, Thomas; Glasmacher, Birgit

    2015-08-01

    Multipotent stromal cells derived from the common marmoset monkey Callithrix jacchus (cjMSCs) possess high phylogenetic similarity to humans, with a great potential for preclinical studies in the field of regenerative medicine. Safe and effective long-term storage of cells is of great significance to clinical and research applications. Encapsulation of such cell types within alginate beads that can mimic an extra-cellular matrix and provide a supportive environment for cells during cryopreservation, has several advantages over freezing of cells in suspension. In this study we have analysed the effect of dimethyl sulfoxide (Me2SO, 2.5-10%, v/v) and pre-freeze loading time of alginate encapsulated cjMSCs in Me2SO (0-45 min) on the viability and metabolic activity of the cells after freezing using a slow cooling rate (-1°C/min). It was found that these parameters affect the stability and homogeneity of alginate beads after thawing. Moreover, the cjMSCs can be frozen in alginate beads with lower Me2SO concentration of 7.5% after 30 min of loading, while retaining high cryopreservation outcome. We demonstrated the maximum viability, membrane integrity and metabolic activity of the cells under optimized, less cytotoxic conditions. The results of this study are another step forward towards the application of cryopreservation for the long-term storage and subsequent applications of transplants in cell-based therapies.

  14. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

    PubMed

    Vicente-Manzanares, M; Rey, M; Jones, D R; Sancho, D; Mellado, M; Rodriguez-Frade, J M; del Pozo, M A; Yáñez-Mó, M; de Ana, A M; Martínez-A, C; Mérida, I; Sánchez-Madrid, F

    1999-10-01

    The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

  15. Stromal cell-derived factor-1 mediates changes of bone marrow stem cells during the bone repair process.

    PubMed

    Okada, Kiyotaka; Kawao, Naoyuki; Yano, Masato; Tamura, Yukinori; Kurashimo, Shinzi; Okumoto, Katsumi; Kojima, Kotarou; Kaji, Hiroshi

    2016-01-01

    Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.

  16. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

    PubMed Central

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells. PMID:26880974

  17. Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells

    PubMed Central

    Eggenschwiler, Reto; Wichmann, Christian; Buhmann, Raymund; Cantz, Tobias

    2017-01-01

    Kindlin-2 is a multidomain intracellular protein that can be recruited to β-integrin domains to activate signaling, initiate transcriptional programs, and bind to E-cadherin. To explore its involvement in cell fate decisions in mesenchymal cells, we studied the effects of Kindlin-2 modification (overexpression/knockdown) in induced pluripotent cell-derived mesenchymal stromal cells (iPSC-MSCs). Kindlin-2 overexpression resulted in increased proliferation and reduced apoptosis of iPSC-MSCs, as well as inhibition of their differentiation towards osteocytes, adipocytes, and chondrocytes. In contrast, siRNA-mediated Kindlin-2 knockdown induced increased apoptosis and increased differentiation response in iPSC-MSCs. The ability of iPSC-MSCs to adhere to VCAM-1/SDF-1α under shear stress and to migrate in a wound scratch assay was significantly increased after Kindlin-2 overexpression. In contrast, inhibition of mixed lymphocyte reaction (MLR) was generally independent of Kindlin-2 modulation in iPSC-MSCs, except for decreased production of interleukin-2 (IL-2) after Kindlin-2 overexpression in iPS-MSCs. Thus, Kindlin-2 upregulates survival, proliferation, stemness, and migration potential in iPSC-MSCs and may therefore be beneficial in optimizing performance of iPSC-MSC in therapies. PMID:28163724

  18. Embryonic stem cell-derived cardiomyocyte heterogeneity and the isolation of immature and committed cells for cardiac remodeling and regeneration.

    PubMed

    Boheler, Kenneth R; Joodi, Robert N; Qiao, Hui; Juhasz, Ondrej; Urick, Amanda L; Chuppa, Sandra L; Gundry, Rebekah L; Wersto, Robert P; Zhou, Rong

    2011-01-01

    Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs) are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction.

  19. Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

    PubMed

    Fafián-Labora, J; Lesende-Rodriguez, I; Fernández-Pernas, P; Sangiao-Alvarellos, S; Monserrat, L; Arntz, O J; Loo, F J Van de; Mateos, J; Arufe, M C

    2017-03-06

    Stem cells possess significant age-dependent differences in their immune-response profile. These differences were analysed by Next-Generation Sequencing of six age groups from bone marrow mesenchymal stem cells. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways. We focused our research on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. Extracellular vesicles extracted from each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, we conducted a functional experiment inhibiting its expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Together, these results indicate for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles.

  20. Species-dependent differences of embryonic stem cell-derived neural stem cells after Interferon gamma treatment

    PubMed Central

    Walter, Janine; Dihné, Marcel

    2012-01-01

    Pluripotent stem cell (pSC)-derived, neural stem cells (NSCs) are actually extensively explored in the field of neuroregeneration and to clarify disease mechanisms or model neurological diseases in vitro. Regarding the latter, proliferation and differentiation of pSC-derived NSCs are investigated under the influence of a variety of different substances among them key players of inflammation. However, results generated on a murine genetic background are not always representative for the human situation which increasingly leads to the application of human cell culture systems derived from human pSCs. We investigated here, if the recently described interferon gamma (IFNγ)-induced dysregulated neural phenotype characterized by simultaneous expression of glial and neuronal markers on murine NSCs (Walter et al., 2011, 2012) can also be found on a human genetic background. For this purpose, we performed experiments with human embryonic stem cell-derived NSCs. We could show that the IFNγ-induced dysregulated neural phenotype cannot be induced in human NSCs. This difference occurs, although typical genes like signal transducers and activators of transcription 1 (Stat 1) or interferon regulatory factor 9 (IRF-9) are similarly regulated by IFNγ in both, murine and human populations. These results illustrate that fundamental differences between murine and human neural populations exist in vitro, independent of anatomical system-related properties. PMID:23162429

  1. Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells

    SciTech Connect

    Zhang Xiaohong; Soda, Yasushi; Takahashi, Kenji; Bai, Yuansong; Mitsuru, Ayako; Igura, Koichi; Satoh, Hitoshi; Yamaguchi, Satoru; Tani, Kenzaburo; Tojo, Arinobu; Takahashi, Tsuneo A. . E-mail: takahasi@ims.u-tokyo.ac.jp

    2006-12-29

    We reported previously that mesenchymal progenitor cells derived from chorionic villi of the human placenta could differentiate into osteoblasts, adipocytes, and chondrocytes under proper induction conditions and that these cells should be useful for allogeneic regenerative medicine, including cartilage tissue engineering. However, similar to human mesenchymal stem cells (hMSCs), though these placental cells can be isolated easily, they are difficult to study in detail because of their limited life span in vitro. To overcome this problem, we attempted to prolong the life span of human placenta-derived mesenchymal cells (hPDMCs) by modifying hTERT and Bmi-1, and investigated whether these modified hPDMCs retained their differentiation capability and multipotency. Our results indicated that the combination of hTERT and Bmi-1 was highly efficient in prolonging the life span of hPDMCs with differentiation capability to osteogenic, adipogenic, and chondrogenic cells in vitro. Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT + Bmi-1. Interestingly, hPDMC/Bmi-1 showed extended proliferation after long-term growth arrest and telomerase was activated in the immortal hPDMC/Bmi-1 cells. However, the differentiation potential was lost in these cells. This study reports a method to extend the life span of hPDMCs with hTERT and Bmi-1 that should become a useful tool for the study of mesenchymal stem cells.

  2. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells.

    PubMed

    Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

    2017-01-04

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great promise for these RPE cell therapies. The aim of the present study was to assess whether a flexible, elastic and biodegradable poly(trimethylene carbonate) (PTMC) film promotes the formation of functional hESC-RPE and performs better than often used biodegradable poly(d,l-lactide) (PDLLA) film. Human ESC-RPE maturation and functionality on PTMC films was assessed by cell proliferation assays, RPE-specific gene and protein expression, phagocytic activity and growth factor secretion. It is demonstrated that the mechanical properties of PTMC films have close resemblance to those of the native Bruch's membrane and support the formation hESC-RPE monolayer in serum-free culture conditions with high degree of functionality. In contrast, use of PDLLA films did not lead to the formation of confluent monolayers of hESC-RPE cells and had unsuitable mechanical properties for retinal application. In conclusion, the present study indicates that flexible and elastic biodegradable PTMC films show potential for retinal tissue engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.

  3. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    PubMed

    Hu, Chenxia; Li, Lanjuan

    2015-08-01

    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  4. Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles

    PubMed Central

    Fafián-Labora, J.; Lesende-Rodriguez, I.; Fernández-Pernas, P.; Sangiao-Alvarellos, S.; Monserrat, L.; Arntz, O. J.; Loo, F. J. Van de; Mateos, J.; Arufe, M. C.

    2017-01-01

    Stem cells possess significant age-dependent differences in their immune-response profile. These differences were analysed by Next-Generation Sequencing of six age groups from bone marrow mesenchymal stem cells. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways. We focused our research on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. Extracellular vesicles extracted from each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, we conducted a functional experiment inhibiting its expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Together, these results indicate for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles. PMID:28262816

  5. Embryonic mouse STO cell-derived xenografts express hepatocytic functions in the livers of nonimmunosuppressed adult rats.

    PubMed

    Zhang, Mingjun; Joseph, Brigid; Gupta, Sanjeev; Guest, I; Xu, Meng; Sell, Stewart; Son, Kyung-Hwa; Koch, Katherine S; Leffert, Hyam L

    2005-02-01

    Cells derived from embryonic mouse STO cell lines differentiate into hepatocytes when transplanted into the livers of nonimmunosuppressed dipeptidylpeptidase IV (DPPIV)-negative F344 rats. Within 1 day after intrasplenic injection, donor cells moved rapidly into the liver and were found in intravascular and perivascular sites; by 1 month, they were intrasinusoidal and also integrated into hepatic plates with approximately 2% efficiency and formed conjoint bile canaliculi. Neither donor cell proliferation nor host inflammatory responses were observed during this time. Detection of intrahepatic mouse COX1 mitochondrial DNA and mouse albumin mRNA in recipient rats indicated survival and differentiation of donor cells for at least 3 months. Mouse COX1 targets were also detected intrahepatically 4-9 weeks after STO cell injection into nonimmunosuppressed wild-type rats. In contrast to STO-transplanted rats, mouse DNA or RNA was not detectable in untreated or mock-transplanted rats or in rats injected with donor cell DNA. In cultured STO donor cells, DPPIV and glucose-6-phosphatase activities were observed in small clusters; in contrast, mouse major histocompatibility complex class I H-2Kq, H-2Dq, and H-2Lq and class II I-Aq markers were undetectable in vitro before or after interferon gamma treatment. Together with H-2K allele typing, which confirmed the Swiss mouse origin of the donor cells, these observations indicate that mouse-derived STO cell lines can differentiate along hepatocytic lineage and engraft into rat liver across major histocompatibility barriers.

  6. Human natural killer cells promote cross-presentation of tumor cell-derived antigens by dendritic cells.

    PubMed

    Deauvieau, Florence; Ollion, Vincent; Doffin, Anne-Claire; Achard, Carole; Fonteneau, Jean-François; Verronese, Estelle; Durand, Isabelle; Ghittoni, Raffaella; Marvel, Jacqueline; Dezutter-Dambuyant, Colette; Walzer, Thierry; Vie, Henri; Perrot, Ivan; Goutagny, Nadège; Caux, Christophe; Valladeau-Guilemond, Jenny

    2015-03-01

    Dendritic cells (DCs) cross-present antigen (Ag) to initiate T-cell immunity against most infections and tumors. Natural killer (NK) cells are innate cytolytic lymphocytes that have emerged as key modulators of multiple DC functions. Here, we show that human NK cells promote cross-presentation of tumor cell-derived Ag by DC leading to Ag-specific CD8(+) T-cell activation. Surprisingly, cytotoxic function of NK cells was not required. Instead, we highlight a critical and nonredundant role for IFN-γ and TNF-α production by NK cells to enhance cross-presentation by DC using two different Ag models. Importantly, we observed that NK cells promote cell-associated Ag cross-presentation selectively by monocytes-derived DC (Mo-DC) and CD34-derived CD11b(neg) CD141(high) DC subsets but not by myeloid CD11b(+) DC. Moreover, we demonstrate that triggering NK cell activation by monoclonal antibodies (mAbs)-coated tumor cells leads to efficient DC cross-presentation, supporting the concept that NK cells can contribute to therapeutic mAbs efficiency by inducing downstream adaptive immunity. Taken together, our findings point toward a novel role of human NK cells bridging innate and adaptive immunity through selective induction of cell-associated Ag cross-presentation by CD141(high) DC, a process that could be exploited to better harness Ag-specific cellular immunity in immunotherapy.

  7. A basis for comparison: sensitive authentication of stem cell derived RPE using physiological responses of intact RPE monolayers

    PubMed Central

    Miyagishima, Kiyoharu J.; Wan, Qin; Miller, Sheldon S.; Bharti, Kapil

    2017-01-01

    The retinal pigment epithelium (RPE) is a monolayer of highly specialized cells that help maintain the chemical composition of its surrounding subretinal and choroidal extracellular spaces. Retinal cells (photoreceptors in particular), RPE, and choroidal endothelial cells together help ensure a homeostatically stable metabolic environment with exquisitely sensitive functional responses to light. Aging and disease of the RPE impairs its supportive functions contributing to the progressive loss of photoreceptors and vision. The prevalence of RPE associated retinal degenerations has prompted researchers to develop new therapies aimed at replacing the affected RPE with induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) derived RPE. Despite recent attempts to characterize stem cell derived RPE and to truly authenticate RPE for clinical applications, there remains a significant unmet need to explore the heterogeneity resulting from donor to donor variation as well as the variations inherent in the current processes of cell manufacture. Additionally, it remains unknown whether the starting cell type influences the resulting RPE phenotype following reprogramming and differentiation. To address these questions, we performed a comprehensive evaluation (genomic, structural, and functional) of 15 iPSC derived RPE originating from different donors and tissues and compiled a reference data set for the authentication of iPSC-derived RPE and RPE derived from other stem cell sources. PMID:28286868

  8. Chemotherapeutic Vulnerability of Triple-negative Breast Cancer Cell-derived Tumors to Pretreatment with Vernonia amygdalina Aqueous Extracts

    PubMed Central

    Howard, Carolyn B.; Mcdowell, Roderick; Feleke, Kidus; Deer, Evangeline; Stamps, Symone; Thames, Easter; Singh, Vikash; Pervin, Shehla

    2016-01-01

    Background Unresponsive to most clinical therapies, triple-negative breast cancer (TNBC) is the dominant biological cause of population-based racioethnic disparities in breast cancer mortality in the United States. We report the chemotherapeutic vulnerability of TNBC cells and stem cell-derived tumors to Vernonia amygdalina aqueous leaf extracts (VA extracts). VA extracts arrest cell proliferation and induce apoptosis in vitro and inhibit growth of implanted tumors and show chemo-preventive efficacy in vivo. Materials and Methods HRAS cells and MDA-MB-468 cells were subcutaneously implanted into nude mice with or without pretreatment with VA extracts before chemotherapeutic treatment with VA extracts and/or paclitaxel to evaluate their ability to inhibit tumor growth. Results The most significant reduction in tumor volume was observed in the MDA-MB-468 cell-induced tumors following VA extract pre-treatment compared to those from HRAS cell implantation. Conclusion VA extracts induce apoptosis, exhibit additive effects, inhibit tumor growth and display chemo-preventive actions against TNBCs. PMID:27466496

  9. B cell-derived transforming growth factor-β1 expression limits the induction phase of autoimmune neuroinflammation

    PubMed Central

    Bjarnadóttir, Kristbjörg; Benkhoucha, Mahdia; Merkler, Doron; Weber, Martin S.; Payne, Natalie L.; Bernard, Claude C. A.; Molnarfi, Nicolas; Lalive, Patrice H.

    2016-01-01

    Studies in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), have shown that regulatory B cells modulate the course of the disease via the production of suppressive cytokines. While data indicate a role for transforming growth factor (TGF)-β1 expression in regulatory B cell functions, this mechanism has not yet been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-β1 expression in B cells (B–TGF-β1−/−) were tested in EAE induced by recombinant mouse myelin oligodendrocyte glycoprotein (rmMOG). In this model, B–TGF-β1−/− mice showed an earlier onset of neurologic impairment compared to their littermate controls. Exacerbated EAE susceptibility in B–TGF-β1−/− mice was associated with augmented CNS T helper (Th)1/17 responses. Moreover, selective B cell TGF-β1–deficiency increased the frequencies and activation of myeloid dendritic cells, potent professional antigen-presenting cells (APCs), suggesting that B cell-derived TGF-β1 can constrain Th1/17 responses through inhibition of APC activity. Collectively our data suggest that B cells can down-regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-β1, findings that may be relevant to B cell-targeted therapies. PMID:27708418

  10. A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

    PubMed

    Lewis, Kimberley J; Silvester, Nicole C; Barberini-Jammaers, Steven; Mason, Sammy A; Marsh, Sarah A; Lipka, Magdalena; George, Christopher H

    2015-03-01

    The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.

  11. Retinal pigment epithelium cell-derived exosomes: Possible relevance to CNV in wet-age related macular degeneration.

    PubMed

    Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

    2016-12-01

    Exosomes are small vesicles that are released by almost every cell type and play a crucial role in many physiological and pathological processes associated with different diseases. Specifically, they promote angiogenesis in the pathogenesis of some diseases. According to previous research, the proteins of exosomes taken from the aqueous humor (AH) of patients with wet-age related macular degeneration (AMD) may function as a new diagnostic biomarker of AMD, suggesting that exosomes may play an important role in the occurrence and development of choroidal neovascularization (CNV). Moreover, additional research has revealed that the levels of some protein makers of exosomes are up-regulated in aged retinal pigment epithelium (RPE) and that drusen and oxidative stress may promote the secretion of exosomes derived from RPE cells. Consequently, we hypothesize that RPE cell-derived exosomes may be relevant to CNV in wet AMD. If this hypothesis is proven correct, future studies based on this link may also help to elucidate the molecular mechanisms of wet AMD and to find new therapeutic targets for the treatment of AMD.

  12. Time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer.

    PubMed

    Nakles, Rebecca E; Millman, Sarah L; Cabrera, M Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S; Schroeder, Timm; Furth, Priscilla A

    2013-02-08

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

  13. Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

    PubMed Central

    Sharma, Ruchi; Greenhough, Sebastian; Medine, Claire N.; Hay, David C.

    2010-01-01

    The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays. PMID:20169088

  14. 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.

  15. Compound K attenuates stromal cell-derived growth factor 1 (SDF-1)-induced migration of C6 glioma cells

    PubMed Central

    Kim, Hyuck; Roh, Hyo Sun; Kim, Jai Eun; Park, Sun Dong; Park, Won Hwan

    2016-01-01

    BACKGROUND/OBJECTIVES Stromal cell-derived growth factor 1 (SDF-1), also known as chemokine ligand 12, and chemokine receptor type 4 are involved in cancer cell migration. Compound K (CK), a metabolite of protopanaxadiol-type ginsenoside by gut microbiota, is reported to have therapeutic potential in cancer therapy. However, the inhibitory effect of CK on SDF-1 pathway-induced migration of glioma has not yet been established. MATERIALS/METHODS Cytotoxicity of CK in C6 glioma cells was determined using an EZ-Cytox cell viability assay kit. Cell migration was tested using the wound healing and Boyden chamber assay. Phosphorylation levels of protein kinase C (PKC)α and extracellular signal-regulated kinase (ERK) were measured by western blot assay, and matrix metallopeptidases (MMP) were measured by gelatin-zymography analysis. RESULTS CK significantly reduced the phosphorylation of PKCα and ERK1/2, expression of MMP9 and MMP2, and inhibited the migration of C6 glioma cells under SDF-1-stimulated conditions. CONCLUSIONS CK is a cell migration inhibitor that inhibits C6 glioma cell migration by regulating its downstream signaling molecules including PKCα, ERK1/2, and MMPs. PMID:27247721

  16. Endothelial cell-derived CD95 ligand serves as a chemokine in induction of neutrophil slow rolling and adhesion

    PubMed Central

    Gao, Liang; Gülcüler, Gülce Sila; Golbach, Lieke; Block, Helena; Zarbock, Alexander; Martin-Villalba, Ana

    2016-01-01

    Integrin activation is crucial for the regulation of leukocyte rolling, adhesion and trans-vessel migration during inflammation and occurs by engagement of myeloid cells through factors presented by inflamed vessels. However, endothelial-dependent mechanisms of myeloid cell recruitment are not fully understood. Here we show using an autoperfused flow chamber assay of whole blood neutrophils and intravital microscopy of the inflamed cremaster muscle that CD95 mediates leukocyte slow rolling, adhesion and transmigration upon binding of CD95-ligand (CD95L) that is presented by endothelial cells. In myeloid cells, CD95 triggers activation of Syk-Btk/PLCγ2/Rap1 signaling that ultimately leads to integrin activation. Excitingly, CD95-deficient myeloid cells exhibit impaired bacterial clearance in an animal model of sepsis induced by cecal ligation and puncture (CLP). Our data identify the cellular and molecular mechanisms underlying the chemoattractant effect of endothelial cell-derived CD95L in induction of neutrophil recruitment and support the use of therapeutic inhibition of CD95’s activity in inflammatory diseases. DOI: http://dx.doi.org/10.7554/eLife.18542.001 PMID:27763263

  17. Tolerance induction and reversal of diabetes in mice transplanted with human embryonic stem cell-derived pancreatic endoderm.

    PubMed

    Szot, Gregory L; Yadav, Mahesh; Lang, Jiena; Kroon, Evert; Kerr, Justin; Kadoya, Kuniko; Brandon, Eugene P; Baetge, Emmanuel E; Bour-Jordan, Hélène; Bluestone, Jeffrey A

    2015-02-05

    Type 1 diabetes (T1D) is an autoimmune disease caused by T cell-mediated destruction of insulin-producing β cells in the islets of Langerhans. In most cases, reversal of disease would require strategies combining islet cell replacement with immunotherapy that are currently available only for the most severely affected patients. Here, we demonstrate that immunotherapies that target T cell costimulatory pathways block the rejection of xenogeneic human embryonic-stem-cell-derived pancreatic endoderm (hESC-PE) in mice. The therapy allowed for long-term development of hESC-PE into islet-like structures capable of producing human insulin and maintaining normoglycemia. Moreover, short-term costimulation blockade led to robust immune tolerance that could be transferred independently of regulatory T cells. Importantly, costimulation blockade prevented the rejection of allogeneic hESC-PE by human PBMCs in a humanized model in vivo. These results support the clinical development of hESC-derived therapy, combined with tolerogenic treatments, as a sustainable alternative strategy for patients with T1D.

  18. Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

    PubMed

    Yeung, A T Y; Hale, C; Xia, J; Tate, P H; Goulding, D; Keane, J A; Mukhopadhyay, S; Forrester, L; Billker, O; Skarnes, W C; Hancock, R E W; Dougan, G

    2015-03-10

    The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

  19. Magnetically Responsive Bone Marrow Mesenchymal Stem Cell-Derived Smooth Muscle Cells Maintain Their Benefits to Augmenting Elastic Matrix Neoassembly.

    PubMed

    Swaminathan, Ganesh; Sivaraman, Balakrishnan; Moore, Lee; Zborowski, Maciej; Ramamurthi, Anand

    2016-04-01

    Abdominal aortic aneurysms (AAA) represent abnormal aortal expansions that result from chronic proteolytic breakdown of elastin and collagen fibers by matrix metalloproteases. Poor elastogenesis by adult vascular smooth muscle cells (SMCs) limits regenerative repair of elastic fibers, critical for AAA growth arrest. Toward overcoming these limitations, we recently demonstrated significant elastogenesis by bone marrow mesenchymal stem cell-derived SMCs (BM-SMCs) and their proelastogenesis and antiproteolytic effects on rat aneurysmal SMCs (EaRASMCs). We currently investigate the effects of super paramagnetic iron oxide nanoparticle (SPION) labeling of BM-SMCs, necessary to magnetically guide them to the AAA wall, on their functional benefits. Our results indicate that SPION-labeling is noncytotoxic and does not adversely impact the phenotype and elastogenesis by BM-SMCs. In addition, SPION-BM-SMCs showed no changes in the ability of the BM-SMCs to stimulate elastin regeneration and attenuate proteolytic activity by EaRASMCs. Together, our results are promising toward the utility of SPIONs for magnetic targeting of BM-SMCs for in situ AAA regenerative repair.

  20. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device.

    PubMed

    van der Meer, Andries D; Orlova, Valeria V; ten Dijke, Peter; van den Berg, Albert; Mummery, Christine L

    2013-09-21

    Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here, we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells, human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h, the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell-cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels, inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity, highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary, we have

  1. Optimization of the tissue source, malignancy, and initial substrate of tumor cell-derived matrices to increase cancer cell chemoresistance against 5-fluorouracil.

    PubMed

    Hoshiba, Takashi; Tanaka, Masaru

    2015-02-13

    The low chemoresistance of in vitro cancer cells inhibits the development of new anti-cancer drugs. Thus, development of a new in vitro culture system is required to increase the chemoresistance of in vitro cancer cells. Tumor cell-derived matrices have been reported to increase the chemoresistance of in vitro cancer cells. However, it remains unclear how tissue sources and the malignancy of cells used for the preparation of matrices affect the chemoresistance of tumor cell-derived matrices. Moreover, it remains unclear how the initial substrates used for the preparation of matrices affect the chemoresistance. In this study, we compared the effects of tissue sources and the malignancy of tumor cells, as well as the effect of the initial substrates on chemoresistance against 5-fluorouracil (5-FU). The chemoresistance of breast and colon cancer cells against 5-FU increased on matrices prepared with cells derived from the corresponding original tissues with higher malignancy. Moreover, the chemoresistance against 5-FU was altered on matrices prepared using different initial substrates that exhibited different characteristics of protein adsorption. Taken together, these results indicated that the appropriate selection of tissue sources, malignancy of tumor cells, and initial substrates used for matrix preparation is important for the preparation of tumor cell-derived matrices for chemoresistance assays.

  2. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells

    SciTech Connect

    Yamada, Yoji; Sakurada, Kazuhiro; Takeda, Yukiji; Gojo, Satoshi; Umezawa, Akihiro . E-mail: umezawa@1985.jukuin.keio.ac.jp

    2007-02-15

    Bone marrow-derived stromal cells can give rise to cardiomyocytes as well as adipocytes, osteocytes, and chondrocytes in vitro. The existence of mesenchymal stem cells has been proposed, but it remains unclear if a single-cell-derived stem cell stochastically commits toward a cardiac lineage. By single-cell marking, we performed a follow-up study of individual cells during the differentiation of 9-15c mesenchymal stromal cells derived from bone marrow cells. Three types of cells, i.e., cardiac myoblasts, cardiac progenitors and multipotent stem cells were differentiated from a single cell, implying that cardiomyocytes are generated stochastically from a single-cell-derived stem cell. We also demonstrated that overexpression of Csx/Nkx2.5 and GATA4, precardiac mesodermal transcription factors, enhanced cardiomyogenic differentiation of 9-15c cells, and the frequency of cardiomyogenic differentiation was increased by co-culturing with fetal cardiomyocytes. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 behaved like cardiac transient amplifying cells, and still retained their plasticity in vivo.

  3. In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

    PubMed

    Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

    2012-05-01

    In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

  4. Body Image

    MedlinePlus

    ... spider veins Body dysmorphic disorder (BDD) Eating disorders Anorexia nervosa Binge eating disorder Bulimia nervosa Over-exercising ... conditions? Visit our Mental health section. Fact sheets Anorexia nervosa Binge eating disorder Bulimia nervosa Cosmetics and ...

  5. Body Piercing

    MedlinePlus

    ... must have a consistent mirror finish. Implant grade stainless steel is least likely to produce a foreign body ... All of these cost more than implant grade stainless steel. Gold jewelry should be at least 14 karat ...

  6. Bog bodies.

    PubMed

    Lynnerup, Niels

    2015-06-01

    In northern Europe during the Iron Age, many corpses were deposited in bogs. The cold, wet and anaerobic environment leads in many cases to the preservation of soft tissues, so that the bodies, when found and excavated several thousand years later, are remarkably intact. Since the 19th century the bog bodies have been studied using medical and natural scientific methods, and recently many bog bodies have been re-examined using especially modern, medical imaging techniques. Because of the preservation of soft tissue, especially the skin, it has been possible to determine lesions and trauma. Conversely, the preservation of bones is less good, as the mineral component has been leached out by the acidic bog. Together with water-logging of collagenous tissue, this means that if the bog body is simply left to dry out when found, as was the case pre-19th century, the bones may literally warp and shrink, leading to potential pitfalls in paleopathological diagnostics. Bog bodies have in several instances been crucial in determining the last meal, as gut contents may be preserved, and thus augment our knowledge on pre-historic diet by adding to, for example, stable isotope analyses. This article presents an overview of our knowledge about the taphomic processes as well as the methods used in bog body research.

  7. Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture.

    PubMed

    Zaniboni, Andrea; Bernardini, Chiara; Alessandri, Marco; Mangano, Chiara; Zannoni, Augusta; Bianchi, Francesca; Sarli, Giuseppe; Calzà, Laura; Bacci, Maria Laura; Forni, Monica

    2014-02-15

    Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aortic-derived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wall.

  8. Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Burridge, Paul W; Metzler, Scott A; Nakayama, Karina H; Abilez, Oscar J; Simmons, Chelsey S; Bruce, Marc A; Matsuura, Yuka; Kim, Paul; Wu, Joseph C; Butte, Manish; Huang, Ngan F; Yang, Phillip C

    2014-01-01

    Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca2+ ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases. PMID:25628783

  9. Alterations in T cell-derived colony-stimulating factors associated with GVH-induced immune deficiency

    SciTech Connect

    Hakim, F.T.; Pluznik, D.H.; Shearer, G.M. )

    1990-04-01

    Injection of parental C57BL/10 spleen cells into unirradiated immune-competent (B10 x B10.BR)F1 hosts has been demonstrated to produce a graft-vs.-host-induced immune deficiency in T cell-mediated functions, including mitogen or alloantigen stimulated proliferation or cytotoxic T cell generation. The production of T cell-derived lymphokines affecting hematopoiesis was also altered during GVH. During the first two weeks of GVH, IL-3 and particularly GM-CSF were produced spontaneously; in subsequent weeks, the spontaneous production dropped to normal or subnormal levels. CSF content in concanavalin A-stimulated splenic supernatants was reduced at weeks 1-2, and declined to less than 5% of normal levels by 3-4 weeks of GVH. This decline in CSF content was correlated with a decrease in immune function as assessed by concanavalin A-stimulated IL-2 production and by generation of cytotoxic T lymphocytes. Concurrent with the recovery of immune function during GVH weeks 8-15, mitogen-stimulated production of CSF returned to normal levels. In addition to the decrease in CSF production identified in acute suppressive GVH, CSF content in concanavalin A-stimulated splenic supernatants was also decreased in chronic stimulatory GVH, generated in the strain combination (B6 x B6bm1)F1----(B6bm1 x B6bm12)F1. This decrease in CSF production correlated with a decrease in self-restricted T helper cell function. Finally, a decrease in both immune function and CSF production capacity was observed in the acute GVH following allogeneic (minor histocompatibility loci) bone marrow transplantation into irradiated hosts.

  10. Mammalian Cell-Derived Respiratory Syncytial Virus-Like Particles Protect the Lower as well as the Upper Respiratory Tract.

    PubMed

    Walpita, Pramila; Johns, Lisa M; Tandon, Ravi; Moore, Martin L

    2015-01-01

    Globally, Respiratory Syncytial Virus (RSV) is a leading cause of bronchiolitis and pneumonia in children less than one year of age and in USA alone, between 85,000 and 144,000 infants are hospitalized every year. To date, there is no licensed vaccine. We have evaluated vaccine potential of mammalian cell-derived native RSV virus-like particles (RSV VLPs) composed of the two surface glycoproteins G and F, and the matrix protein M. Results of in vitro testing showed that the VLPs were functionally assembled and immunoreactive, and that the recombinantly expressed F protein was cleaved intracellularly similarly to the virus-synthesized F protein to produce the F1 and F2 subunits; the presence of the F1 fragment is critical for vaccine development since all the neutralizing epitopes present in the F protein are embedded in this fragment. Additional in vitro testing in human macrophage cell line THP-1 showed that both virus and the VLPs were sensed by TLR-4 and induced a Th1-biased cytokine response. Cotton rats vaccinated with RSV VLPs adjuvanted with alum and monophosphoryl lipid A induced potent neutralizing antibody response, and conferred protection in the lower as well as the upper respiratory tract based on substantial virus clearance from these sites. To the best of our knowledge, this is the first VLP/virosome vaccine study reporting protection of the lower as well as the upper respiratory tract: Prevention from replication in the nose is an important consideration if the target population is infants < 6 months of age. This is because continued virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers. In conclusion, these results taken together suggest that our VLPs show promise to be a safe and effective vaccine for RSV.

  11. Hyperthermia Influences the Effects of Sodium Channel Blocking Drugs in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    El-Battrawy, Ibrahim; Lang, Siegfried; Zhao, Zhihan; Akin, Ibrahim; Yücel, Gökhan; Meister, Sophie; Patocskai, Bence; Behnes, Michael; Rudic, Boris; Tülümen, Erol; Liebe, Volker; Tiburcy, Malte; Dworacek, Jennifer; Zimmermann, Wolfram-Hubertus; Utikal, Jochen; Wieland, Thomas; Borggrefe, Martin; Zhou, Xiao-Bo

    2016-01-01

    Introduction Fever can increase the susceptibility to supraventricular and ventricular arrhythmias, in which sodium channel dysfunction has been implicated. Whether fever influences the efficacy of sodium channel blocking drugs is unknown. The current study was designed to investigate the temperature dependent effects of distinct sodium channel blocking drugs on the sodium currents in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and Results hiPSC-CMs were generated from human skin fibroblasts of a healthy donor. The peak and late sodium currents (INa), steady-state activation, inactivation and recovery from inactivation of INa in hiPSC-CMs were analyzed using the whole-cell patch clamp technique. The effects of different concentrations of the antiarrhythmic drugs flecainide, lidocaine, ajmaline and the antianginal drug ranolazine on INa were tested at 36°C and 40°C. Increasing the temperature of the bath solution from 36°C to 40°C enhanced the inhibition of peak INa but reduced the inhibition of late INa by flecainide and lidocaine. By contrast, increasing the temperature reduced the effect of ajmaline and ranolazine on the peak INa but not late INa. None of the tested drugs showed temperature-dependent effects on the steady-state activation and inactivation as well as on the recovery from inactivation of INa in hiPSC-CMs. Conclusions Temperature variation from the physiological to the febrile range apparently influences the effects of sodium channel blockers on the sodium currents. This may influence their antiarrhythmic efficacy in patients suffering from fever. PMID:27829006

  12. Mathematically-Engineered Stromal Cell-Derived Factor 1alpha Stem Cell Cytokine Analogue Enhances Mechanical Properties of Infarcted Myocardium

    PubMed Central

    Jr, John W. MacArthur; Trubelja, Alen; Shudo, Yasuhiro; Hsiao, Philip; Fairman, Alex; Yang, Elaine; Hiesinger, William; Atluri, Pavan; Woo, Y Joseph

    2014-01-01

    Background The biomechanical response to a myocardial infarction consists of ventricular remodeling that leads to dilation, loss of contractile function, abnormal stress patterns and ultimately heart failure. We hypothesized that intramyocardial injection of our previously designed pro-angiogenic chemokine, an engineered stromal cell derived factor 1alpha analogue(ESA), improves mechanical properties of the heart post-infarction. Methods Male rats (n=54) underwent either sham surgery (n=17) with no coronary artery ligation or ligation of the LAD (n=37). Rats in the MI group were then randomized to receive either saline (0.1cc, n=18) or ESA (6μg/kg, n=19) injected into the myocardium at 4 predetermined spots around the borderzone. Echocardiograms were performed preoperatively and before the terminal surgery. After 4 weeks the hearts were explanted and longitudinally sectioned. Uniaxial tensile testing was completed using an Instron 5543 Microtester. Optical strain was evaluated utilizing custom image acquisition software, Digi-Velpo, and analyzed in MATLAB. Results Compared to the saline control group at 4 weeks, the ESA injected hearts had higher ejection fractions (71.8% ± 9.0 vs. 55.3% ± 12.6, p= 0.0004) smaller end-diastolic left ventricular internal dimensions (0.686cm ± 0.110 vs. 0.763cm ± 0.160, p= 0.04), higher cardiac output (36ml/min ± 11.6 vs. 26.9ml/min ± 7.3, p= 0.05) and the tensile modulus was lower(251kPa ± 56 vs. 301kPa ± 81, p= 0.04). The tensile modulus for the sham group was 195kPa ± 56, indicating ESA injection results in a less stiff ventricle. Conclusions Direct injection of ESA alters the biomechanical response to MI, improving the mechanical properties in the post-infarct heart. PMID:23244259

  13. Cardiomyocyte MEA data analysis (CardioMDA)--a novel field potential data analysis software for pluripotent stem cell derived cardiomyocytes.

    PubMed

    Pradhapan, Paruthi; Kuusela, Jukka; Viik, Jari; Aalto-Setälä, Katriina; Hyttinen, Jari

    2013-01-01

    Cardiac safety pharmacology requires in-vitro testing of all drug candidates before clinical trials in order to ensure they are screened for cardio-toxic effects which may result in severe arrhythmias. Micro-electrode arrays (MEA) serve as a complement to current in-vitro methods for drug safety testing. However, MEA recordings produce huge volumes of data and manual analysis forms a bottleneck for high-throughput screening. To overcome this issue, we have developed an offline, semi-automatic data analysis software, 'Cardiomyocyte MEA Data Analysis (CardioMDA)', equipped with correlation analysis and ensemble averaging techniques to improve the accuracy, reliability and throughput rate of analysing human pluripotent stem cell derived cardiomyocyte (CM) field potentials. With the program, true field potential and arrhythmogenic complexes can be distinguished from one another. The averaged field potential complexes, analysed using our software to determine the field potential duration, were compared with the analogous values obtained from manual analysis. The reliability of the correlation analysis algorithm, evaluated using various arrhythmogenic and morphology changing signals, revealed a mean sensitivity and specificity of 99.27% and 94.49% respectively, in determining true field potential complexes. The field potential duration of the averaged waveforms corresponded well to the manually analysed data, thus demonstrating the reliability of the software. The software has also the capability to create overlay plots for signals recorded under different drug concentrations in order to visualize and compare the magnitude of response on different ion channels as a result of drug treatment. Our novel field potential analysis platform will facilitate the analysis of CM MEA signals in semi-automated way and provide a reliable means of efficient and swift analysis for cardiomyocyte drug or disease model studies.

  14. Proarrhythmia Risk Assessment in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Using the Maestro MEA Platform.

    PubMed

    Qu, Yusheng; Vargas, Hugo M

    2015-09-01

    Evaluation of stem cell-derived cardiomyocytes (SC-CM) using multi-electrode array (MEA) has attracted attention as a novel model to detect drug-induced arrhythmia. An experiment was conducted to determine if MEA recording from human induced pluripotent SC-CM (hiPSC-CM) could assess proarrhythmic risk. Ten hERG blockers, 4 Na(+) blockers, and 1 IKs blocker were evaluated blindly. Eight drugs are associated with Torsades de Pointes (TdP) and 4 are not. Multiple parameters, including field potential duration (FPD), Na(+) slope, Na(+) amplitude, beat rate (BR), and early after-depolarization (EAD) were recorded. Minimum effective concentrations (MEC) that elicited a significant change were calculated. Our results determined that FPD and EAD were unable to distinguish torsadogenic from benign compounds, Na(+) slope and amplitude could not differentiate Na(+) channel blockade from hERG blockade, BR had an inconsistent response to pharmacological treatment, and that hiPSC-CM were, in general, insensitive to IKs inhibition. A ratio was calculated that relates MEC for evoking FPD prolongation, or triggering EAD, to the human therapeutic unbound Cmax (MEC/Cmax). The key finding was that the ratio was sensitive, but specificity was low. Consistently, the ratio had high positive predictive value and low negative predictive value. In conclusion, MEA recordings of hiPSC-CM were sensitive for FPD and EAD detection, but unable to distinguish agents with low- and high-risk for TdPs. Although some published reports suggested great potential for MEA recordings in hSC-CM to assess preclinical cardiac toxicity, the current evaluation implies that this model would have a high false-positive rate in regard to proarrhythmic risk.

  15. Accelerated intoxication of GABAergic synapses by botulinum neurotoxin A disinhibits stem cell-derived neuron networks prior to network silencing

    PubMed Central

    Beske, Phillip H.; Scheeler, Stephen M.; Adler, Michael; McNutt, Patrick M.

    2015-01-01

    Botulinum neurotoxins (BoNTs) are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses, preventing neurotransmitter release. Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo. Consequently, the dynamic effects of intoxication on synaptic behaviors are not well-understood. We have reported that mouse embryonic stem cell-derived neurons (ESNs) are highly sensitive to BoNT based on molecular readouts of intoxication. Here we study the time-dependent changes in synapse- and network-level behaviors following addition of BoNT/A to spontaneously active networks of glutamatergic and GABAergic ESNs. Whole-cell patch-clamp recordings indicated that BoNT/A rapidly blocked synaptic neurotransmission, confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism. Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses, which was consistent with the selective accumulation of cleaved SNAP-25 at GAD1+ pre-synaptic terminals at early timepoints. Different latencies of intoxication resulted in complex network responses to BoNT/A addition, involving rapid disinhibition of stochastic firing followed by network silencing. Synaptic activity was found to be highly sensitive to SNAP-25 cleavage, reflecting the functional consequences of the localized cleavage of the small subpopulation of SNAP-25 that is engaged in neurotransmitter release in the nerve terminal. Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT. PMID:25954159

  16. Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

    PubMed Central

    Katsman, Diana; Stackpole, Emma J.; Domin, Daniel R.; Farber, Debora B.

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation. PMID:23226281

  17. Comparison of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes with Human Mesenchymal Stem Cells following Acute Myocardial Infarction

    PubMed Central

    Hassan, Fatemat; Kuppusamy, M. Lakshmi; Kuppusamy, Periannan; Angelos, Mark G.; Khan, Mahmood

    2014-01-01

    Introduction Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently been shown to express key cardiac proteins and improve in vivo cardiac function when administered following myocardial infarction. However, the efficacy of hiPSC-derived cell therapies, in direct comparison to current, well-established stem cell-based therapies, is yet to be elucidated. The goal of the current study was to compare the therapeutic efficacy of human mesenchymal stem cells (hMSCs) with hiPSC-CMs in mitigating myocardial infarction (MI). Methods Male athymic nude hyrats were subjected to permanent ligation of the left-anterior-descending (LAD) coronary artery to induce acute MI. Four experimental groups were studied: 1) control (non-MI), 2) MI, 3) hMSCs (MI+MSC), and 4) hiPSC-CMs (MI+hiPSC-derived cardiomyocytes). The hiPSC-CMs and hMSCs were labeled with superparamagnetic iron oxide (SPIO) in vitro to track the transplanted cells in the ischemic heart by high-field cardiac MRI. These cells were injected into the ischemic heart 30-min after LAD ligation. Four-weeks after MI, cardiac MRI was performed to track the transplanted cells in the infarct heart. Additionally, echocardiography (M-mode) was performed to evaluate the cardiac function. Immunohistological and western blot studies were performed to assess the cell tracking, engraftment and cardiac fibrosis in the infarct heart tissues. Results Echocardiography data showed a significantly improved cardiac function in the hiPSC-CMs and hMSCs groups, when compared to MI. Immunohistological studies showed expression of connexin-43, α-actinin and myosin heavy chain in engrafted hiPSC-CMs. Cardiac fibrosis was significantly decreased in hiPSC-CMs group when compared to hMSCs or MI groups. Overall, this study demonstrated improved cardiac function with decreased fibrosis with both hiPSC-CMs and hMSCs groups when compared with MI group. PMID:25551230

  18. Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.

    PubMed

    Böcker, Wolfgang; Yin, Zhanhai; Drosse, Inga; Haasters, Florian; Rossmann, Oliver; Wierer, Matthias; Popov, Cvetan; Locher, Melanie; Mutschler, Wolf; Docheva, Denitsa; Schieker, Matthias

    2008-08-01

    Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

  19. Alpha-Latrotoxin Rescues SNAP-25 from BoNT/A-Mediated Proteolysis in Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Mesngon, Mariano; McNutt, Patrick

    2011-01-01

    The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K+ had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca2+ influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery. PMID:22069721

  20. [Cancer vaccine therapy using genetically modified induced pluripotent stem cell-derived dendritic cells expressing the TAA gene].

    PubMed

    Iwamoto, Hiromitsu; Ojima, Toshiyasu; Nakamori, Mikihito; Nakamura, Masaki; Hayata, Keiji; Katsuda, Masahiro; Iida, Takeshi; Miyazawa, Motoki; Iwahashi, Makoto; Yamaue, Hiroki

    2013-11-01

    It is generally accepted that the difficulty in obtaining a sufficient number of functional dendritic cells (DCs) poses a serious problem in DC-based immunotherapy. Therefore, we used induced pluripotent stem (iPS) cell-derived DCs (iPSDCs) instead. If the therapeutic efficacy of iPSDCs was equivalent to that of bone marrow-derived DCs( BMDCs), then the above-mentioned problems may be solved. In this study, we generated iPSDCs from iPS cells and compared their capacity to mature and migrate to the regional lymph nodes with that of BMDCs. We adenovirally transduced the hgp100 gene, which codes for a natural tumor antigen, into the DCs and immunized the mice with these genetically modified DCs. The cytotoxic activity of CD8( +) cytotoxic T lymphocytes( CTLs) was assayed using a 51Cr-release assay. The therapeutic efficacy of the vaccination was examined in a subcutaneous tumor model. Our results demonstrated that iPSDCs equaled BMDCs in terms of their maturation and migration capacity. Furthermore, hgp100-specific CTLs were generated in mice that were immunized with the genetically modified iPSDCs. These CTLs exhibited a high level of cytotoxicity against B16 cells, which is similar to that exhibited by CTLs generated in BMDCs immunized mice. Moreover, vaccination with genetically modified iPSDCs elicited a high level of therapeutic efficacy equaling that of vaccination with BMDCs. This study clarified experimentally that genetically modified iPSDCs are equivalent to BMDCs in terms of tumor-associated antigen-specific therapeutic antitumor immunity. This vaccination strategy may therefore be useful for future clinical application as a cancer vaccine.

  1. Role of mast cell- and non-mast cell-derived inflammatory mediators in immunologic induction of synaptic plasticity.

    PubMed

    Albuquerque, A A; Leal-Cardoso, J H; Weinreich, D

    1997-07-01

    We have previously discovered a long-lasting enhancement of synaptic transmission in mammal autonomic ganglia caused by immunological activation of ganglionic mast cells. Subsequent to mast cell activation, lipid and peptide mediators are released which may modulate synaptic function. In this study we determined whether some mast cell-derived mediators, prostaglandin D2 (PGD2; 1.0 microM), platelet aggregating factor (PAF; 0.3 microM) and U44619 (a thromboxane analogue; 1.0 microM), and also endothelin-1 (ET-1; 0.5 microM) induce synaptic potentiation in the guinea pig superior cervical ganglion (SCG), and compared their effects on synaptic transmission with those induced by a sensitizing antigen, ovalbumin (OVA; 10 micrograms/ml). The experiments were carried out on SCGs isolated from adult male guinea pigs (200-250 g) actively sensitized to OVA, maintained in oxygenated Locke solution at 37 degrees C. Synaptic potentiation was measured through alterations of the integral of the post-ganglionic compound action potential (CAP). All agents tested caused long-term (LTP; duration > or = 30 min) or short-term (STP; < 30 min) potentiation of synaptic efficacy, as measured by the increase in the integral of the post-ganglionic CAP. The magnitude of mediator-induced potentiation was never the same as the antigen-induced long-term potentiation (A-LTP). The agent that best mimicked the antigen was PGD2, which induced a 75% increase in CAP integral for LTP (antigen: 94%) and a 34% increase for STP (antigen: 91%). PAF-, U44619-, and ET-1-induced increases in CAP integral ranged for LTP from 34 to 47%, and for STP from 0 to 26%. These results suggest that the agents investigated may participate in the induction of A-LTP.

  2. The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions*

    PubMed Central

    Saini, Vikas; Staren, Daniel M.; Ziarek, Joshua J.; Nashaat, Zayd N.; Campbell, Edward M.; Volkman, Brian F.; Marchese, Adriano; Majetschak, Matthias

    2011-01-01

    Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gαi-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands. PMID:21757744

  3. Functional and phenotypic differences of pure populations of stem cell-derived astrocytes and neuronal precursor cells.

    PubMed

    Kleiderman, Susanne; Sá, João V; Teixeira, Ana P; Brito, Catarina; Gutbier, Simon; Evje, Lars G; Hadera, Mussie G; Glaab, Enrico; Henry, Margit; Sachinidis, Agapios; Alves, Paula M; Sonnewald, Ursula; Leist, Marcel

    2016-05-01

    Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, preactivated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, nonproliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to tumor necrosis factor and interleukin 1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved marker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features.

  4. Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

    PubMed Central

    Cusella-De Angelis, Maria Gabriella; Laino, Gregorio; Piattelli, Adriano; Pacifici, Maurizio; De Rosa, Alfredo; Papaccio, Gianpaolo

    2007-01-01

    Background Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. Methodology and Findings In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. Conclusion In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and

  5. Surface Modified Biodegradable Electrospun Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

    PubMed

    Sorkio, Anni; Porter, Patrick J; Juuti-Uusitalo, Kati; Meenan, Brian J; Skottman, Heli; Burke, George A

    2015-09-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications.

  6. In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic-like neurons.

    PubMed

    Alizadeh, Rafieh; Hassanzadeh, Gholamreza; Joghataei, Mohammad Taghi; Soleimani, Mansoureh; Moradi, Fatemeh; Mohammadpour, Shahram; Ghorbani, Jahangir; Safavi, Ali; Sarbishegi, Maryam; Pirhajati Mahabadi, Vahid; Alizadeh, Leila; Hadjighassem, Mahmoudreza

    2017-03-01

    This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain-death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell-derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l-amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co-positive cells (microtubule-associated protein 2 and tyrosine hydroxylase; aromatic l-amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell-based therapeutic strategy for Parkinson

  7. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

    PubMed Central

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

    2016-01-01

    The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

  8. Evaluation of the Cardiotoxicity of Mitragynine and Its Analogues Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Wu, Jianjun; Jamil, Mohd Fadzly Amar; Tan, Mei Lan; Adenan, Mohd Ilham; Wong, Philip; Shim, Winston

    2014-01-01

    Introduction Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and Results The rapid delayed rectifier potassium current (IKr), L-type Ca2+ current (ICa,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed IKr in hiPSC-CMs by 67% ∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression,and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine. Conclusions Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes. PMID:25535742

  9. Enhanced survival of retinal ganglion cells is mediated by Müller glial cell-derived PEDF.

    PubMed

    Unterlauft, Jan Darius; Claudepierre, Thomas; Schmidt, Manuela; Müller, Katja; Yafai, Yousef; Wiedemann, Peter; Reichenbach, Andreas; Eichler, Wolfram

    2014-10-01

    The death of retinal ganglion cells (RGC) leads to visual impairment and blindness in ocular neurodegenerative diseases, primarily in glaucoma and diabetic retinopathy; hence, mechanisms that contribute to protecting RGC from ischemia/hypoxia are of great interest. We here address the role of retinal glial (Müller) cells and of pigment-epithelium-derived factor (PEDF), one of the main neuroprotectants released from the glial cells. We show that the hypoxia-induced loss in the viability of cultured purified RGC is due to apoptosis, but that the number of viable RGC increases when co-cultured with Müller glial cells suggesting that glial soluble mediators attenuate the death of RGC. When PEDF was ablated from Müller cells a significantly lower number of RGC survived in RGC-Müller cell co-cultures indicating that PEDF is a major survival factor allowing RGC to escape cell death. We further found that RGC express a PEDF receptor known as patatin-like phospholipase domain-containing protein 2 (PNPLA2) and that PEDF exposure, as well as the presence of Müller cells, leads to an activation of nuclear factor (NF)-κB in RGC. Furthermore, adding an NF-κB inhibitor (SN50) to PEDF-treated RGC cultures reduced the survival of RGC. These findings strongly suggest that NF-κB activation in RGC is critically involved in the pro-survival action of Müller-cell derived PEDF and plays an important role in maintaining neuronal survival.

  10. Nanofiber Matrices Promote the Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors In Vitro

    PubMed Central

    Lim, Shawn H.; Christopherson, Gregory T.; Xu, Leyan; Nasonkin, Igor; Yu, Christopher; Mao, Hai-Quan; Koliatsos, Vassilis E.

    2011-01-01

    The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs. PMID:20973749

  11. Increasing the physical size and nucleation status of human pluripotent stem cell-derived ventricular cardiomyocytes by cell fusion.

    PubMed

    Kong, Chi-Wing; Chen, Shuxun; Geng, Lin; Shum, Angie Man-Yee; Sun, Dong; Li, Ronald A

    2017-03-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide an unlimited source of donor cells for potential cardiac regenerative therapies. However, hPSC-CMs are immature. For instance, hPSC-CMs are only 1/10 of the physical size of their adult counterparts; the majority are mono- rather than bi- or multi-nucleated, which is an evolutionary adaptive feature in metabolically active cells such as adult CMs. Here, we attempted to increase the physical size and nucleation status of hPSC-derived ventricular (V) cardiomyocytes (hPSC-VCMs) using chemically-induced cell fusion, and examined the subsequent functional effects. Polyethylene glycol (PEG) was employed to fuse a 1:1 mixture of lentiviral vectors LV-MLC2v-GFP- or -tdTomato-labeled hPSC-VCMs, such that hPSC-VCMs fused syncytia (FS) were identified as doubly GFP(+)/tdTomato(+) multi-nucleated cells. These microscopically-identified FS were doubled in size as gauged by their capacitance when compared to the control mononucleated hPSC-VCMs using patch-clamp analysis. Reduced automaticity or action potential (AP) firing rate and moderately prolonged AP duration were observed in FS from day 6 post-fusion induction. However, Ca(2+) handling, mitochondrial biogenesis and the extent of apoptosis were not significantly altered. We conclude that larger, multi-nucleated hPSC-VCMs FS can be created by chemically-induced cell fusion but global maturation requires additional triggering cues.

  12. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation

    SciTech Connect

    Nozaki, Yumiko; Honda, Yayoi; Tsujimoto, Shinji; Watanabe, Hitoshi; Kunimatsu, Takeshi; Funabashi, Hitoshi

    2014-07-01

    Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K{sup +} channel and Ca{sup 2+} channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2 min every 10 min for 30 min after drug exposure for the vehicle and each drug concentration. I{sub Kr} and I{sub Ks} blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca{sup 2+} channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the I{sub Kr} blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. - Highlights: • We focused on hiPS-CMs to replace in vitro assays in preclinical screening studies. • hiPS-CMs FPD is useful as an indicator to predict drug potential for QT prolongation. • MEA assay can help detect EAD for drugs with TdP potentials. • MEA assay in hiPS-CMs is useful for accurately predicting drug TdP risk in humans.

  13. Embryonic stem cell-derived granulosa cells participate in ovarian follicle formation in vitro and in vivo.

    PubMed

    Woods, Dori C; White, Yvonne A R; Niikura, Yuichi; Kiatpongsan, Sorapop; Lee, Ho-Joon; Tilly, Jonathan L

    2013-05-01

    Differentiating embryonic stem cells (ESCs) can form ovarian follicle-like structures in vitro, consisting of an oocyte-like cell surrounded by somatic cells capable of steroidogenesis. Using a dual-fluorescence reporter system in which mouse ESCs express green fluorescent protein (GFP) under the control of a germ cell-specific Pou5f1 gene promoter and red fluorescent protein (Discosoma sp red [DsRed]) driven by the granulosa cell-specific Forkhead box L2 (Foxl2) gene promoter, we first confirmed in vitro formation of follicle-like structures containing GFP-positive cells surrounded by DsRed-positive cells. Isolated DsRed-positive cells specified from ECSs exhibited a gene expression profile consistent with granulosa cells, as revealed by the detection of messenger RNAs (mRNAs) for Foxl2, follistatin (Fst), anti-Müllerian hormone (Amh), and follicle-stimulating hormone receptor (Fshr) as well as by production of both progesterone and estradiol. In addition, treatment of isolated DsRed-expressing cells with follicle-stimulating hormone (FSH) significantly increased estradiol production over basal levels, confirming the presence of functional FSH receptors in these cells. Last, ESC-derived DsRed-positive cells injected into neonatal mouse ovaries became incorporated within the granulosa cell layer of immature follicles. These studies demonstrate that Foxl2-expressing ovarian somatic cells derived in vitro from differentiating ESCs express granulosa cell markers, actively associate with germ cells in vitro, synthesize steroids, respond to FSH, and participate in folliculogenesis in vivo.

  14. Potent Paracrine Effects of human induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells Attenuate Doxorubicin-induced Cardiomyopathy

    PubMed Central

    Zhang, Yuelin; Liang, Xiaoting; Liao, Songyan; Wang, Weixin; Wang, Junwen; Li, Xiang; Ding, Yue; Liang, Yingmin; Gao, Fei; Yang, Mo; Fu, Qingling; Xu, Aimin; Chai, Yuet-Hung; He, Jia; Tse, Hung-Fat; Lian, Qizhou

    2015-01-01

    Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) can protect cardiomyocytes against anthracycline-induced cardiomyopathy (AIC) through paracrine effects. Nonetheless the paracrine effects of human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) on AIC are poorly understood. In vitro studies reveal that doxorubicin (Dox)-induced reactive oxidative stress (ROS) generation and cell apoptosis in neonatal rat cardiomyocytes (NRCMs) are significantly reduced when treated with conditioned medium harvested from BM-MSCs (BM-MSCs-CdM) or iPSC-MSCs (iPSC-MSCs-CdM). Compared with BM-MSCs-CdM, NRCMs treated with iPSC-MSCs-CdM exhibit significantly less ROS and cell apoptosis in a dose-dependent manner. Transplantation of BM-MSCs-CdM or iPSC-MSCs-CdM into mice with AIC remarkably attenuated left ventricular (LV) dysfunction and dilatation. Compared with BM-MSCs-CdM, iPSC-MSCs-CdM treatment showed better alleviation of heart failure, less cardiomyocyte apoptosis and fibrosis. Analysis of common and distinct cytokines revealed that macrophage migration inhibitory factor (MIF) and growth differentiation factor-15 (GDF-15) were uniquely overpresented in iPSC-MSC-CdM. Immunodepletion of MIF and GDF-15 in iPSC-MSCs-CdM dramatically decreased cardioprotection. Injection of GDF-15/MIF cytokines could partially reverse Dox-induced heart dysfunction. We suggest that the potent paracrine effects of iPSC-MSCs provide novel “cell-free” therapeutic cardioprotection against AIC, and that MIF and GDF-15 in iPSC-MSCs-CdM are critical for these enhanced cardioprotective effects. PMID:26057572

  15. Stromal cell-derived factor-1 promotes human adipose tissue-derived stem cell survival and chronic wound healing

    PubMed Central

    LI, QIANG; GUO, YANPING; CHEN, FEIFEI; LIU, JING; JIN, PEISHENG

    2016-01-01

    Adipose tissue-derived stem cells (ADSCs) hold great potential for the stem cell-based therapy of cutaneous wound healing. Stromal cell-derived factor-1 (SDF-1) activates CXC chemokine receptor (CXCR)4+ and CXCR7+ cells and plays an important role in wound healing. Increasing evidence suggests a critical role for SDF-1 in cell apoptosis and the survival of mesenchymal stem cells. However, the function of SDF-1 in the apoptosis and wound healing ability of ADSCs is not well understood. The aim of this study was to analyze the effect of SDF-1 on the apoptosis and therapeutic effect of ADSCs in cutaneous chronic wounds in vitro and in vivos. By flow cytometric analysis, it was found that hypoxia and serum free promoted the apoptosis of ADSCs. When pretreated with SDF-1, the apoptosis of ADSCs induced by hypoxia and serum depletion was partly recovered. Furthermore, in vivo experiments established that the post-implantation cell survival and chronic wound healing ability of ADSCs were increased following pretreatment with SDF-1 in a diabetic mouse model of chronic wound healing. To explore the potential mechanism underlying the effect of SDF-1 on ADSC apoptosis, western blot analysis was employed and the results indicate that SDF-1 may protect against cell apoptosis in hypoxic and serum-free conditions through activation of the caspase signaling pathway in ADSCs. This study provides evidence that SDF-1 pretreatment can increase the therapeutic effect of ADSCs in cutaneous chronic wounds in vitro and in vivo. PMID:27347016

  16. Nucleus pulposus cells derived IGF-1 and MCP-1 enhance osteoclastogenesis and vertebrae disruption in lumbar disc herniation

    PubMed Central

    Zhu, Zhongjiao; Huang, Peng; Chong, Yanxue; George, Suraj K; Wen, Bingtao; Han, Na; Liu, Zhiqiang; Kang, Lixin; Lin, Nie

    2014-01-01

    Study design: Chronic strained lumbar disc herniation (LDH) cases were classified into bulging LDH, herniated LDH and prolapse LDH types according to imaging examination, and vertebrae disruptions were evaluated. Cytokines derived from the nucleus pulposus cells were detected, and their effects on osteoclastogenesis, as well as the mechanisms involved, were studied via an in vitro osteoclast differentiation system. Objective: To clarify the mechanisms of lumbar vertebrae resorption induced by lumbar herniation. Summary and background data: Chronic strained lumbar disc herniation induced vertebrae erosion exacerbates quality of patients’ life and clinical outcome. Although nucleus pulposus cells derived cytokines were reported to play an important role in this pathogenesis, the fundamental mechanisms underlying this process are still unclear. Methods: Chronic strained lumbar disc herniation patients were diagnosed with CT scan and T2-weighted magnetic resonance imaging. RNA was extracted from 192 surgical specimens of the herniated lumbar disc and 29 surgical excisions of the lumbar disc from spinal injury patients. The expressions of osteoclastogenesis related cytokines and chemokines were examined using real time PCR. Monocytes were induced into osteoclast with M-CSF and RANKL in vitro, while the IGF-1 and MCP-1 were added into the differentiation procedure in order to evaluate the effects and explore the molecular mechanisms. Results: Vertebrae erosion had a positive relationship with lumbar disc herniation severity types. In all of the osteoclastogenesis related cytokines, the IGF-1 and MCP-1 were the most highly expressed in the nucleus pulposus cells. IGF-1 enhances activation of NF-kB signaling directly, but MCP-1 upregulated the expression of RANK, so that enhanced cellular sensitivity to RANKL resulted in increasing osteoclastogenesis and activity. Conclusion: Lumbar herniation induced overexpression of IGF-1 and MCP-1 in nucleus pulposus cells aggravated

  17. In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer

    PubMed Central

    Veluchamy, John P.; Lopez-Lastra, Silvia; Spanholtz, Jan; Bohme, Fenna; Kok, Nina; Heideman, Daniëlle A. M.; Verheul, Henk M. W.; Di Santo, James P.; de Gruijl, Tanja D.; van der Vliet, Hans J.

    2017-01-01

    Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RASwt metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR− RASwt, EGFR+ RASmut, and EGFR+ BRAFmut cells, A-PBNK were able to initiate lysis of EGFR+ colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR+ colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR−RASwt (42 ± 8 versus 67 ± 7%), EGFR+ RASmut (20 ± 2 versus 37 ± 6%), and EGFR+ BRAFmut (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR+ RASmut colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered. PMID:28220124

  18. Embryonic stem cell-derived microvesicles induce gene expression changes in Müller cells of the retina.

    PubMed

    Katsman, Diana; Stackpole, Emma J; Domin, Daniel R; Farber, Debora B

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  19. Isolation and Transplantation of Corneal Endothelial Cell–Like Cells Derived from In-Vitro-Differentiated Human Embryonic Stem Cells

    PubMed Central

    Zhang, Kai; Pang, Kunpeng

    2014-01-01

    The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell–conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)–based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future. PMID:24499373

  20. Recording axonal conduction to evaluate the integration of pluripotent cell-derived neurons into a neuronal network.

    PubMed

    Shimba, Kenta; Sakai, Koji; Takayama, Yuzo; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-10-01

    Stem cell transplantation is a promising therapy to treat neurodegenerative disorders, and a number of in vitro models have been developed for studying interactions between grafted neurons and the host neuronal network to promote drug discovery. However, methods capable of evaluating the process by which stem cells integrate into the host neuronal network are lacking. In this study, we applied an axonal conduction-based analysis to a co-culture study of primary and differentiated neurons. Mouse cortical neurons and neuronal cells differentiated from P19 embryonal carcinoma cells, a model for early neural differentiation of pluripotent stem cells, were co-cultured in a microfabricated device. The somata of these cells were separated by the co-culture device, but their axons were able to elongate through microtunnels and then form synaptic contacts. Propagating action potentials were recorded from these axons by microelectrodes embedded at the bottom of the microtunnels and sorted into clusters representing individual axons. While the number of axons of cortical neurons increased until 14 days in vitro and then decreased, those of P19 neurons increased throughout the culture period. Network burst analysis showed that P19 neurons participated in approximately 80% of the bursting activity after 14 days in vitro. Interestingly, the axonal conduction delay of P19 neurons was significantly greater than that of cortical neurons, suggesting that there are some physiological differences in their axons. These results suggest that our method is feasible to evaluate the process by which stem cell-derived neurons integrate into a host neuronal network.

  1. Circulating Stromal Cell-Derived Factor 1α Levels in Heart Failure: A Matter of Proper Sampling

    PubMed Central

    Baerts, Lesley; Waumans, Yannick; Brandt, Inger; Jungraithmayr, Wolfgang; Van der Veken, Pieter; Vanderheyden, Marc; De Meester, Ingrid

    2015-01-01

    Background The chemokine Stromal cell-derived factor 1α (SDF1α, CXCL12) is currently under investigation as a biomarker for various cardiac diseases. The correct interpretation of SDF1α levels is complicated by the occurrence of truncated forms that possess an altered biological activity. Methodology We studied the immunoreactivities of SDF1α forms and evaluated the effect of adding a DPP4 inhibitor in sampling tubes on measured SDF1α levels. Using optimized sampling, we measured DPP4 activity and SDF1α levels in patients with varying degrees of heart failure. Results The immunoreactivities of SDF1α and its degradation products were determined with three immunoassays. A one hour incubation of SDF1α with DPP4 at 37°C resulted in 2/3 loss of immunoreactivity in each of the assays. Incubation with serum gave a similar result. Using appropriate sampling, SDF1α levels were found to be significantly higher in those heart failure patients with a severe loss of left ventricular function. DPP4 activity in serum was not altered in the heart failure population. However, the DPP4 activity was found to be significantly decreased in patients with high SDF1α levels Conclusions We propose that all samples for SDF1α analysis should be collected in the presence of at least a DPP4 inhibitor. In doing so, we found higher SDF1α levels in subgroups of patients with heart failure. Our work supports the need for further research on the clinical relevance of SDF1α levels in cardiac disease. PMID:26544044

  2. Stem cell-derived nodal-like cardiomyocytes as a novel pharmacologic tool: insights from sinoatrial node development and function.

    PubMed

    Barbuti, Andrea; Robinson, Richard B

    2015-01-01

    Since the first reports on the isolation and differentiation of stem cells, and in particular since the early success in driving these cells down a cardiac lineage, there has been interest in the potential of such preparations in cardiac regenerative therapy. Much of the focus of such research has been on improving mechanical function after myocardial infarction; however, electrophysiologic studies of these preparations have revealed a heterogeneous mix of action potential characteristics, including some described as "pacemaker" or "nodal-like," which in turn led to interest in the therapeutic potential of these preparations in the treatment of rhythm disorders; several proof-of-concept studies have used these cells to create a biologic alternative to electronic pacemakers. Further, there are additional potential applications of a preparation of pacemaker cells derived from stem cells, for example, in high-throughput screens of new chronotropic agents. All such applications require reasonably efficient methods for selecting or enriching the "nodal-like" cells, however, which in turn depends on first defining what constitutes a nodal-like cell since not all pacemaking cells are necessarily of nodal lineage. This review discusses the current state of the field in terms of characterizing sinoatrial-like cardiomyocytes derived from embryonic and induced pluripotent stem cells, markers that might be appropriate based on the current knowledge of the gene program leading to sinoatrial node development, what functional characteristics might be expected and desired based on studies of the sinoatrial node, and recent efforts at enrichment and selection of nodal-like cells.

  3. In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model.

    PubMed

    Sirenko, Oksana; Grimm, Fabian A; Ryan, Kristen R; Iwata, Yasuhiro; Chiu, Weihsueh A; Parham, Frederick; Wignall, Jessica A; Anson, Blake; Cromwell, Evan F; Behl, Mamta; Rusyn, Ivan; Tice, Raymond R

    2017-03-01

    An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30min or 24h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca(2+) flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30min of exposure. In contrast, after 24h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes.

  4. Developmental stage-dependent effects of cardiac fibroblasts on function of stem cell-derived engineered cardiac tissues

    PubMed Central

    Liau, Brian; Jackman, Christopher P.; Li, Yanzhen; Bursac, Nenad

    2017-01-01

    We investigated whether the developmental stage of mouse cardiac fibroblasts (CFs) influences the formation and function of engineered cardiac tissues made of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs). Engineered cardiac tissue patches were fabricated by encapsulating pure mESC-CMs, mESC-CMs + adult CFs, or mESC-CMs + fetal CFs in fibrin-based hydrogel. Tissue patches containing fetal CFs exhibited higher velocity of action potential propagation and contractile force amplitude compared to patches containing adult CFs, while pure mESC-CM patches did not form functional syncytium. The functional improvements in mESC-CM + fetal CF patches were associated with differences in structural remodeling and increased expression of proteins involved in cardiac function. To determine role of paracrine signaling, we cultured pure mESC-CMs within miniature tissue “micro-patches” supplemented with media conditioned by adult or fetal CFs. Fetal CF-conditioned media distinctly enhanced CM spreading and contractile activity, which was shown by pathway inhibitor experiments and Western blot analysis to be mediated via MEK-ERK signaling. In mESC-CM monolayers, CF-conditioned media did not alter CM spreading or MEK-ERK activation. Collectively, our studies show that 3D co-culture of mESC-CMs with embryonic CFs is superior to co-culture with adult CFs for in vitro generation of functional myocardium. Ensuring consistent developmental stages of cardiomyocytes and supporting non-myocytes may be a critical factor for promoting functional maturation of engineered cardiac tissues. PMID:28181589

  5. Body Imaging

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The high-tech art of digital signal processing (DSP) was pioneered at NASA's Jet Propulsion Laboratory (JPL) in the mid-1960s for use in the Apollo Lunar Landing Program. Designed to computer enhance pictures of the Moon, this technology became the basis for the Landsat Earth resources satellites and subsequently has been incorporated into a broad range of Earthbound medical and diagnostic tools. DSP is employed in advanced body imaging techniques including Computer-Aided Tomography, also known as CT and CATScan, and Magnetic Resonance Imaging (MRI). CT images are collected by irradiating a thin slice of the body with a fan-shaped x-ray beam from a number of directions around the body's perimeter. A tomographic (slice-like) picture is reconstructed from these multiple views by a computer. MRI employs a magnetic field and radio waves, rather than x-rays, to create images. In this photograph, a patient undergoes an open MRI.

  6. Body Imaging

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The high-tech art of digital signal processing (DSP) was pioneered at NASA's Jet Propulsion Laboratory (JPL) in the mid-1960s for use in the Apollo Lunar Landing Program. Designed to computer enhance pictures of the Moon, this technology became the basis for the Landsat Earth resources satellites and subsequently has been incorporated into a broad range of Earthbound medical and diagnostic tools. DSP is employed in advanced body imaging techniques including Computer-Aided Tomography, also known as CT and CATScan, and Magnetic Resonance Imaging (MRI). CT images are collected by irradiating a thin slice of the body with a fan-shaped x-ray beam from a number of directions around the body's perimeter. A tomographic (slice-like) picture is reconstructed from these multiple views by a computer. MRI employs a magnetic field and radio waves, rather than x-rays, to create images.

  7. Body parts

    NASA Astrophysics Data System (ADS)

    Ayiter, Elif

    2010-01-01

    In this project, the artist wishes to examine corporeality in the virtual realm, through the usage of the (non)-physical body of the avatar. An art installation created in the virtual world of Second Life, which is meant to be accessed with site specific avatars, will provide the creative platform whereby this investigation is undertaken. Thus, "body parts" seeks to challenge the residents of virtual environments into connecting with the virtual manifestations, i.e., avatars of others in an emotionally expressive/intimate manner.

  8. Body Imaging

    NASA Technical Reports Server (NTRS)

    1990-01-01

    Magnetic Resonance Imaging (MRI) and Computer-aided Tomography (CT) images are often complementary. In most cases, MRI is good for viewing soft tissue but not bone, while CT images are good for bone but not always good for soft tissue discrimination. Physicians and engineers in the Department of Radiology at the University of Michigan Hospitals are developing a technique for combining the best features of MRI and CT scans to increase the accuracy of discriminating one type of body tissue from another. One of their research tools is a computer program called HICAP. The program can be used to distinguish between healthy and diseased tissue in body images.

  9. Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

    EPA Science Inventory

    During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

  10. Phospholipid-transfer activities in cytosols from lung, isolated alveolar type II cells and alveolar type II cell-derived adenomas.

    PubMed Central

    Pool, G L; Bubacz, D G; Lumb, R H; Mason, R J

    1983-01-01

    We have examined phospholipid-transfer activities in cytosols from rat and mouse whole lung, isolated rat alveolar type II cells and alveolar type II cell-derived mouse pulmonary adenomas. We report an enrichment in phosphatidylcholine and phosphatidylglycerol (but not phosphatidylinositol) protein-catalysed transfer in the type II cell and adenoma cytosols compared with the whole-lung cytosols. The activities from these cytosols were resolved using column chromatofocusing, which clearly demonstrated the presence of a phosphatidylcholine-specific transfer protein in each of the four tissues. In addition, two proteins (rat) or three proteins (mouse) catalysing both phosphatidylcholine and phosphatidylglycerol transfer were resolved from whole lung, whereas in both the rat isolated alveolar type II cells and the mouse type II cell-derived adenomas one of these less specific proteins is not present. PMID:6661189

  11. Grafted Human iPS Cell-Derived Oligodendrocyte Precursor Cells Contribute to Robust Remyelination of Demyelinated Axons after Spinal Cord Injury

    PubMed Central

    Kawabata, Soya; Takano, Morito; Numasawa-Kuroiwa, Yuko; Itakura, Go; Kobayashi, Yoshiomi; Nishiyama, Yuichiro; Sugai, Keiko; Nishimura, Soraya; Iwai, Hiroki; Isoda, Miho; Shibata, Shinsuke; Kohyama, Jun; Iwanami, Akio; Toyama, Yoshiaki; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

    2015-01-01

    Summary Murine- and human-induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NS/PCs) promote functional recovery following transplantation into the injured spinal cord in rodents and primates. Although remyelination of spared demyelinated axons is a critical mechanism in the regeneration of the injured spinal cord, human iPSC-NS/PCs predominantly differentiate into neurons both in vitro and in vivo. We therefore took advantage of our recently developed protocol to obtain human-induced pluripotent stem cell-derived oligodendrocyte precursor cell-enriched neural stem/progenitor cells and report the benefits of transplanting these cells in a spinal cord injury (SCI) model. We describe how this approach contributes to the robust remyelination of demyelinated axons and facilitates functional recovery after SCI. PMID:26724902

  12. Cell survival and differentiation with nanocrystalline glass-like carbon using substantia nigra dopaminergic cells derived from transgenic mouse embryos

    PubMed Central

    Romero, Pablo; Estivill-Torrús, Guillermo; Guzmán de Villoria, Roberto

    2017-01-01

    Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells. Because the interest in carbon nanomaterials has grown within the last decade in light of a wide variety of applications, the aim of this study was to test and evaluate the suitability and cytocompatibility of a particular nanometer-thin nanocrystalline glass-like carbon film (NGLC) composed of curved graphene flakes joined by an amorphous carbon matrix. This material is a disordered structure with high transparency and electrical conductivity. For this purpose, we used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryos. Cells were cultured either in a powder of increasing concentrations of NGLC microflakes (82±37μm) in the medium or on top of nanometer-thin films bathed in the same culture medium. The metabolism activity of SN4741 cells in presence of NGLC was assessed using methylthiazolyldiphenyl-tetrazolium (MTT) and apoptosis/necrosis flow cytometry assay respectively. Growth and proliferation as well as senescence were demonstrated by western blot (WB) of proliferating cell nuclear antigen (PCNA), monoclonal phosphorylate Histone 3 (serine 10) (PH3) and SMP30 marker. Specific dopaminergic differentiation was confirmed by the WB analysis of tyrosine hydroxylase (TH). Cell maturation and neural capability were characterized using specific markers (SYP: synaptophysin and GIRK2: G-protein-regulated inward-rectifier potassium channel 2 protein) via immunofluorescence and coexistence measurements. The results demonstrated cell positive biocompatibility with different concentrations of NGLC. The cells underwent a process of adaptation of SN4741 cells to NGLC where their metabolism decreases. This process is related to a decrease of PH3 expression and

  13. Cell survival and differentiation with nanocrystalline glass-like carbon using substantia nigra dopaminergic cells derived from transgenic mouse embryos.

    PubMed

    Rodriguez-Losada, Noela; Romero, Pablo; Estivill-Torrús, Guillermo; Guzmán de Villoria, Roberto; Aguirre, Jose A

    2017-01-01

    Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells. Because the interest in carbon nanomaterials has grown within the last decade in light of a wide variety of applications, the aim of this study was to test and evaluate the suitability and cytocompatibility of a particular nanometer-thin nanocrystalline glass-like carbon film (NGLC) composed of curved graphene flakes joined by an amorphous carbon matrix. This material is a disordered structure with high transparency and electrical conductivity. For this purpose, we used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryos. Cells were cultured either in a powder of increasing concentrations of NGLC microflakes (82±37μm) in the medium or on top of nanometer-thin films bathed in the same culture medium. The metabolism activity of SN4741 cells in presence of NGLC was assessed using methylthiazolyldiphenyl-tetrazolium (MTT) and apoptosis/necrosis flow cytometry assay respectively. Growth and proliferation as well as senescence were demonstrated by western blot (WB) of proliferating cell nuclear antigen (PCNA), monoclonal phosphorylate Histone 3 (serine 10) (PH3) and SMP30 marker. Specific dopaminergic differentiation was confirmed by the WB analysis of tyrosine hydroxylase (TH). Cell maturation and neural capability were characterized using specific markers (SYP: synaptophysin and GIRK2: G-protein-regulated inward-rectifier potassium channel 2 protein) via immunofluorescence and coexistence measurements. The results demonstrated cell positive biocompatibility with different concentrations of NGLC. The cells underwent a process of adaptation of SN4741 cells to NGLC where their metabolism decreases. This process is related to a decrease of PH3 expression and

  14. Texture Descriptors Ensembles Enable Image-Based Classification of Maturation of Human Stem Cell-Derived Retinal Pigmented Epithelium

    PubMed Central

    Caetano dos Santos, Florentino Luciano; Skottman, Heli; Juuti-Uusitalo, Kati; Hyttinen, Jari

    2016-01-01

    showed that the developed ensembles of texture descriptors are able to classify the RPE cell maturation stage. Moreover, we proved that preprocessing and region-based decomposition improves many descriptors’ accuracy in biological dataset classification. Finally, we built the first public dataset of stem cell-derived RPE cells, which is publicly available to the scientific community for classification studies. The proposed tool is available at https://www.dei.unipd.it/node/2357 and the RPE dataset at http://www.biomeditech.fi/data/RPE_dataset/. Both are available at https://figshare.com/s/d6fb591f1beb4f8efa6f. PMID:26895509

  15. Differential clearance and immune responses to tick cell-derived versus macrophage culture-derived Ehrlichia chaffeensis in mice.

    PubMed

    Ganta, Roman R; Cheng, Chuanmin; Miller, Elizabeth C; McGuire, Bridget L; Peddireddi, Lalitha; Sirigireddy, Kamesh R; Chapes, Stephen K

    2007-01-01

    Human monocytic ehrlichiosis is caused by a tick-transmitted rickettsia, Ehrlichia chaffeensis. We recently reported that E. chaffeensis grown in tick cells expresses different proteins than bacteria grown in macrophages. Therefore, we tested the hypothesis that immune responses against E. chaffeensis would be different if the mice are challenged with bacteria grown in macrophages or tick cells. We assessed the E. chaffeensis clearance from the peritoneum, spleen, and liver by C57BL/6J mice using a TaqMan-based real-time reverse transcription-PCR assay. Macrophage-grown E. chaffeensis was cleared in 2 weeks from the peritoneum, whereas the pathogen from tick cells persisted for nine additional days and included three relapses of increasing bacterial load separated by three-day intervals. Tick cell-grown bacteria also persisted in the livers and spleens with higher bacterial loads compared to macrophage-grown bacteria and fluctuated over a period of 35 days. Three-day periodic cycles were detected in T-cell CD62L/CD44 ratios in the spleen and bone marrow in response to infections with both tick cell- and macrophage-grown bacteria and were accompanied by similar periodic cycles of spleen cell cytokine secretions and nitric oxide and interleukin-6 by peritoneal macrophages. The E. chaffeensis-specific immunoglobulin G response was considerably higher and steadily increased in mice infected with the tick cell-derived E. chaffeensis compared to DH82-grown bacteria. In addition, antigens detected by the immunoglobulins were significantly different between mice infected with the E. chaffeensis originating from tick cells or macrophages. The differences in the immune response to tick cell-grown bacteria compared to macrophage-grown bacteria reflected a delay in the shift of gene expression from the tick cell-specific Omp 14 gene to the macrophage-specific Omp 19 gene. These data suggest that the host response to E. chaffeensis depends on the source of the bacteria and that

  16. Ca2+-Currents in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Effects of Two Different Culture Conditions

    PubMed Central

    Uzun, Ahmet U.; Mannhardt, Ingra; Breckwoldt, Kaja; Horváth, András; Johannsen, Silke S.; Hansen, Arne; Eschenhagen, Thomas; Christ, Torsten

    2016-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca2+-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were two- to three-fold smaller than adult CM, independently of culture format [capacitance ML 45 ± 1 pF (n = 289), EHT 45 ± 1 pF (n = 460), atrial CM 87 ± 3 pF (n = 196), ventricular CM 126 ± 8 pF (n = 50)]. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10 ± 1 pA/pF (n = 207) for ML and 12 ± 1 pA/pF (n = 361) for EHT and was larger than in adult CM [7 ± 1 pA/pF (p < 0.05, n = 196) for atrial CM and 6 ± 1 pA/pF (p < 0.05, n = 47) for ventricular CM]. However, ML and EHT showed robust T-type Ca2+-currents (ICa,T). While (−)-Bay K 8644, that activates ICa,L directly, increased ICa,Lto the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (−)-Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (−logEC50: 5.9 and 6.1 for norepinephrine (NE) and epinephrine (Epi), respectively), but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (−logEC50: 5.3 and 5.3 respectively for NE and Epi). Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation. PMID:27672365

  17. Transplantation of stem cell-derived astrocytes for the treatment of amyotrophic lateral sclerosis and spinal cord injury

    PubMed Central

    Nicaise, Charles; Mitrecic, Dinko; Falnikar, Aditi; Lepore, Angelo C

    2015-01-01

    Neglected for years, astrocytes are now recognized to fulfill and support many, if not all, homeostatic functions of the healthy central nervous system (CNS). During neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI), astrocytes in the vicinity of degenerating areas undergo both morphological and functional changes that might compromise their intrinsic properties. Evidence from human and animal studies show that deficient astrocyte functions or loss-of-astrocytes largely contribute to increased susceptibility to cell death for neurons, oligodendrocytes and axons during ALS and SCI disease progression. Despite exciting advances in experimental CNS repair, most of current approaches that are translated into clinical trials focus on the replacement or support of spinal neurons through stem cell transplantation, while none focus on the specific replacement of astroglial populations. Knowing the important functions carried out by astrocytes in the CNS, astrocyte replacement-based therapies might be a promising approach to alleviate overall astrocyte dysfunction, deliver neurotrophic support to degenerating spinal tissue and stimulate endogenous CNS repair abilities. Enclosed in this review, we gathered experimental evidence that argue in favor of astrocyte transplantation during ALS and SCI. Based on their intrinsic properties and according to the cell type transplanted, astrocyte precursors or stem cell-derived astrocytes promote axonal growth, support mechanisms and cells involved in myelination, are able to modulate the host immune response, deliver neurotrophic factors and provide protective molecules against oxidative or excitotoxic insults, amongst many possible benefits. Embryonic or adult stem cells can even be genetically engineered in order to deliver missing gene products and therefore maximize the chance of neuroprotection and functional recovery. However, before broad clinical translation, further preclinical

  18. Resveratrol-Induced Apoptosis and Increased Radiosensitivity in CD133-Positive Cells Derived From Atypical Teratoid/Rhabdoid Tumor

    SciTech Connect

    Kao, C.-L.; Huang, P.-I; Tsai, P.-H.; Tsai, M.-L.; Lo, J.-F.; Lee, Y.-Y.; Chen, Y.-J.; Chen, Y.-W.; Chiou, S.-H.

    2009-05-01

    Purpose: CD133 has recently been proposed as a marker for cancer stem-like cells (CSC) in brain tumors. The aim of the present study was to investigate the possible role of resveratrol (RV) in radiosensitivity of CD133-positive/-negative cells derived from atypical teratoid/rhabdoid tumors (AT/RT-CD133{sup +/-}). Materials and Methods: AT/RT-CD133{sup +/-} were isolated and characterized by flow cytometry and quantitative real-time reverse transcription-polymerase chain reaction, and then treated with RV at different doses. Migratory ability, colony formation, apoptotic activity, and xenotransplantation were assessed for RV alone, ionizing radiation (IR) alone, and IR with RV conditions. Results: AT/RT-CD133{sup +} displayed enhanced self-renewal and highly coexpressed 'stem cell' genes and drug-resistant genes, in addition to showing significant resistance to chemotherapeutic agents and radiotherapy as compared with CD133{sup -} cells. After treatment with 200 {mu}M RV, the in vitro proliferation rates and in vivo tumor restoration abilities of ATRT-CD133{sup +} were dramatically inhibited. Importantly, treatment with 150 {mu}M RV can effectively inhibit the expression of drug-resistant genes in AT/RT-CD133{sup +}, and further facilitate to the differentiation of CD133{sup +} into CD133{sup -}. In addition, treatment with 150 {mu}M RV could significantly enhance the radiosensitivity and IR-mediated apoptosis in RV-treated ATRT-CD133{sup +/-}. Kaplan-Meier survival analysis indicated that the mean survival rate of mice with ATRT-CD133{sup +} that were treated with IR could be significantly improved when IR was combined with 150 {mu}M RV treatment. Conclusions: AT/RT-CD133{sup +} exhibit CSC properties and are refractory to IR treatment. Our results suggest that RV treatment plays crucial roles in antiproliferative, proapoptotic, and radiosensitizing effects on treated-CD133{sup +/-}; RV may therefore improve the clinical treatment of AT/RT.

  19. In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.

    PubMed

    Veluchamy, John P; Lopez-Lastra, Silvia; Spanholtz, Jan; Bohme, Fenna; Kok, Nina; Heideman, Daniëlle A M; Verheul, Henk M W; Di Santo, James P; de Gruijl, Tanja D; van der Vliet, Hans J

    2017-01-01

    Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RAS(wt) metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR(-) RAS(wt), EGFR(+) RAS(mut), and EGFR(+) BRAF(mut) cells, A-PBNK were able to initiate lysis of EGFR(+) colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR(+) colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR(-)RAS(wt) (42 ± 8 versus 67 ± 7%), EGFR(+) RAS(mut) (20 ± 2 versus 37 ± 6%), and EGFR(+) BRAF(mut) (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR(+) RAS(mut) colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered.

  20. Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

    SciTech Connect

    Gardner, Carol R.; Hankey, Pamela; Mishin, Vladimir; Francis, Mary; Yu, Shan; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-07-15

    Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK{sup −/−} mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK{sup −/−} mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK{sup −/−} mice. Whereas F4/80{sup +} macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK{sup −/−} mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK{sup −/−} mice

  1. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    NASA Technical Reports Server (NTRS)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; Topol, Eric J.; Penn, Marc S.

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, p<0.02) resulting in greater left-ventricular mass (1.24 [0.29] vs 1.57 [0.27] g) and better cardiac function (shortening fraction 9.2 [4.9] vs 17.2 [4.2]%, n=8 per group, p<0.05). INTERPRETATION: These findings show that SDF-1 is sufficient to induce therapeutic stem-cell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  2. The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars.

    PubMed

    Chen, Bo; Sun, Hai-Hua; Wang, Han-Guo; Kong, Hui; Chen, Fa-Ming; Yu, Qing

    2012-07-01

    Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) in the large-scale expansion of dental pulp stem cells (DPSCs). However, the biological effects and the optimal concentrations of PL for the proliferation and differentiation of human DPSCs remain unexplored. We isolated and expanded stem cells from the dental pulp of extracted third molars and evaluated the effects of PL on the cells' proliferative capacity and differentiation potential in vitro and in vivo. Before testing, immunocytochemical staining and flow cytometry-based cell sorting showed that the cells derived from human dental pulp contained mesenchymal stem cell populations. Cells were grown on tissue culture plastic or on hydroxyapatite-tricalcium phosphate (HA/TCP) biomaterials and were incubated with either normal or odontogenic/osteogenic media in the presence or absence of various concentrations of human PL for further investigation. The proliferation of DPSCs was significantly increased when the cells were cultured in 5% PL under all testing conditions (P < 0.05). However, this enhancement was inconsistent when the cells were cultured in 1% PL or in 10% PL; 10% PL significantly inhibited cell proliferation and was therefore excluded from further differentiation testing. Culture medium containing 5% PL also significantly promoted the mineralized differentiation of DPSCs, as indicated by the measurement of alkaline phosphatase activity and calcium deposition under mineral-conditioned media (P < 0.05). Scanning electron microscopy and modified Ponceau trichrome staining showed that the cells treated with 5% PL and mineralizing media were highly capable of integrating with the HA/TCP biomaterials and had fully covered the surface of the scaffold with an extensive sheet-like structure 14 d after seeding. In addition, 5% PL showed significantly positive effects on tissue regeneration in two in vivo transplantation models. We conclude that the appropriate

  3. Ca(2+)-Currents in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Effects of Two Different Culture Conditions.

    PubMed

    Uzun, Ahmet U; Mannhardt, Ingra; Breckwoldt, Kaja; Horváth, András; Johannsen, Silke S; Hansen, Arne; Eschenhagen, Thomas; Christ, Torsten

    2016-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca(2+)-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were two- to three-fold smaller than adult CM, independently of culture format [capacitance ML 45 ± 1 pF (n = 289), EHT 45 ± 1 pF (n = 460), atrial CM 87 ± 3 pF (n = 196), ventricular CM 126 ± 8 pF (n = 50)]. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10 ± 1 pA/pF (n = 207) for ML and 12 ± 1 pA/pF (n = 361) for EHT and was larger than in adult CM [7 ± 1 pA/pF (p < 0.05, n = 196) for atrial CM and 6 ± 1 pA/pF (p < 0.05, n = 47) for ventricular CM]. However, ML and EHT showed robust T-type Ca(2+)-currents (ICa,T). While (-)-Bay K 8644, that activates ICa,L directly, increased ICa,Lto the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (-)-Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE) and epinephrine (Epi), respectively), but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi). Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation.

  4. Ion channelopathies in human induced pluripotent stem cell derived cardiomyocytes: a dynamic clamp study with virtual IK1

    PubMed Central

    Meijer van Putten, Rosalie M. E.; Mengarelli, Isabella; Guan, Kaomei; Zegers, Jan G.; van Ginneken, Antoni C. G.; Verkerk, Arie O.; Wilders, Ronald

    2015-01-01

    Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs—and consequently the profile of individual membrane currents active during that action potential—differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to “normalize” the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4–6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near −80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen–Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac

  5. Reprogramming of mesenchymal stem cells derived from iPSCs seeded on biofunctionalized calcium phosphate scaffold for bone engineering

    PubMed Central

    Liu, Jun; Chen, Wenchuan; Zhao, Zhihe; Xu, Hockin H. K.

    2013-01-01

    Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a promising choice of patient-specific stem cells with superior capability of cell expansion. There has been no report on bone morphogenic protein 2 (BMP2) gene modification of iPSC-MSCs for bone tissue engineering. The objectives of this study were to: (1) genetically modify iPSC-MSCs for BMP2 delivery; and (2) to seed BMP2 gene-modified iPSC-MSCs on calcium phosphate cement (CPC) immobilized with RGD for bone tissue engineering. iPSC-MSCs were infected with green fluorescence protein (GFP-iPSC-MSCs), or BMP2 lentivirus (BMP2-iPSC-MSCs). High levels of GFP expression were detected and more than 68% of GFP-iPSC-MSCs were GFP positive. BMP2-iPSC-MSCs expressed higher BMP2 levels than iPSC-MSCs in quantitative RT-PCR and ELISA assays (p < 0.05). BMP2-iPSC-MSCs did not compromise growth kinetics and cell cycle stages compared to iPSC-MSCs. After 14 d in osteogenic medium, ALP activity of BMP2-iPSC-MSCs was 1.8 times that of iPSC-MSCs (p < 0.05), indicating that BMP2 gene transduction of iPSC-MSCs enhanced osteogenic differentiation. BMP2-iPSC-MSCs were seeded on CPC scaffold biofunctionalized with RGD (RGD-CPC). BMP2-iPSC-MSCs attached well on RGD-CPC. At 14 d, COL1A1 expression of BMP2-iPSC-MSCs was 1.9 times that of iPSC-MSCs. OC expression of BMP2-iPSC-MSCs was 2.3 times that of iPSC-MSCs. Bone matrix mineralization by BMP2-iPSC-MSCs was was 1.8 times that of iPSC-MSCs at 21 d. In conclusion, iPSC-MSCs seeded on CPC were suitable for bone tissue engineering. BMP2 gene-modified iPSC-MSCs on RGD-CPC underwent osteogenic differentiation, and the overexpression of BMP2 in iPSC-MSCs enhanced differentiation and bone mineral production on RGD-CPC. BMP2-iPSC-MSC seeding on RGD-CPC scaffold is promising to enhance bone regeneration efficacy. PMID:23891395

  6. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    SciTech Connect

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja; Milatovic, Dejan; Fan, Guo-Huang; Richmond, Ann

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP-2 or

  7. Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment.

    PubMed

    Xiao, Deyi; Barry, Samantha; Kmetz, Daniel; Egger, Michael; Pan, Jianmin; Rai, Shesh N; Qu, Jifu; McMasters, Kelly M; Hao, Hongying

    2016-07-01

    The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma.

  8. A role for the Rho-p160 Rho coiled-coil kinase axis in the chemokine stromal cell-derived factor-1alpha-induced lymphocyte actomyosin and microtubular organization and chemotaxis.

    PubMed

    Vicente-Manzanares, Miguel; Cabrero, José Román; Rey, Mercedes; Pérez-Martínez, Manuel; Ursa, Angeles; Itoh, Kazuyuki; Sánchez-Madrid, Francisco

    2002-01-01

    The possible involvement of the Rho-p160ROCK (Rho coiled-coil kinase) pathway in the signaling induced by the chemokine Stromal cell-derived factor (SDF)-1alpha has been studied in human PBL. SDF-1alpha induced activation of RhoA, but not that of Rac. RhoA activation was followed by p160ROCK activation mediated by RhoA, which led to myosin light chain (MLC) phosphorylation, which was dependent on RhoA and p160ROCK activities. The kinetics of MLC activation was similar to that of RhoA and p160ROCK. The role of this cascade in overall cell morphology and functional responses to the chemokine was examined employing different chemical inhibitors. Inhibition of either RhoA or p160ROCK did not block SDF-1alpha-induced short-term actin polymerization, but induced the formation of long spikes arising from the cell body, which were found to be microtubule based. This morphological change was associated with an increase in microtubule instability, which argues for an active microtubule polymerization in the formation of these spikes. Inhibition of the Rho-p160ROCK-MLC kinase signaling cascade at different steps blocked lymphocyte migration and the chemotaxis induced by SDF-1alpha. Our results indicate that the Rho-p160ROCK axis plays a pivotal role in the control of the cell shape as a step before lymphocyte migration toward a chemotactic gradient.

  9. Lewy bodies

    PubMed Central

    Shults, Clifford W.

    2006-01-01

    Lewy bodies (LB) in the substantia nigra are a cardinal pathological feature of Parkinson's disease, but they occur in a number of neurodegenerative diseases and can be widespread in the nervous system. The characteristics, locations, and composition of LB are reviewed, with particular attention to α-synuclein (α-SYN), which appears to be the major component of LB. The propensity for α-SYN, a presynaptic protein widely expressed in the brain, to aggregate is because of an amyloidogenic central region. The factors that favor the aggregation of α-SYN and mechanisms of toxicity are examined, and a mechanism through which aggregates of α-SYN could induce mitochondrial dysfunction and/or release of proapoptotic molecules is proposed. PMID:16449387

  10. A Multiplex High-Throughput Gene Expression Assay to Simultaneously Detect Disease and Functional Markers in Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

    PubMed Central

    Ferrer, Marc; Corneo, Barbara; Davis, Janine; Wan, Qin; Miyagishima, Kiyoharu Joshua; King, Rebecca; Maminishkis, Arvydas; Marugan, Juan; Sharma, Ruchi; Shure, Michael; Temple, Sally; Miller, Sheldon

    2014-01-01

    There is continuing interest in the development of lineage-specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into “adult-like” cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage-specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained “adult-like” phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high-throughput format. Here, we use a multiplex high-throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell-derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell-derived RPE that was then g