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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. Characterization of tubular liquid crystal structure in embryonic stem cell derived embryoid bodies.

    PubMed

    Xu, MengMeng; Jones, Odell D; Wang, Liyang; Zhou, Xin; Davis, Harry G; Bryant, Joseph L; Ma, Jianjie; Isaacs, Willian B; Xu, Xuehong

    2017-01-01

    Massive liquid crystal droplets have been found during embryonic development in more than twenty different tissues and organs, including the liver, brain and kidney. Liquid crystal deposits have also been identified in multiple human pathologies, including vascular disease, liver dysfunction, age-related macular degeneration, and other chronic illnesses. Despite the involvement of liquid crystals in such a large number of human processes, this phenomenon is poorly understood and there are no in vitro systems to further examine the function of liquid crystals in biology. We report the presence of tubular birefringent structures in embryoid bodies (EBs) differentiated in culture. These birefringent tubular structures initiate at the EB surface and penetrated the cortex at a variety of depths. Under crossed polarized light, these tubules are seen as a collection of birefringent Maltese crosses and tubules with birefringent walls and a non-birefringent lumen. The fluidity of these birefringent structures under pressure application led to elongation and widening, which was partially recoverable with pressure release. These birefringent structures also displayed heat triggered phase transition from liquid crystal to isotropic status that is partially recoverable with return to ambient temperature. These pressure and temperature triggered changes confirm the birefringent structures as liquid crystals. The first report of liquid crystal so early in development. The structure of the liquid crystal tubule network we observed distributed throughout the differentiated embryoid bodies may function as a transportation network for nutrients and metabolic waste during EB growth, and act as a precursor to the vascular system. This observation not only reveals the involvement of liquid crystals earlier than previously known, but also provides a method for studying liquid crystals in vitro.

  3. TGFbeta inhibition of yolk-sac-like differentiation of human embryonic stem-cell-derived embryoid bodies illustrates differences between early mouse and human development.

    PubMed

    Poon, Ellen; Clermont, Frederic; Firpo, Meri T; Akhurst, Rosemary J

    2006-02-15

    Transforming growth factor beta (TGFbeta) plays an important role in development and maintenance of murine yolk sac vascular development. Targeted deletions of Tgfb1 and other components of this signaling pathway, such as Acvrl1, Tgfbr1 and Tgfbr2, result in abnormal vascular development especially of the yolk sac, leading to embryonic lethality. There are significant differences between murine and primate development that limit interpretation of studies from mouse models. Thus, to examine the role of TGFbeta in early human vascular development we used the model of differentiating human embryonic stem cell-derived embryoid bodies to recapitulate early stages of embryonic development. TGFbeta was applied for different time frames after initiation of embryoid body cultures to assess its effect on differentiation. TGFbeta inhibited the expression of endodermal, endothelial and hematopoietic markers, which contrasts with findings in the mouse in which TGFbeta reduced the level of endodermal markers but increased endothelial marker expression. The inhibition observed was not due to changes in proliferation or apoptosis. This marked contrast between the two species may reflect the different origins of the yolk sac hemangiogenic lineages in mouse and human. TGFbeta effects on the hypoblast, from which these cell lineages are derived in human, would decrease subsequent differentiation of hematopoietic, endothelial and endodermal cells. By contrast, TGFbeta action on murine hypoblast, while affecting endoderm would not affect the hemangiogenic lineages that are epiblast-derived in the mouse. This study highlights important differences between early human and mouse embryonic development and suggests a role of TGFbeta in human hypoblast differentiation.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Vascular development in embryoid bodies: quantification of transgenic intervention and antiangiogenic treatment.

    PubMed

    Evans, Amanda Lisabeth; Bryant, James; Skepper, Jeremy; Smith, Stephen K; Print, Cristin G; Charnock-Jones, D Stephen

    2007-01-01

    It has become increasingly clear that the investigation of vascular development is best considered in the context of a whole tissue environment since in vivo endothelial cells interact closely with other cell types. Murine embryoid bodies have been used as a model for the early development of a vascular network and are amenable to genetic manipulation and treatment with soluble modulators. However, quantifying morphological changes in these complex three-dimensional structures is challenging. In this paper we describe protocols to culture embryoid bodies on a large scale to study vascular development together with methods to quantify changes seen when antiangiogenic agents or endothelial cell-specific transgenes are introduced.

  12. 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. ©AlphaMed Press.

  13. 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.

  14. 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

  15. [Cytotoxic effects of etoposide at different stages of differentiation of embryoid bodies formed by mouse embryonic stem cells].

    PubMed

    2013-01-01

    The initial stages of in vitro differentiation of embryonic stem cells are considered as unique three-dimensional models of early development of mammals for basic, pharmacological, and toxicological studies. It has been previously shown (Gordeeva, 2012) that the assessment of embryotoxicity in the model of undifferentiated embryonic stem cells can be insufficiently accurate in predicting toxic effects on mammalian embryos. In view of this, we performed a comparative study of the damaging effects of the cytostatic etoposide in undifferentiated embryonic stem cells and embryoid bodiesof different stages of differentiation that have similar three-dimensional structures with early embryos. The analysis of growth, cell death, and dynamics of differentiation of embryonic stem cells and embryoid bodies exposed to etoposide showed that the cytostatic and cytotoxic effects of etoposide are stage-specific. The damaging effects of etoposide were maximum in the undifferentiated embryonic stem cells and decreased with growth and differentiation of embryoid bodies. We assume that the increase in the cell volume of embryoid bodies and the development of the hypertrophic we suggest that the increase of embryoid body volume and overgrowth of extraembryonic endoderm layer lead to a decrease in the diffusion, transport, and metabolism of chemical and bioactive substances and prevent the damaging effects.

  16. Development of an embryoid body-based screening strategy for assessing the hepatocyte differentiation potential of human embryonic stem cells following single-cell dissociation.

    PubMed

    Greenhough, Sebastian; Bradburn, Helen; Gardner, John; Hay, David C

    2013-02-01

    We have devised an embryoid body-based screening method for the selection of human embryonic stem cell (hESC) lines capable of forming functional hepatocyte-like cells (HLCs) after single-cell dissociation. The screening method highlighted one cell line from a panel of five that produced albumin-positive cells during embryoid body (EB) formation. Cell lines that did not produce albumin-positive cells during EB formation were shown to respond less well to directed differentiation following single-cell replating. Additionally, the seeding density of the pluripotent populations prior to differentiation was shown to exert a significant effect on the hepatic function of the final population of cells. In summary, we have developed a simple procedure that facilitates the identification of human hESC lines that tolerate single-cell replating and are capable of differentiating to HLCs. Although the hepatic function of cells produced by this method is ∼10-fold lower than our current gold standard stem cell-derived models, we believe that these findings represent an incremental step toward producing HLCs at scale.

  17. 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

  18. A PDMS-Based Microfluidic Hanging Drop Chip for Embryoid Body Formation.

    PubMed

    Wu, Huei-Wen; Hsiao, Yi-Hsing; Chen, Chih-Chen; Yet, Shaw-Fang; Hsu, Chia-Hsien

    2016-07-06

    The conventional hanging drop technique is the most widely used method for embryoid body (EB) formation. However, this method is labor intensive and limited by the difficulty in exchanging the medium. Here, we report a microfluidic chip-based approach for high-throughput formation of EBs. The device consists of microfluidic channels with 6 × 12 opening wells in PDMS supported by a glass substrate. The PDMS channels were fabricated by replicating polydimethyl-siloxane (PDMS) from SU-8 mold. The droplet formation in the chip was tested with different hydrostatic pressures to obtain optimal operation pressures for the wells with 1000 μm diameter openings. The droplets formed at the opening wells were used to culture mouse embryonic stem cells which could subsequently developed into EBs in the hanging droplets. This device also allows for medium exchange of the hanging droplets making it possible to perform immunochemistry staining and characterize EBs on chip.

  19. 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

  20. 3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body.

    PubMed

    Kim, Choong; Lee, Kang Sun; Bang, Jae Hoon; Kim, Young Eyn; Kim, Min-Cheol; Oh, Kwang Wook; Lee, Soo Hyun; Kang, Ji Yoon

    2011-03-07

    This paper proposes a microfluidic device for the on-chip differentiation of an embryoid body (EB) formed in a microwell via 3-dimensional cultures of mouse embryonic carcinoma (EC) cells. The device adjusted the size of the EB by fluid volume, differentiated the EB by chemical treatment, and evaluated its effects in EC cells by on-chip immunostaining. A microfluidic resistance network was designed to control the size of the embryoid body. The duration time and flow rate into each microwell regulated the initial number of trapped cells in order to adjust the size of the EB. The docked cells were aggregated and formed a spherical EB on the non-adherent surface of the culture chip for 3 days. The EC cells in the EB were then differentiated into diverse cell lineages without attachment for an additional 4 days; meanwhile, retinoic acid (RA) was applied without serum to direct the cells into early neuronal lineage. On-chip immunostaining of the EB in the microwell with a neuronal marker was conducted to assess the differentiation-inducing ability of RA. The effect of RA on neuronal differentiation was analyzed with confocal microscopic images of the TuJ1 marker. The RA-treated cells expressed more neuronal markers and appeared as mature neuronal cells with long neurites. The fluorescence intensity of the TuJ1 in the RA-treated EB was twice that observed in the non-treated EB on day 5. It was demonstrated that the pre-screening of inducing chemicals on the early neuronal differentiation of EC cells in a single microfluidic chip was indeed feasible. This chip is expected to constitute a useful tool for assessing the early differentiation of ES cells without attachment, and is also expected to prove useful as an anti-cancer drug test platform for the cytotoxicity assay with cellular spheroids.

  1. 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

  2. Meniscus induced self organization of multiple deep concave wells in a microchannel for embryoid bodies generation.

    PubMed

    Jeong, Gi Seok; Jun, Yesl; Song, Ji Hoon; Shin, Soo Hyun; Lee, Sang-Hoon

    2012-01-07

    Embryonic stem cells (ESCs) have attracted great interest in the fields of tissue engineering, regenerative medicine, and organogenesis for their pluripotency and ability to self-renew. ESC aggregation, which produces an embryoid body (EB), has been widely utilized as a trigger of in vitro directed differentiation. In this paper, we propose a novel method for constructing large numbers of deep concave wells in PDMS microfluidic chips using the meniscus induced by the surface tension of a liquid PDMS prepolymer, and applied this chip for the mass production of uniform sized EBs. To investigate if the microenvironment in the deep concave well is suitable for ES cells, the oxygen diffusion to the deep concave well was analyzed by CFD simulation. Murine EBs were successfully formed in the deep concave wells without loss of cells and laborious careful intervention to refresh culture media. The size of the EBs was uniform, and retrieving of EBs was done just by flipping over the chip. All the processes including EB formation and harvest are easy and safe to cells, and their viability after completion of all processes was over 95%. The basic properties of the EBs were generated and their capacity to differentiate into 3 germ layers was investigated by analyzing the gene expression profile. The harvested EBs were found to differentiate into cardiac cells and neurons, and neurofilaments formed branches of elongated extensions more than 1.0 mm in length.

  3. Osteogenic Embryoid Body-Derived Material Induces Bone Formation In Vivo

    PubMed Central

    Sutha, Ken; Schwartz, Zvi; Wang, Yun; Hyzy, Sharon; Boyan, Barbara D.; McDevitt, Todd C.

    2015-01-01

    The progressive loss of endogenous regenerative capacity that accompanies mammalian aging has been attributed at least in part to alterations in the extracellular matrix (ECM) composition of adult tissues. Thus, creation of a more regenerative microenvironment, analogous to embryonic morphogenesis, may be achieved via pluripotent embryonic stem cell (ESC) differentiation and derivation of devitalized materials as an alternative to decellularized adult tissues, such as demineralized bone matrix (DBM). Transplantation of devitalized ESC materials represents a novel approach to promote functional tissue regeneration and reduce the inherent batch-to-batch variability of allograft-derived materials. In this study, the osteoinductivity of embryoid body-derived material (EBM) was compared to DBM in a standard in vivo ectopic osteoinduction assay in nude mice. EBM derived from EBs differentiated for 10 days with osteogenic media (+β-glycerophosphate) exhibited similar osteoinductivity to active DBM (osteoinduction score = 2.50 ± 0.27 vs. 2.75 ± 0.16) based on histological scoring, and exceeded inactive DBM (1.13 ± 0.13, p < 0.005). Moreover, EBM stimulated formation of new bone, ossicles, and marrow spaces, similar to active DBM. The potent osteoinductivity of EBM demonstrates that morphogenic factors expressed by ESCs undergoing osteogenic differentiation yield a novel devitalized material capable of stimulating de novo bone formation in vivo. PMID:25961152

  4. 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.

  5. 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

  6. 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

  7. Molecular analysis of LEFTY-expressing cells in early human embryoid bodies.

    PubMed

    Dvash, Tamar; Sharon, Nadav; Yanuka, Ofra; Benvenisty, Nissim

    2007-02-01

    Human ESCs (HESCs) are self-renewing pluripotent cell lines that are derived from the inner cell mass of blastocyst-stage embryos. These cells can produce terminally differentiated cells representing the three embryonic germ layers. We thus hypothesized that during the course of in vitro differentiation of HESCs, progenitor-like cells are transiently formed. We demonstrated that LEFTY proteins, which are known to play a major role during mouse gastrulation, are transiently expressed during HESC differentiation. Moreover, LEFTY proteins seemed to be exclusively expressed by a certain population of cells in the early human embryoid bodies that does not overlap with the population expressing the ESC marker OCT4. We also showed that LEFTY expression is regulated at the cellular transcription level by molecular labeling of LEFTY-positive cells. A DNA microarray analysis of LEFTY-overexpressing cells revealed a signature of cell surface markers such as CADHERIN 2 and 11. Expression of LEFTY controlled by NODAL appears to have a substantial role in mesodermal origin cell population establishment, since inhibition of NODAL activity downregulated expression not only of LEFTY A and LEFTY B but also of BRACHYURY, an early mesodermal marker. In addition, other mesodermal lineage-related genes were downregulated, and this was accompanied by an upregulation in ectoderm-related genes. We propose that during the initial step of HESC differentiation, mesoderm progenitor-like cells appear via activation of the NODAL pathway. Our analysis suggests that in vitro differentiation of HESCs can model early events in human development.

  8. 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.

  9. Characterization of gastrulation-stage progenitor cells and their inhibitory crosstalk in human embryoid bodies.

    PubMed

    Kopper, Oded; Giladi, Odil; Golan-Lev, Tamar; Benvenisty, Nissim

    2010-01-01

    Human embryoid bodies (HEBs) are cell aggregates that are produced during the course of embryonic stem cell differentiation in suspension. Mature HEBs have been shown to contain derivatives of the three embryonic germ layers. In this study, using a combination of laser capture microscopy followed by DNA microarray analysis and cell sorting, we demonstrate that early HEBs are composed of three major cell populations. These cell populations can be defined by the expression of specific cell markers, namely: (i) OCT4(+), REX1(-); (ii) NCAD(+), OCT4(-); and (iii) EPOR(+), OCT4(-). By analyzing gene expression in embryonic tissues, these cell populations could respectively be assigned to the embryonic ectoderm, mesendoderm, and extraembryonic endoderm lineages. We show that the extraembryonic endoderm, which selectively expresses platelet-derived growth factor B (PDGF-B), negatively affects the mesendoderm lineage, which selectively expresses the receptor PDGFRA. Our analysis suggests that early HEBs are spatially patterned and that cell differentiation is governed by interactions between the different cell types.

  10. 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.

  11. Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

    PubMed

    Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

    2016-12-01

    Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

  12. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.

    PubMed

    Zhou, Jin; Zhang, Ye; Lin, Qiuxia; Liu, Zhiqiang; Wang, Haibin; Duan, Cuimi; Wang, Yanmeng; Hao, Tong; Wu, Kuiwu; Wang, Changyong

    2010-07-01

    Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

  14. 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.

  15. Embryoid body morphology influences diffusive transport of inductive biochemicals: a strategy for stem cell differentiation.

    PubMed

    Sachlos, Eleftherios; Auguste, Debra T

    2008-12-01

    Differentiation of human embryonic stem (hES) cells into cells for regenerative medicine is often initiated by embryoid body (EB) formation. EBs may be treated with soluble biochemicals such as cytokines, growth factors and vitamins to induce differentiation. A scanning electron microscopy analysis, conducted over 14 days, revealed time-dependent changes in EB structure which led to the formation of a shell that significantly reduced the diffusive transport of a model molecule (374 Da) by >80%. We found that the shell consists of 1) an extracellular matrix (ECM) comprised of collagen type I; 2) a squamous cellular layer with tight cell-cell adhesions associated with E-cadherin; and 3) a collagen type IV lining indicative of a basement membrane. Disruption of the basement membrane, by either inhibiting its formation with noggin or permeabilizing it with collagenase, resulted in recovery of diffusive transport. Increasing the diffusive transport of retinoic acid (RA) and serum in EBs by a 15-min collagenase digestion on days 4, 5, 6 and 7 promoted neuronal differentiation. Flow cytometry and quantitative RT-PCR analysis of collagenase-treated EBs revealed 68% of cells expressing neural cell adhesion molecule (NCAM) relative to 28% for untreated EBs. Our results suggest that limitations in diffusive transport of biochemicals need to be considered when formulating EB differentiation strategies.

  16. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells.

    PubMed

    Kurosawa, Hiroshi

    2007-05-01

    When cultured in suspension without antidifferentiation factors, embryonic stem (ES) cells spontaneously differentiate and form three-dimensional multicellular aggregates called embryoid bodies (EBs). EBs recapitulate many aspects of cell differentiation during early embryogenesis, and play an important role in the differentiation of ES cells into a variety of cell types in vitro. There are several methods for inducing the formation of EBs from ES cells. The three basic methods are liquid suspension culture in bacterial-grade dishes, culture in methylcellulose semisolid media, and culture in hanging drops. Recently, the methods using a round-bottomed 96-well plate and a conical tube are adopted for forming EBs from predetermined numbers of ES cells. For the production of large numbers of EBs, stirred-suspension culture using spinner flasks and bioreactors is performed. Each of these methods has its own peculiarity; thus, the features of formed EBs depending on the method used. Therefore, we should choose an appropriate method for EB formation according to the objective to be attained. In this review, we summarize the studies on in vitro differentiation of ES cells via EB formation and highlight the EB formation methods recently developed including the techniques, devices, and procedures involved.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. [Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

    PubMed

    Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

    2008-10-14

    To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

  2. Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors.

    PubMed

    Rungarunlert, Sasitorn; Techakumphu, Mongkol; Pirity, Melinda K; Dinnyes, Andras

    2009-12-31

    Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation. For standardized mass EB production, a well defined scale-up platform is necessary. Recently, novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems (spinner flasks), rotating cell culture system and rotary orbital culture have allowed large-scale EB formation. Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods. This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems. Furthermore, an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently, new insights in induced pluripotent stem (iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research. These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity. Hence, culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells, although direct evidence of their use for iPS cells is still limited.

  3. Paramagnetic Beads and Magnetically Mediated Strain Enhance Cardiomyogenesis in Mouse Embryoid Bodies

    PubMed Central

    Geuss, Laura R.; Wu, Douglas C.; Ramamoorthy, Divya; Alford, Corinne D.; Suggs, Laura J.

    2014-01-01

    Mechanical forces play an important role in proper embryologic development, and similarly such forces can directly impact pluripotency and differentiation of mouse embryonic stem cells (mESC) in vitro. In addition, manipulation of the embryoid body (EB) microenvironment, such as by incorporation of microspheres or microparticles, can similarly influence fate determination. In this study, we developed a mechanical stimulation regimen using permanent neodymium magnets to magnetically attract cells within an EB. Arginine-Glycine-Aspartic Acid (RGD)-conjugated paramagnetic beads were incorporated into the interior of the EBs during aggregation, allowing us to exert force on individual cells using short-term magnetization. EBs were stimulated for one hour at different magnetic field strengths, subsequently exerting a range of force intensity on the cells at different stages of early EB development. Our results demonstrated that following exposure to a 0.2 Tesla magnetic field, ESCs respond to magnetically mediated strain by activating Protein Kinase A (PKA) and increasing phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression. The timing of stimulation can also be tailored to guide ESC differentiation: the combination of bone morphogenetic protein 4 (BMP4) supplementation with one hour of magnetic attraction on Day 3 enhances cardiomyogenesis by increasing contractile activity and the percentage of sarcomeric α-actin-expressing cells compared to control samples with BMP4 alone. Interestingly, we also observed that the beads alone had some impact on differentiation by increasingly slightly, albeit not significantly, the percentage of cardiomyocytes. Together these results suggest that magnetically mediated strain can be used to enhance the percentage of mouse ESC-derived cardiomyocytes over current differentiation protocols. PMID:25501004

  4. 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.

  5. Intercellular cancer collisions generate an ejected crystal comet tail effect with fractal interface embryoid body reassembly transformation.

    PubMed

    Díaz, Jairo A; Murillo, Mauricio F; Barrero, Alvaro

    2011-01-01

    We have documented self-assembled geometric triangular chiral crystal complexes (GTCHC) and a framework of collagen vascular invariant geometric attractors in cancer tissues. This article shows how this system evolves in time. These structures are incorporated together and evolve in different ways. When the geometric core is stable, and the tissue architecture collapses, fragmented components emerge, which reveal a hidden interior identifying how each molecule is reassembled into the original mold, using one common connection, ie, a fractal self-similarity that guided the system from the beginning. GTCHC complexes generate ejected crystal comet tail effects and produce strange helicity states that arise in the form of spin domain interactions. As the crystal growth vibration stage progresses, biofractal echo images converge in a master-built construction of embryoid bodies with enolase-selective immunopositivity in relation to clusters of triangular chiral cell organization. In our electro-optic collision model, we were able to predict and replicate all the characteristics of this complex geometry that connects a physical phenomenon with the signal patterns that generate biologic chaos. Intrinsically, fractal geometry makes spatial correction errors embrace the chaotic system in a way that permits new structures to emerge, and as a result, an ordered self-assembly of embryoid bodies with neural differentiation at the final stage of cancer development is a predictable process. We hope that further investigation of these structures will lead not only to a new way of thinking about physics and biology, but also to a rewarding area in cancer research.

  6. 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.

  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. Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

    PubMed

    Novosadova, E V; Manuilova, E S; Arsen'eva, E L; Khaidarova, N V; Dolotov, O V; Inozemtseva, L S; Kozachenkov, K Yu; Tarantul, V Z; Grivennikov, I A

    2005-07-01

    The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

  9. 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.

  10. Differentiation of mouse iPS cells is dependent on embryoid body size in microwell chip culture.

    PubMed

    Miyamoto, Daisuke; Nakazawa, Kohji

    2016-10-01

    A microwell chip possessing microwells of several hundred micrometers is a promising platform for generating embryoid bodies (EBs) of stem cells. Here, we investigated the effects of initial EB size on the growth and differentiation of mouse iPS cells in microwell chip culture. We fabricated a chip that contained 195 microwells in a triangular arrangement at a diameter of 600 μm. To evaluate the effect of EB size, four similar conditions were designed with different seeding cell densities of 100, 500, 1000, and 2000 cells/EB. The cells in each microwell gradually aggregated and then spontaneously formed a single EB within 1 d of culture, and EB size increased with further cell proliferation. EB growth was regulated by the initial EB size, and the growth ability of smaller EBs was higher than that of larger EBs. Furthermore, stem cell differentiation also depended on the initial EB size, and the EBs at more than 500 cells/EB promoted hepatic and cardiac differentiations, but the EBs at 100 cells/EB preferred vascular differentiation. These results indicated that the initial EB size was one of the important factors controlling the proliferation and differentiation of stem cells in the microwell chip culture. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. The Human Embryoid Body Cystic Core Exhibits Architectural Complexity Revealed by use of High Throughput Polymer Microarrays.

    PubMed

    Tomov, Martin L; Olmsted, Zachary T; Paluh, Janet L

    2015-07-01

    In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 μm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 μm EBs are monocystic versus 500 μm multicystic EBs that contain macro, meso and microsized cysts. In adherent differentiation of 500 μm EBs, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. 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.

  13. Nonlinear 3D Projection Printing of Concave Hydrogel Microstructures for Long-Term Multicellular Spheroid and Embryoid Body Culture

    PubMed Central

    Hribar, K.C; Finlay, D.; Ma, X.; Qu, X.; Ondeck, M. G.; Chung, P. H.; Zanella, F.; Engler, A. J.; Sheikh, F.; Vuori, K.; Chen, S.

    2015-01-01

    Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propogation. Here, we used a continuous 3D projection printing approach – with an important modification of nonlinear exposure — to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs. PMID:25900329

  14. 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.

  15. 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.

  16. Assessment of differentiation aspects by the morphological classification of embryoid bodies derived from human embryonic stem cells.

    PubMed

    Kim, Jung Mo; Moon, Sung-Hwan; Lee, Sung Geum; Cho, Youn Jeong; Hong, Ki Sung; Lee, Jae Ho; Lee, Hey Jin; Chung, Hyung-Min

    2011-11-01

    In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; ∼10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.

  17. In vitro direct osteogenesis of murine embryonic stem cells without embryoid body formation.

    PubMed

    Hwang, Yu-Shik; Polak, Julia M; Mantalaris, Athanasios

    2008-10-01

    Embryonic stem cells (ESCs) posses the ability to self-renew and differentiate into a multitude of lineages, including the osteogenic lineage in vitro. Currently, most approaches have focused on embryonic body (EB)-mediated osteogenic differentiation, which relies on formation of all three germ layers resulting in limited yields and labour-intensive culture processes. Our study aimed at developing an efficient culture strategy resulting in the upregulated in vitro osteogenic differentiation of murine ESCs (mESCs), which completely avoided EB formation. Specifically, mESCs were cultured in HepG2 conditioned medium for 3 days and then directed into osteogenic differentiation for 21 days without prior EB formation. The mineralised bone nodules generated were characterized by Alizarin red S-staining, phenotypic alkaline phosphatase expression, time-course analysis of ALPase activity, the presence of type I collagen and osteopontin, and osteocalcin, cbfa-1/runx-2, and osterix gene expression. Our method of direct osteogenic differentiation of mESCs represents a novel and efficient approach that results in enhanced yields and could have significant applications in bone tissue engineering.

  18. Neural Commitment of Embryonic Stem Cells through the Formation of Embryoid Bodies (EBs)

    PubMed Central

    LIYANG, Gao; ABDULLAH, Syahril; ROSLI, Rozita; NORDIN, Norshariza

    2014-01-01

    An embryonic stem cell (ESC) is a good tool to generate neurons in vitro and can be used to mimic neural development in vivo. It has been widely used in research to examine the role of cell signalling during neuronal development, test the effects of drugs on neurons, and generate a large population of functional neurons. So far, a number of protocols have been established to promote the differentiation of ESCs, such as direct and indirect differentiation. One of the widely used protocols to generate neurons is through the spontaneous formation of multicellular aggregates known as embryonic bodies (EBs). However, for some, it is not clear why EB protocol could be the protocol of choice. EB also is known to mimic an early embryo; hence, knowing the similarities between EB and an early embryo is essential, particularly the information on the players that promote the formation of EBs or the aggregation of ESCs. This review paper focuses on these issues and discusses further the generation of neural cells from EBs using a well-known protocol, the 4−/4+ protocol. PMID:25977628

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Herpes simplex virus-mediated expression of Pax3 and MyoD in embryoid bodies results in lineage-Related alterations in gene expression profiles.

    PubMed

    Craft, April M; Krisky, David M; Wechuck, James B; Lobenhofer, Edward K; Jiang, Ying; Wolfe, Darren P; Glorioso, Joseph C

    2008-12-01

    The ability of embryonic stem cells to develop into multiple cell lineages provides a powerful resource for tissue repair and regeneration. Gene transfer offers a means to dissect the complex events in lineage determination but is limited by current delivery systems. We designed a high-efficiency replication-defective herpes simplex virus gene transfer vector (JDbetabeta) for robust and transient expression of the transcription factors Pax3 and MyoD, which are known to be involved in skeletal muscle differentiation. JDbetabeta-mediated expression of each gene in day 4 embryoid bodies (early-stage mesoderm) resulted in the induction of unique alterations in gene expression profiles, including the upregulation of known target genes relevant to muscle and neural crest development, whereas a control enhanced green fluorescent protein expression vector was relatively inert. This vector delivery system holds great promise for the use of gene transfer to analyze the impact of specific genes on both regulatory genetic events and commitment of stem cells to particular lineages.

  8. 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.

  9. Retinoic acid influences the embryoid body formation in mouse embryonic stem cells by induction of caspase and p38 MAPK/JNK-mediated apoptosis.

    PubMed

    Huang, Fu-Jen; Lan, Kuo-Chung; Kang, Hong-Yo; Lin, Pin-Yao; Chan, Wen-Hsiung; Hsu, Yu-Cheng; Liu, Yen-Chih; Huang, Ko-En

    2013-04-01

    Although all-trans retinoic acid (RA), the oxidative metabolite of vitamin A, is essential for normal development, high levels are teratogenic in many species. RA results in immediate effects on the preimplantation embryo and on blastocyst development in vitro and in vivo. To further elucidate the cellular mechanisms of early postimplantation embryo development induced by RA, we present an embryonic cell line, B5, as a candidate system for the investigation of these processes. We used undifferentiated ES cells as the model, which is from the undifferentiated status to differentiated status [embryoid body (EB) formation] mimicking postimplantation embryo development (egg-cylinder stage of embryo formation) to clarify the cellular mechanism of action of RA in the implanted blastocysts and cell apoptosis following the series of exposures to differing RA concentrations. Using an in vitro model, we identified the impact of RA on undifferentiated embryonic stem (ES) cells, including inhibition of cell proliferation and induction of cell apoptosis. JNK, P-38 and caspase activation were shown in the nature of RA-triggered apoptotic signaling in ES cells. The carry-on influences of RA on the ES cell were shown in the formation of EB from the pretreated ES cells. RA resulted in apparent impact on undifferentiated ES cells in vitro, with increased numbers of apoptotic cells initially and inhibited cell proliferation, which led to decreased size of EB. The process of EB formation (mimicking the early postimplantation embryo development) is regulated by RA-induced apoptosis through the activation of caspase and P38 MAPK/JNK pathway. Copyright © 2011 Wiley Periodicals, Inc.

  10. 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

    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.

  11. 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

  12. Concentration-dependent effects of spermine on apoptosis and consequent generation of multilayer myotube sheets from mouse embryoid bodies in vitro.

    PubMed

    Saito, Mikako; Abe, Natsuki; Ishida, Ayano; Nakagawa, Shota; Matsuoka, Hideaki

    2014-12-01

    The concentration-dependent effect of spermine was investigated on the spermine-induced generation of multilayer myotube sheets (MMTS) from mouse embryoid bodies (EBs). During spermine treatment for 24 h, a monolayer cell sheet that had already grown radially from the periphery of an EB was exfoliated. The exfoliation was inhibited by z-VAD.fmk, indicating the occurrence of apoptosis, and inhibited also by aminoguanidine, indicating the involvement of amine oxidase. Following the exfoliation, the cell growth restarted from the fresh periphery of EB in a spermine-free medium and finally formed MMTS. To analyze the contribution of apoptosis to the cell death causing exfoliation, the numbers of apoptotic, necrotic, and 2nd apoptotic cells were counted by staining with Annexin V-Cyanine-3 (AVC3) and 7-aminoactinomycin (7AAC). AVC3-positive, 7AAC-positive, and AVC3/7AAC doubly positive cells were assigned as apoptotic, necrotic, and 2nd necrotic cells, respectively. The relative number of apoptotic and 2nd necrotic cells (N A + N A/7) to the total number of dying cells (N T) was 84 ∼ 94%, which was independent of spermine concentration in the range from 0.1 to 2.0 mM. The MMTS generation rate at the final stage, however, was dependent on the spermine concentration. It was 60 ∼ 80% in the range from 0.1 to 1.5 mM, while it decreased sharply to 1% at 2 mM. This suggests another role of spermine in the MMTS generation in addition to the induction of apoptosis. This 2nd role seems to be inhibited at a spermine concentration higher than a critical limit between 1.5 and 2.0 mM.

  13. 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

  14. Epigenetic regulation of histone modifications and Gata6 gene expression induced by maternal diet in mouse embryoid bodies in a model of developmental programming.

    PubMed

    Sun, Congshan; Denisenko, Oleg; Sheth, Bhavwanti; Cox, Andy; Lucas, Emma S; Smyth, Neil R; Fleming, Tom P

    2015-01-21

    Dietary interventions during pregnancy alter offspring fitness. We have shown mouse maternal low protein diet fed exclusively for the preimplantation period (Emb-LPD) before return to normal protein diet (NPD) for the rest of gestation, is sufficient to cause adult offspring cardiovascular and metabolic disease. Moreover, Emb-LPD blastocysts sense altered nutrition within the uterus and activate compensatory cellular responses including stimulated endocytosis within extra-embryonic trophectoderm and primitive endoderm (PE) lineages to protect fetal growth rate. However, these responses associate with later disease. Here, we investigate epigenetic mechanisms underlying nutritional programming of PE that may contribute to its altered phenotype, stabilised during subsequent development. We use embryonic stem (ES) cell lines established previously from Emb-LPD and NPD blastocysts that were differentiated into embryoid bodies (EBs) with outer PE-like layer. Emb-LPD EBs grow to a larger size than NPD EBs and express reduced Gata6 transcription factor (regulator of PE differentiation) at mRNA and protein levels, similar to Emb-LPD PE derivative visceral yolk sac tissue in vivo in later gestation. We analysed histone modifications at the Gata6 promoter in Emb-LPD EBs using chromatin immunoprecipitation assay. We found significant reduction in histone H3 and H4 acetylation and RNA polymerase II binding compared with NPD EBs, all markers of reduced transcription. Other histone modifications, H3K4Me2, H3K9Me3 and H3K27Me3, were unaltered. A similar but generally non-significant histone modification pattern was found on the Gata4 promoter. Consistent with these changes, histone deacetylase Hdac-1, but not Hdac-3, gene expression was upregulated in Emb-LPD EBs. First, these data demonstrate ES cells and EBs retain and propagate nutritional programming adaptations in vitro, suitable for molecular analysis of mechanisms, reducing animal use. Second, they reveal maternal diet

  15. 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

  16. PlGF Knockdown Decreases Tumorigenicity and Stemness Properties of Spheroid Body Cells Derived from Gastric Cancer Cells.

    PubMed

    Mahmoodi, Fatemeh; Akrami, Hassan

    2017-04-01

    Placental growth factor (PlGF) a member of the vascular endothelial growth factor family regulates some cell processes such as survival, growth of vascular endothelial cells, invasiveness, and also involves in pathological angiogenesis and metastasis in most cancers. Cancer stem cells are believed to be the main reason for the tumor relapse and resistance to therapy. These cells have various characteristics as same as normal tissue-specific adult stem cells including self-renewability and potent to differentiate into various cell types. However, the function of PlGF in gastric cancer stem cells is not well understood. We have investigated the effect of PlGF knockdown on the tumorigenicity and stem cell properties of spheroid body cells derived from two human gastric cancer cell lines. In this study, we isolated spheroid body cells which have stemness properties from MKN-45 and AGS without using growth factors. Validation of spheroid body cells was confirmed by various methods. Then the effects of PlGF knockdown were investigated on in vitro tumorigenicity, differentiation, migration, angiogenesis, and transcription levels of stemness markers of spheroid body cells. Our findings indicated that isolation of spheroid body cells from MKN-45 and AGS cells without using growth factors is an easy and inexpensive method to isolate cancer stem cells and knockdown of PlGF in spheroid body cells reduced in vitro tumorigenicity and stemness properties of spheroid body cells such as Self-renewal ability, colony forming, migratory, and MMPs activities and decreased ability to differentiation and angiogenesis. J. Cell. Biochem. 118: 851-859, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells.

    PubMed

    Fu, Qiuli; Qin, Zhenwei; Jin, Xiuming; Zhang, Lifang; Chen, Zhijian; He, Jiliang; Ji, Junfeng; Yao, Ke

    2017-01-01

    The pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells. Isolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter "X," respectively. We developed a "fried egg" differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers αA-, αB-, β-, and γ-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, αA-, and αB-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.7×) of the LBs generated from UiPSCs. Our study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.

  18. 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.

  19. Musashi1 and hairy and enhancer of split 1 high expression cells derived from embryonic stem cells enhance the repair of small-intestinal injury in the mouse.

    PubMed

    Yu, Tao; Lan, Shao-Yang; Wu, Bin; Pan, Qiu-Hui; Shi, Liu; Huang, Kai-Hong; Lin, Ying; Chen, Qi-Kui

    2011-05-01

    Embryonic stem cells have great plasticity. In this study, we repaired impaired small intestine by transplanting putative intestinal epithelial stem cells (Musashi1 and hairy and enhancer of split 1 high expression cells) derived from embryonic stem cells. The differentiation of definitive endoderm in embryoid bodies, derived from male ES-E14TG2a cells by the hanging-drop method, was monitored to define a time point for maximal induction of putative intestinal epithelial stem cells by epidermal growth factor. Furthermore, to evaluate the regenerative potential of intestinal epithelium, these putative stem cells were engrafted into NOD/SCID mice and female mice with enteritis. Donor cells were located by SRY DNA in situ hybridization. The results revealed that definitive endodermal markers were highly expressed in 5-day embryoid bodies. These embryoid body cells were induced into putative intestinal epithelial stem cells on the 5th day of epidermal growth factor administration. Grafts from these cells consisted of adenoid structures and nonspecific structural cells with strong expression of small-intestinal epithelial cell markers. In situ hybridization revealed that the donor cells could specifically locate in damaged intestinal epithelium, contribute to epithelial structures, and enhance regeneration. In conclusion, the Musashi1 and hairy and enhancer of split 1 high expression cells, derived from mouse embryonic stem cells, locate predominantly in impaired small-intestinal epithelium after transplantation and contribute to epithelial regeneration.

  20. 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

  1. 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.

  2. 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.

  3. Static magnetic fields increase cardiomyocyte differentiation of Flk-1+ cells derived from mouse embryonic stem cells via Ca2+ influx and ROS production.

    PubMed

    Bekhite, Mohamed M; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2013-08-10

    To investigate the effects of static magnetic fields (MFs) on cardiomyogenesis of mouse embryonic stem (ES) cell-derived embryoid bodies and Flk-1(+) cardiac progenitor cells and to assess the impact of cytosolic calcium [Ca(2+)]c and reactive oxygen species (ROS). Embryoid bodies and ES cell-derived Flk-1(+) cardiovascular progenitor cells were exposed to static MFs. The expression of cardiac genes was evaluated by RT-PCR; sarcomeric structures were assessed by immunohistochemistry; intracellular ROS and [Ca(2+)]c of ES cells were examined by H2DCF-DA- and fluo-4-based microfluorometry. Treatment of embryoid bodies with MFs dose-dependent increased the number of contracting foci and cardiac areas as well as mRNA expression of the cardiac genes MLC2a, MLC2v, α-MHC and β-MHC. In Flk-1(+) cells MFs (1 mT) elevated both [Ca(2+)]c and ROS, increased expression of the cardiogenic transcription factors Nkx-2.5 and GATA-4 as well as cardiac genes. This effect was due to Ca(2+) influx, since extracellular Ca(2+) chelation abrogated ROS production and MF-induced cardiomyogenesis. Furthermore absence of extracellular calcium impaired sarcomere structures. Neither the phospholipase C inhibitor U73122 nor thapsigargin inhibited MF-induced increase in [Ca(2+)]c excluding involvement of intracellular calcium stores. ROS were generated through NAD(P)H oxidase, since NOX-4 but not NOX-1 and NOX-2 mRNA was upregulated upon MF exposure. Ablation of NOX-4 by sh-RNA and treatment with the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI) totally abolished MF-induced cardiomyogenesis. The ability of static MFs to enhance cardiomyocyte differentiation of ES cells allows high throughput generation of cardiomyocytes without pharmacological or genetic modification. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  4. 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

  5. [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.

  6. 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.

  7. Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine.

    PubMed

    Hodge, Alexander J; Zhong, Juming; Lipke, Elizabeth A

    2016-04-01

    The development of cell-based treatments for heart disease relies on the creation of functionally mature stem cell-derived cardiomyocytes employing in vitro culture suspension systems, a process which remains a formidable and expensive endeavor. The use of nitric oxide as a signaling molecule during differentiation has demonstrated the potential for creating increased numbers of spontaneously contracting embryoid bodies in culture; however, the effects of nitric oxide signaling on the function and maturation of stem cell-derived cardiomyocytes is not well understood. In this study, the effects of nitric oxide on mouse embryonic stem cell-derived cardiomyocyte contractile activity, protein, and gene expression, and calcium handling were quantified. Embryoid bodies (EBs) formed using the hanging drop method, were treated with the soluble nitric oxide donor S-nitrosocysteine (CysNO) over a period of 18 days in suspension culture and spontaneous contractile activity was assessed. On day 8, selected EBs were dissociated to form monolayers for electrophysiological characterization using calcium transient mapping. Nitric oxide treatment led to increased numbers of stem cell-derived cardiomyocytes (SC-CMs) relative to non-treated EBs after 8 days in suspension culture. Increased incidence of spontaneous contraction and frequency of contraction were observed from days 8-14 in EBs receiving nitric oxide treatment in comparison to control. Expression of cardiac markers and functional proteins was visualized using immunocytochemistry and gene expression was assessed using qPCR. Cardiac-specific proteins were present in both CysNO-treated and control SC-CMs; however, CysNO treatment during differentiation significantly increased βMHC gene expression in SC-CMs relative to control SC-CMs. Furthermore, increased calcium transient velocity and decreased calcium transient duration was observed for CysNO-treated SC-CMs in comparison to control SC-CMs. Soluble nitric oxide donors

  8. Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation.

    PubMed

    Zhang, Peng; Wu, Xinglong; Hu, Chunchao; Wang, Pengbo; Li, Xiangyun

    2012-01-01

    Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.

  9. β-globin-expressing definitive erythroid progenitor cells generated from embryonic and induced pluripotent stem cell-derived sacs

    PubMed Central

    Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J.; Winkler, Thomas; Tisdale, John

    2016-01-01

    Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ε-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ~120 erythroid cells per single ES cell. Both primitive (ε-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of GPA or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. PMID:26866725

  10. 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.

  11. 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

  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. Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

    PubMed Central

    Lamas, Nuno Jorge; Johnson-Kerner, Bethany; Roybon, Laurent; Kim, Yoon A.; Garcia-Diaz, Alejandro; Wichterle, Hynek; Henderson, Christopher E.

    2014-01-01

    Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC50 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening. PMID:25337699

  14. 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

  15. 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.

  16. Efficient Generation of Human Embryonic Stem Cell-Derived Cardiac Progenitors Based on Tissue-Specific Enhanced Green Fluorescence Protein Expression

    PubMed Central

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

    2015-01-01

    Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFPhigh rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFPhigh rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFPhigh rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications. PMID:24734786

  17. 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.

  18. 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

  19. [Generation of high proliferative potential hematopoietic progenitor cells from embryonic stem cell-derived BL-CFC].

    PubMed

    Yao, Hui-Yu; Liu, Bing; Yuan, Ye; Mao, Ning

    2003-08-01

    The blast colony-forming cells (BL-CFC), which are detected within embryoid bodies derived from embryonic stem cells (ES cells) differentiated for 2.5-3.5 days, have dual-potential of differentiation to hematopoietic and endothelial cells. In this investigation the culture method of BL-CFC was established and colony forming assay, immunofluorescent technique as well as nested RT-PCR was employed to identify the differentiation capacity of adherent and nonadherent cells derived from individual blast colony. The results showed that the adherent cells could intake DiI-Ac-LDL and expressed the endothelium-specific surface markers including CD31, UEA-I and VE-cadherin. In addition, nonadherent cells were capable of developing primitive or/and definitive hematopoiesis potential. High proliferative potential colony-forming cells (HPP-CFC) bearing self-renewal capacity was found in 20% of BL-CFC. It is concluded that BL-CFC derived from embryonic stem cells can generate high proliferative potential hematopoietic progenitor cells. However, the whether BL-CFC can reconstitute the adult bone marrow hematopoiesis in long-term remains to be further determined.

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

    PubMed Central

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

    2013-01-01

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

  1. Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

    PubMed Central

    Matsuura, Katsuhisa; Wada, Masanori; Konishi, Kanako; Sato, Michi; Iwamoto, Ushio; Sato, Yuko; Tachibana, Aki; Kikuchi, Tetsutaro; Iwamiya, Takahiro; Shimizu, Tatsuya; Yamashita, Jun K.; Yamato, Masayuki; Hagiwara, Nobuhisa; Okano, Teruo

    2012-01-01

    Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×108 cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×108. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue. PMID:23284924

  2. Fabrication of mouse embryonic stem cell-derived layered cardiac cell sheets using a bioreactor culture system.

    PubMed

    Matsuura, Katsuhisa; Wada, Masanori; Konishi, Kanako; Sato, Michi; Iwamoto, Ushio; Sato, Yuko; Tachibana, Aki; Kikuchi, Tetsutaro; Iwamiya, Takahiro; Shimizu, Tatsuya; Yamashita, Jun K; Yamato, Masayuki; Hagiwara, Nobuhisa; Okano, Teruo

    2012-01-01

    Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1 × 10(8) cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5 × 10(8). Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.

  3. 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. © 2014 AlphaMed Press.

  4. 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.

  5. 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.

  6. [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.

  7. 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.

  8. 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.

  9. Phenotype-dependent role of the L-type calcium current in embryonic stem cell derived cardiomyocytes

    PubMed Central

    Dan, Pauline; Zeng, Zheng; Li, Ying; Qu, Yang; Hove-Madsen, Leif; Tibbits, Glen F

    2014-01-01

    Although the L-type Ca2+ current (ICa,L) plays an important role in cardiac contractility and pacemaking, its role in embryonic stem-cell derived cardiomyocytes (ESC-CMs) has not yet been explored in detail. We used patch-clamp techniques to characterize ICa,L, action potential properties, and nifedipine (an ICa,L blocker) sensitivity on spontaneously contracting embryoid bodies (EBs) or isolated ESC-CMs. Cellular preparations exhibited differential sensitivity to nifedipine, with substantial variation in the dose required to abolish automaticity. Isolated ESC-CMs expressing nodal-like action potentials were highly sensitive to nifedipine; 1 nM significantly decreased firing rate, diastolic depolarization rate (DDR), and upstroke velocity, and 10 nM completely abolished spontaneous activity. In contrast, ESC-CMs expressing atrial-like action potentials were relatively nifedipine-resistant, requiring 10 μM to arrest automaticity; 1 μM significantly decreased upstroke velocity while the firing rate and DDR were unaffected. Nodal-like cells exhibited a more negative voltage for half-maximal ICa activation (-30 ± 1 mV vs. -20 ± 3 mV; p<0.05) and slower inactivation (71 ± 10 ms vs. 43 ± 3 ms; p<0.05) than atrial-like cells. Our data indicate that ICa,L differentially regulates automaticity and chronotropy in nodal-like ESC-CMs, and primarily links excitation to contraction in atrial-like ESC-CMs by contributing to the upstroke phase of the action potential. PMID:24660113

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr).

    PubMed

    Doss, Michael Xavier; Di Diego, José M; Goodrow, Robert J; Wu, Yuesheng; Cordeiro, Jonathan M; Nesterenko, Vladislav V; Barajas-Martínez, Héctor; Hu, Dan; Urrutia, Janire; Desai, Mayurika; Treat, Jacqueline A; Sachinidis, Agapios; Antzelevitch, Charles

    2012-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2), at concentrations that selectively block I(K1) (50-100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied, but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells, but robust expression of I(Kr.) In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the

  1. Identification and characterization of a transient outward K+ current in human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Cordeiro, Jonathan M.; Nesterenko, Vladislav V.; Sicouri, Serge; Goodrow, Robert J.; Treat, Jacqueline A.; Desai, Mayurika; Wu, Yuesheng; Doss, Michael Xavier; Antzelevitch, Charles; Di Diego, José M.

    2013-01-01

    Background The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4, KChIP2 and Kv4.3 were quantified from BCs. Results BCs exhibited spontaneous beating (60.5 ± 2.6 bpm) and maximum-diastolic-potential (MDP) of 67.8 ± 0.8 mV (n = 155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7 ± 1.9 pA/pF at +40 mV; n = 14). Recovery of Ito from inactivation (at −80 mV) showed slow kinetics (τ1 = 200 ± 110 ms (12%) and τ2 = 2380 ± 240 ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype. PMID:23542310

  2. Delivery of definable number of drug or growth factor loaded poly(DL-lactic acid-co-glycolic acid) microparticles within human embryonic stem cell derived aggregates.

    PubMed

    Qutachi, Omar; Shakesheff, Kevin M; Buttery, Lee D K

    2013-05-28

    Embryoid bodies (EBs) generated from embryonic stem cells are used to study processes of differentiation within a three dimensional (3D) cell environment. In many instances however, EBs are dispersed to single cell suspensions with a subsequent monolayer culture. Moreover, where the 3D integrity of an EB is maintained, cytokines or drugs of interest to stimulate differentiation are often added directly to the culture medium at fixed concentrations and effects are usually limited to the outer layers of the EB. The aim of this study was to create an EB model with localised drug and or growth factor delivery directly within the EB. Using poly(DL-lactic acid-co-glycolic acid) microparticles (MPs) with an average diameter of 13μm, we have demonstrated controllable incorporation of defined numbers of MPs within human ES cell derived EBs, down to 1 MP per EB. This was achieved by coating MPs with human ES cell lysate and centrifugation of specific ratios of ES cells and MPs to form 3D aggregates. Using MPs loaded with simvastatin (pro or active drug) or BMP-2, we have demonstrated osteogenic differentiation within the 3D aggregates, maintained in culture for up to 21days, and quantified by real time QPCR for osteocalcin. Immunostaining for RUNX2 and osteocalcin, and also histochemical staining with picrosirius red to demonstrate collage type 1 and Alizarin red to demonstrate calcium/mineralisation further demonstrated osteogenic differentiation and revealed regional staining associated with the locations of MPs within the aggregates. We also demonstrated endothelial differentiation within human ES cell-derived aggregates using VEGF loaded MPs. In conclusion, we demonstrate an effective and reliable approach for engineering stem aggregates with definable number of MPs within the 3D cellular structure. We also achieved localised osteogenic and endothelial differentiation associated with MPs releasing encapsulated drug molecules or cytokines directly within the cell

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Cell-derived microparticles and the lung.

    PubMed

    Nieri, Dario; Neri, Tommaso; Petrini, Silvia; Vagaggini, Barbara; Paggiaro, Pierluigi; Celi, Alessandro

    2016-09-01

    Cell-derived microparticles are small (0.1-1 μm) vesicles shed by most eukaryotic cells upon activation or during apoptosis. Microparticles carry on their surface, and enclose within their cytoplasm, molecules derived from the parental cell, including proteins, DNA, RNA, microRNA and phospholipids. Microparticles are now considered functional units that represent a disseminated storage pool of bioactive effectors and participate both in the maintenance of homeostasis and in the pathogenesis of diseases. The mechanisms involved in microparticle generation include intracellular calcium mobilisation, cytoskeleton rearrangement, kinase phosphorylation and activation of the nuclear factor-κB. The role of microparticles in blood coagulation and inflammation, including airway inflammation, is well established in in vitro and animal models. The role of microparticles in human pulmonary diseases, both as pathogenic determinants and biomarkers, is being actively investigated. Microparticles of endothelial origin, suggestive of apoptosis, have been demonstrated in the peripheral blood of patients with emphysema, lending support to the hypothesis that endothelial dysfunction and apoptosis are involved in the pathogenesis of the disease and represent a link with cardiovascular comorbidities. Microparticles also have potential roles in patients with asthma, diffuse parenchymal lung disease, thromboembolism, lung cancer and pulmonary arterial hypertension. Copyright ©ERS 2016.

  12. Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

    PubMed Central

    Fridley, Krista M.; Nair, Rekha

    2014-01-01

    During development, cell fate specification and tissue development are orchestrated by the sequential presentation of soluble growth factors (GF) and extracellular matrix (ECM) molecules. Similarly, differentiation of stem cells in vitro relies upon the temporal presence of extracellular cues within the microenvironment. Hydrodynamic culture systems are not limited by volume restrictions and therefore offer several practical advantages for scalability over static cultures; however, hydrodynamic cultures expose cells to physical parameters not present in static culture, such as fluid shear stress and mass transfer through convective forces. In this study, the differences between static and hydrodynamic culture conditions on the expression of ECM and GF molecules during the differentiation of mouse embryonic stem cells were examined at both the gene and protein level. The expression of ECM and GF genes exhibited an early decrease in static cultures based on heat map and hierarchical clustering analysis and a relative delayed increase in hydrodynamic cultures. Although the temporal patterns of specific ECM and GF protein expression were comparable between static and hydrodynamic cultures, several notable differences in the magnitudes of expression were observed at similar time points. These results describe the establishment of an analytical framework that can be used to examine the expression patterns of ECM and GF molecules expressed by pluripotent stem cells undergoing differentiation as 3D multicellular aggregates under different culture conditions, and suggest that physical parameters of stem cell microenvironments can alter endogenous ECM and GF expression profiles that may, in turn, influence cell fate decisions. PMID:25423310

  13. 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.

  14. 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.

  15. 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.

  16. Single cell derived murine embryonic stem cell clones stably express Rex1-specific green fluorescent protein and their differentiation study

    SciTech Connect

    Chen Xiaopan; Wu Rongrong; Feng Shumei; Gu Bin; Dai Licheng; Zhang Ming; Zhao Xiaoli

    2007-10-19

    Embryonic stem cells (ESCs) often display high rates of apoptosis and spontaneous differentiation in routine culture, thus bring the proliferation of these cells highly inefficient. Moreover, little is known about the factors that are indispensable for sustaining self-renewal. To surmount these issues, we established transgenic mES cell lines expressing the enhanced green fluorescent protein (EGFP) under the control of the Rex1 promoter which is a key regulator of pluripotency in ES cells. In addition, we provided a simplified and improved protocol to derive transgenic mESCs from single cell. Finally, we showed that embryoid body (EB) development was faster than adherent differentiation in terms of differentiation ratio by real-time tracking of the EGFP expression. Therefore, these cell lines can be tracked and selected both in vitro and in vivo and should be invaluable for studying the factors that are indispensable for maintaining pluripotency.

  17. 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.

  18. Enrichment of Pluripotent Stem Cell-Derived Hepatocyte-Like Cells by Ammonia Treatment

    PubMed Central

    Tomotsune, Daihachiro; Hirashima, Kanji; Fujii, Masako; Yue, Fengming; Matsumoto, Ken; Takizawa-Shirasawa, Sakiko; Yokoyama, Tadayuki; Sasaki, Katsunori

    2016-01-01

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are potential resources for the regeneration of defective organs, including the liver. However, some obstacles must be overcome before this becomes reality. Undifferentiated cells that remain following differentiation have teratoma-forming potential. Additionally, practical applications require a large quantity of differentiated cells, so the differentiation process must be economical. Here we describe a DNA microarray-based global analysis of the gene expression profiles of differentiating human pluripotent stem cells. We identified differences and commonalities among six human pluripotent stem cell lines: the hESCs KhES1, KhES2, KhES3, and H1, and the iPSCs 201B7 and 243G1. Embryoid bodies (EBs) formed without requiring supplementation with inducing factors. EBs also expressed some liver-specific metabolic genes including the ammonia-metabolizing enzymes glutamine synthetase and carbamoyl-phosphate synthase 1. Real-time PCR analysis revealed hepatocyte-like differentiation of EBs treated with ammonia in Lanford medium. Analysis of DNA microarray data suggested that hepatocyte-like cells were the most abundant population in ammonia-treated cells. Furthermore, expression levels of undifferentiated pluripotent stem cell markers were drastically reduced, suggesting a reduced teratoma-forming capacity. These results indicate that treatment of EBs with ammonia in Lanford medium may be an effective inducer of hepatic differentiation in absence of expensive inducing factors. PMID:27632182

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. Immunological Properties of Murine Parthenogenetic Stem Cell-Derived Cardiomyocytes and Engineered Heart Muscle.

    PubMed

    Didié, Michael; Galla, Satish; Muppala, Vijayakumar; Dressel, Ralf; Zimmermann, Wolfram-Hubertus

    2017-01-01

    Pluripotent parthenogenetic stem cells (pSCs) can be derived by pharmacological activation of unfertilized oocytes. Homozygosity of the major histocompatibility complex (MHC) in pSCs makes them an attractive cell source for applications in allogeneic tissue repair. This was recently demonstrated for pSC-based tissue-engineered heart repair. A detailed analysis of immunological properties of pSC-derived cardiomyocytes and engineered heart muscle (EHM) thereof is, however, lacking. The aim of this study was to determine baseline and cytokine-inducible MHC class I and MHC class II as well as programmed death ligand-1 (PDL-1) and co-stimulatory protein (CD40, CD80, CD86) expression in pSC-derived cardiomyocytes and pSC-EHM in vitro and in vivo. Cardiomyocytes from an MHC-homologous (H2(d/d)) pSC-line were enriched to ~90% by making use of a recently developed cardiomyocyte-specific genetic selection protocol. MHC class I and MHC class II expression in cardiomyocytes could only be observed after stimulation with interferon gamma (IFN-γ). PDL-1 was markedly upregulated under IFN-γ. CD40, CD80, and CD86 were expressed at low levels and not upregulated by IFN-γ. EHM constructed from H2(d/d) cardiomyocytes expressed similarly low levels of MHC class I, MHC class II, and costimulatory molecules under basal conditions. However, in EHM only MHC class I, but not MHC class II, molecules were upregulated after IFN-γ-stimulation. We next employed a cocultivation system with MHC-matched and MHC-mismatched splenocytes and T-cells to analyze the immune stimulatory properties of EHMs. Despite MHC-mismatched conditions, EHM did not induce splenocyte or T-cell proliferation in vitro. To evaluate the immunogenicity of pSC-derived cardiomyocytes in vivo, we implanted pSC-derived embryoid bodies after elimination of non-cardiomyocytes (cardiac bodies) under the kidney capsules of MHC-matched and -mismatched mice. Spontaneous beating of cardiac bodies could be observed for 28 days in

  11. 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.

  12. 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.

  13. Red blood cell-derived microparticles: An overview.

    PubMed

    Westerman, Maxwell; Porter, John B

    2016-07-01

    The red blood cell (RBC) is historically the original parent cell of microparticles (MPs). In this overview, we describe the discovery and the early history of red cell-derived microparticles (RMPs) and present an overview of the evolution of RMP. We report the formation, characteristics, effects of RMP and factors which may affect RMP evaluation. The review examines RMP derived from both normal and pathologic RBC. The pathologic RBC studies include sickle cell anemia (SCA), sickle cell trait (STr), thalassemia intermedia (TI), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary stomatocytosis (HSt) and glucose-6-phosphate dehydrogenase deficiency (G6PD). Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Isolation and characterization of alveolar epithelial type II cells derived from mouse embryonic stem cells.

    PubMed

    Sun, Huanhuan; Quan, Yuan; Yan, Qing; Peng, Xinmiao; Mao, Zhengmei; Wetsel, Rick A; Wang, Dachun

    2014-06-01

    The use of embryonic stem cells (ESCs) to regenerate distal lung epithelia damaged by injuries or diseases requires development of safe and efficient methodologies that direct ESC differentiation into transplantable distal lung epithelial progenitors. Time-consuming culture procedure and low differentiation efficiency are major problems that are associated with conventional differentiation approaches via embryoid body formation. The use of a growth factor cocktail or a lung-specific cell-conditioned medium to enrich definitive endoderm for efficient differentiation of mouse ESCs (mESC) into alveolar epithelial progenitor type II cells (ATIICs) has been reported, but not yet successful for generating a homogenous population of ATIICs for tissue regeneration purpose, and it remains unclear whether or not those mESC-derived ATIICs possess normal biological functions. Here, we report a novel method using a genetically modified mESC line harboring an ATIIC-specific neomycin(R) transgene in Rosa 26 locus. We showed that ATIICs can be efficiently differentiated from mESCs as early as day 7 by culturing them directly on Matrigel-coated plates in DMEM containing 15% knockout serum replacement. With this culture condition, the genetically modified mESCs can be selectively differentiated into a homogenous population (>99%) of ATIICs. Importantly, the mESC-derived ATIICs (mESC-ATIICs) exhibited typical lamellar bodies and expressed surfactant protein A, B, and C as normal control ATIICs. When cultured with an air-liquid-interface culture system in Small Airway Epithelial Cell Growth Medium, the mESC-ATIICs can be induced to secrete surfactant proteins after being treated with dibutyryl cAMP+dexamethasone. These mESC-ATIICs can synthesize and secrete surfactant lipid in response to secretagogue, demonstrating active surfactant metabolism in mESC-ATIICs as that seen in normal control ATIICs. In addition, we demonstrated that the selected mESC-ATIICs can be maintained on Matrigel

  15. 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

  16. Equivalency of Buffalo (Bubalus Bubalis) Embryonic Stem Cells Derived From Fertilized, Parthenogenetic, and Hand-Made Cloned Embryos

    PubMed Central

    Muzaffar, Musharifa; Selokar, Naresh L.; Singh, Karn P.; Zandi, Mohammad; Singh, Manoj K.; Shah, Riaz A.; Chauhan, Manmohan S.; Singla, Suresh K.; Palta, Prabhat

    2012-01-01

    Abstract This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production. PMID:22582863

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

    PubMed

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

    2017-07-01

    In this review we aim to provide an overview of the most important ethical pros and cons of stem cell derived gametes (SCD-gametes), as a contribution to the debate about reproductive tissue engineering. Derivation of gametes from stem cells holds promising applications both for research and for clinical use in assisted reproduction. We explore the ethical issues connected to gametes derived from embryonic stem cells (both patient specific and non-patient specific) as well as those related to gametes derived from induced pluripotent stem cells. The technology of SCD-gametes raises moral concerns of how reproductive autonomy relates to issues of embryo destruction, safety, access, and applications beyond clinical infertility.

  18. 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

  19. iPS cell derived neuronal cells for drug discovery.

    PubMed

    Heilker, Ralf; Traub, Stefanie; Reinhardt, Peter; Schöler, Hans R; Sterneckert, Jared

    2014-10-01

    Owing to the inherent disconnect between drug pharmacology in heterologous cellular models and drug efficacy in vivo, the quest for more predictive in vitro systems is one of the most urgent challenges of modern drug discovery. An improved pharmacological in vitro profiling would employ primary samples of the proper drug-targeted human tissue or the bona fide human disease-relevant cells. With the advent of induced pluripotent stem (iPS) cell technology the facilitated access to a variety of disease-relevant target cells is now held out in prospect. In this review, we focus on the use of human iPS cell derived neurons for high throughput pharmaceutical drug screening, employing detection technologies that are sufficiently sensitive to measure signaling in cells with physiological target protein expression levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Cartilage Repair Using Human Embryonic Stem Cell-Derived Chondroprogenitors

    PubMed Central

    Kapacee, Zoher; Peng, Jiang; Lu, Shibi; Lucas, Robert J.; Hardingham, Timothy E.

    2014-01-01

    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. PMID:25273540

  2. Stem cell-derived systems in toxicology assessment.

    PubMed

    Suter-Dick, Laura; Alves, Paula M; Blaauboer, Bas J; Bremm, Klaus-Dieter; Brito, Catarina; Coecke, Sandra; Flick, Burkhard; Fowler, Paul; Hescheler, Jürgen; Ingelman-Sundberg, Magnus; Jennings, Paul; Kelm, Jens M; Manou, Irene; Mistry, Pratibha; Moretto, Angelo; Roth, Adrian; Stedman, Donald; van de Water, Bob; Beilmann, Mario

    2015-06-01

    Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and

  3. Stem cell-derived astrocytes: are they physiologically credible?

    PubMed

    Hill, Eric; Nagel, David; Parri, Rheinallt; Coleman, Michael

    2016-11-15

    Astrocytes are now increasingly acknowledged as having fundamental and sophisticated roles in brain function and dysfunction. Unravelling the complex mechanisms that underlie human brain astrocyte-neuron interactions is therefore an essential step on the way to understanding how the brain operates. Insights into astrocyte function to date have almost exclusively been derived from studies conducted using murine or rodent models. Whilst these have led to significant discoveries, preliminary work with human astrocytes has revealed a hitherto unknown range of astrocyte types with potentially greater functional complexity and increased neuronal interaction with respect to animal astrocytes. It is becoming apparent, therefore, that many important functions of astrocytes will only be discovered by direct physiological interrogation of human astrocytes. Recent advancements in the field of stem cell biology have provided a source of human-based models. These will provide a platform to facilitate our understanding of normal astrocyte functions as well as their role in CNS pathology. A number of recent studies have demonstrated that stem cell-derived astrocytes exhibit a range of properties, suggesting that they may be functionally equivalent to their in vivo counterparts. Further validation against in vivo models will ultimately confirm the future utility of these stem cell-based approaches in fulfilling the need for human-based cellular models for basic and clinical research. In this review we discuss the roles of astrocytes in the brain and highlight the extent to which human stem cell-derived astrocytes have demonstrated functional activities that are equivalent to those observed in vivo. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  4. 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.

  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 Central

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

    2011-01-01

    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. PMID:21965376

  7. 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.

  8. 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.

  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. T Cell-Derived Lymphotoxin Regulates Liver Regeneration

    PubMed Central

    TUMANOV, ALEXEI V.; KOROLEVA, EKATERINA P.; CHRISTIANSEN, PETER A.; KHAN, MEHTAB A.; RUDDY, MATTHEW J.; BURNETTE, BYRON; PAPA, SALVATORE; FRANZOSO, GUIDO; NEDOSPASOV, SERGEI A.; FU, YANG-XIN; ANDERS, ROBERT A.

    2011-01-01

    Background & Aims The ability of the liver to regenerate hepatic mass is essential to withstanding liver injury. The process of liver regeneration is tightly regulated by distinct signaling cascades involving components of the innate immune system, cytokines, and growth factors. However, the role of the adaptive immune system in regulation of liver regeneration is not well-defined. The role of adaptive immune system in liver regeneration was investigated in lymphocyte-deficient mice and in conditional lymphotoxin-deficient mice. Methods A model of liver regeneration after 70% partial hepatectomy was used, followed by examination of liver pathology, survival, DNA synthesis, and cytokine expression. Results We found that mice deficient in T cells show a reduced capacity for liver regeneration following partial hepatectomy. Furthermore, surface lymphotoxin, provided by T cells, is critical for liver regeneration. Mice specifically deficient in T-cell lymphotoxin had increased liver damage and a reduced capacity to initiate DNA synthesis after partial hepatectomy. Transfer of splenocytes from wild-type but not lymphotoxin-deficient mice improved liver regeneration in T cell-deficient mice. We found that an agonistic antibody against the lymphotoxin β receptor was able to facilitate liver regeneration by reducing liver injury, increasing interleukin-6 production, hepatocyte DNA synthesis, and survival of lymphocyte-deficient (Rag) mice after partial hepatectomy. Conclusions The adaptive immune system directly regulates liver regeneration via a T cell-derived lymphotoxin axis, and pharmacological stimulation of lymphotoxin β receptor might represent a novel therapeutic approach to improve liver regeneration. PMID:18952083

  11. 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.

  12. 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.

  13. Analysis of side population cells derived from dental pulp tissue.

    PubMed

    Kenmotsu, M; Matsuzaka, K; Kokubu, E; Azuma, T; Inoue, T

    2010-12-01

    To investigate the characteristics of side population (SP) cells derived from the dental pulp of young and aged rats. Maxillary and mandibular incisors were extracted from 5-week-old (young) rats and 60- to 80-week-old (aged) rats. Coronal pulp tissue was removed mechanically, and single-cell suspensions were prepared using collagenase and dispase. Cells were stained with Hoechst 33342 and sorted with an fluorescence-activated cell sorter (FACS). Isolated SP and main population (MP) cells were analysed by real-time reverse transcription polymerase chain reaction, immunohistochemical localization and cell cycle determination. Two-way analysis of variance and the multiple comparison Scheffè test were used for statistical analysis (P<0.05). Approximately 0.40% of pulp cells in young rats and 0.11% in aged rats comprised SP cells. SP cells expressed a higher mRNA level of ATP-binding cassette transporter G2 (ABCG2), but lower mRNA levels of nestin, alkaline phosphatase, p16 and p57 than MP cells in both age groups. Immunohistochemical observation revealed ABCG2-positive cells localized in the cell-rich zone and nestin in the odontoblastic layer in both groups. Furthermore, the majority of both young and aged SP and MP cells were in growth arrest of the G(0) /G(1) phase. The FACS analysis revealed a decrease in the proportion of SP cells with age, whilst p16 mRNA expression indicated an increase in cell senescence. The cell cycles of SP and MP cells from both young and aged dental pulp were generally in the G0/G1 phase. © 2010 International Endodontic Journal.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

    PubMed

    Wang, Dachun; Morales, John E; Calame, Daniel G; Alcorn, Joseph L; Wetsel, Rick A

    2010-03-01

    Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

  19. 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.

  20. The ROCK inhibitor Y-26732 enhances the survival and proliferation of human embryonic stem cell-derived neural progenitor cells upon dissociation.

    PubMed

    Rungsiwiwut, Ruttachuk; Manolertthewan, Chirawattana; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Virutamasen, Pramuan; Techakumphu, Mongkol; Pruksananonda, Kamthorn

    2013-01-01

    Human neural progenitor cells (hNPCs) are the starting material required for neuronal subtype differentiation. Proliferation of hNPCs allows researchers to study the mechanistic complexities and microenvironments present during neural differentiation and to explore potential applications for hNPCs in cell therapies. The use of enzymatic dissociation during hNPC proliferation causes dissociation-induced apoptosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum-free culture medium supplemented with noggin. The established hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrated that supplementation of the culture media with 10 μM Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was applied either at (i) 24 h before dissociation of the cells and at 24 h after plating the cells or (ii) at 24 h after plating of the cells only. In addition to reducing apoptosis, both supplementation conditions with Y-26732 enhanced the proliferation of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in respect to apoptosis and cell proliferation. © 2013 S. Karger AG, Basel.

  1. Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

    PubMed Central

    LIANG, YIN-KU; PING, WEI; BIAN, LIU-JIAO

    2015-01-01

    Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with the recombinant plasmid pCMV-SPORT6-hSDF1 as the template, and the prokaryotic expression vector pET15b-hSDF-1α was constructed. This hSDF-1α was successfully expressed as an inclusion body in Escherichia coli BL21(DE3). The recombinant hSDF-1α was refolded in vitro and separated by cation exchange chromatography. Following these two steps the purity of the hSDF-1α was able to reach >85%. The recombinant hSDF-1α was then purified by size-exclusion chromatography. SDS-PAGE analysis demonstrated that the purity of the hSDF-1α was >95%, which meets almost all the requirements of a protein experiment. Chemotactic activity of the recombinant hSDF-1α was analyzed by Transwell migration assay and it was found that the recombinant hSDF-1α was able to stimulate THP-1 cell migration. These data suggest that the procedure of producing recombinant hSDF-1α proteins with chemotactic activity was feasible and the N-terminal signal peptide of hSDF-1α has little effect on the chemotactic activity of hSDF-1α. PMID:26136888

  2. 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

  3. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    PubMed Central

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  4. Different Angiogenic Potentials of Mesenchymal Stem Cells Derived from Umbilical Artery, Umbilical Vein, and Wharton's Jelly

    PubMed Central

    Zhou, Jianjun; Liu, Jingyu; Liu, Yong; Wang, Lei; Jiang, Ruiwei; Diao, Zhenyu; Yan, Guijun; Pèault, Bruno

    2017-01-01

    Human mesenchymal stem cells derived from the umbilical cord (UC) are a favorable source for allogeneic cell therapy. Here, we successfully isolated the stem cells derived from three different compartments of the human UC, including perivascular stem cells derived from umbilical arteries (UCA-PSCs), perivascular stem cells derived from umbilical vein (UCV-PSCs), and mesenchymal stem cells derived from Wharton's jelly (WJ-MSCs). These cells had the similar phenotype and differentiation potential toward adipocytes, osteoblasts, and neuron-like cells. However, UCA-PSCs and UCV-PSCs had more CD146+ cells than WJ-MSCs (P < 0.05). Tube formation assay in vitro showed the largest number of tube-like structures and branch points in UCA-PSCs among the three stem cells. Additionally, the total tube length in UCA-PSCs and UCV-PSCs was significantly longer than in WJ-MSCs (P < 0.01). Microarray, qRT-PCR, and Western blot analysis showed that UCA-PSCs had the highest expression of the Notch ligand Jagged1 (JAG1), which is crucial for blood vessel maturation. Knockdown of Jagged1 significantly impaired the angiogenesis in UCA-PSCs. In summary, UCA-PSCs are promising cell populations for clinical use in ischemic diseases.

  5. Regulation of cell adhesion and migration by cell-derived matrices.

    PubMed

    Kutys, Matthew L; Doyle, Andrew D; Yamada, Kenneth M

    2013-10-01

    Three-dimensional in vitro extracellular matrix models provide a physiological alternative to regular two-dimensional cell culture, though they lack the full diversity of molecular composition and physical properties of whole-animal systems. Cell-derived matrices are extracellular matrices that are the product of matrix secretion and assembly by cells cultured at high density in vitro. After the removal of the cells that produced the matrix, an assembled matrix scaffold is left that closely mimics native stromal fiber organization and molecular content. Cell-derived matrices have been shown to impart in vivo-like responses to cells cultured in these matrices. In this review, we focus on mechanisms through which the distinct molecular and topographical composition of cell-derived matrices directs cellular behavior, specifically through regulation of cell-matrix adhesions and subsequent contributions to the process of cell migration. Published by Elsevier Inc.

  6. Solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation: piece by piece

    PubMed Central

    Lundy, David J.; Lee, Desy S.

    2017-01-01

    There is a growing need for in vitro models which can serve as platforms for drug screening and basic research. Human adult cardiomyocytes cannot be readily obtained or cultured, and so pluripotent stem cell-derived cardiomyocytes appear to be an attractive option. Unfortunately, these cells are structurally and functionally immature—more comparable to foetal cardiomyocytes than adult. A recent study by Ruan et al., provides new insights into accelerating the maturation process and takes us a step closer to solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation. PMID:28462223

  7. 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.

  8. A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles

    PubMed Central

    Ståhl, Anne-lie; Arvidsson, Ida; Johansson, Karl E.; Chromek, Milan; Rebetz, Johan; Loos, Sebastian; Kristoffersson, Ann-Charlotte; Békássy, Zivile D.; Mörgelin, Matthias; Karpman, Diana

    2015-01-01

    Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system. PMID:25719452

  9. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    PubMed

    Ståhl, Anne-lie; Arvidsson, Ida; Johansson, Karl E; Chromek, Milan; Rebetz, Johan; Loos, Sebastian; Kristoffersson, Ann-Charlotte; Békássy, Zivile D; Mörgelin, Matthias; Karpman, Diana

    2015-02-01

    Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  10. 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.

  11. 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

  12. Tumour cell-derived exosomes endow mesenchymal stromal cells with tumour-promotion capabilities.

    PubMed

    Lin, L Y; Du, L M; Cao, K; Huang, Y; Yu, P F; Zhang, L Y; Li, F Y; Wang, Y; Shi, Y F

    2016-11-17

    Mesenchymal stromal cells (MSCs) are a major component of the tumour microenvironment. A plethora of elegant studies focusing on tumour-derived MSCs have shown that they, unlike normal MSCs in other tissue, exhibit a strong ability to promote tumour progression. However, the mechanisms underlying the conversion of normal MSCs into tumour-associated MSCs are unknown. We report here a critical role of tumour cell-derived exosomes in endowing bone marrow-derived MSCs (BM-MSCs) with a tumour-favourable phenotype. Tumour cell-derived exosomes affected neither the growth factor production nor the immunosuppressive property of MSCs; rather, they endowed MSCs with a strong ability to promote macrophage infiltration into B16-F0 melanoma or EL-4 lymphoma. Ablation of macrophages by clodronate liposome administration reversed the tumour-promoting effect of MSCs educated by tumour cell-derived exosomes (TE-MSCs) on the tumour growth. By comparing the chemokine profile of BM-MSCs with that of TE-MSCs, we found that TE-MSCs produced a large amount of CCR2 ligands, CCL2 and CCL7, which are responsible for macrophage recruitment. CCR2-specific inhibitor was found to block the tumour-promoting effect of TE-MSCs. Thus, our investigations demonstrated that tumour cell-derived exosomes confer BM-MSCs the ability to enhance tumour growth. Therefore, we uncovered a novel mechanism underlying the conversion of normal MSCs to tumour-associated MSCs.

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

    USDA-ARS?s Scientific Manuscript database

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

  14. 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. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. 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.

  16. 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

  17. 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.

  18. Human embryonic stem cell-derived mesenchymal stem cell seeding on calcium phosphate cement-chitosan-RGD scaffold for bone repair.

    PubMed

    Chen, Wenchuan; Zhou, Hongzhi; Weir, Michael D; Tang, Minghui; Bao, Chongyun; Xu, Hockin H K

    2013-04-01

    Calcium phosphate cement (CPC) has in situ-setting ability and excellent osteoconductivity. Human embryonic stem cells (hESCs) are exciting for regenerative medicine due to their strong proliferative ability and multilineage differentiation capability. However, there has been no report on hESC seeding with CPC. The objectives of this study were to obtain hESC-derived mesenchymal stem cells (hESCd-MSCs), and to investigate hESCd-MSC proliferation and osteogenic differentiation on novel CPC with chitosan immobilized with RGD (CPC-chitosan-RGD). RGD was covalently bonded with chitosan, which was then incorporated into CPC. The CPC-chitosan-RGD scaffold had higher strength and toughness than CPC-chitosan control without RGD (p<0.05). hESCs were cultured to form embryoid bodies (EBs), and the MSCs were then migrated out of the EBs. Flow cytometry indicated that the hESCd-MSCs expressed typical surface antigen profile of MSCs. hESCd-MSCs had good viability when seeded on CPC scaffolds. The percentage of live cells and the cell density were significantly higher on CPC-chitosan-RGD than CPC-chitosan control. Scanning electron microscope examination showed hESCd-MSCs with a healthy spreading morphology adherent to CPC. hESCd-MSCs expressed high levels of osteogenic markers, including alkaline phosphatase, osteocalcin, collagen I, and Runx2. The mineral synthesis by the hESCd-MSCs on the CPC-chitosan-RGD scaffold was twice that for CPC-chitosan control. In conclusion, hESCs were successfully seeded on CPC scaffolds for bone tissue engineering. The hESCd-MSCs had good viability and osteogenic differentiation on the novel CPC-chitosan-RGD scaffold. RGD incorporation improved the strength and toughness of CPC, and greatly enhanced the hESCd-MSC attachment, proliferation, and bone mineral synthesis. Therefore, the hESCd-MSC-seeded CPC-chitosan-RGD construct is promising to improve bone regeneration in orthopedic and craniofacial applications.

  19. Therapeutic potential of lung epithelial progenitor cells derived from embryonic and induced pluripotent stem cells.

    PubMed

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

    2011-01-01

    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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. hESCCO: development of good practice models for hES cell derivation.

    PubMed

    Franklin, Sarah B; Hunt, Charles; Cornwell, Glenda; Peddie, Valerie; Desousa, Paul; Livie, Morag; Stephenson, Emma L; Braude, Peter R

    2008-01-01

    One response of the UK research community to the public sensitivity and logistical complexity of embryo donation to stem cell research has been the formation of a national network of 'human embryonic stem cell coordinators' (hESCCO). The aim of hESCCO is to contribute to the formation and implementation of national standards for hES cell derivation and banking, in particular the ethical protocols for patient information and informed consent. The hESCCO project is an innovative practical intervention within the broader attempt to establish greater transparency, consistency, efficiency and standardization of hES derivation in the UK. A major outcome of the hESCCO initiative has been the drafting and implementation of a national consent form. The lessons learned in this context may be relevant to other practitioners and regulators as a model of best practice in hES cell derivation.

  7. 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.

  8. The atopic march: current insights into skin barrier dysfunction and epithelial cell-derived cytokines.

    PubMed

    Han, Hongwei; Roan, Florence; Ziegler, Steven F

    2017-07-01

    Atopic dermatitis often precedes the development of other atopic diseases. The atopic march describes this temporal relationship in the natural history of atopic diseases. Although the pathophysiological mechanisms that underlie this relationship are poorly understood, epidemiological and genetic data have suggested that the skin might be an important route of sensitization to allergens. Animal models have begun to elucidate how skin barrier defects can lead to systemic allergen sensitization. Emerging data now suggest that epithelial cell-derived cytokines such as thymic stromal lymphopoietin (TSLP), IL-33, and IL-25 may drive the progression from atopic dermatitis to asthma and food allergy. This review focuses on current concepts of the role of skin barrier defects and epithelial cell-derived cytokines in the initiation and maintenance of allergic inflammation and the atopic march. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. 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.

  10. 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.

  11. Transplantation of human stem cell-derived hepatocytes in an animal model of acute liver failure.

    PubMed

    Ramanathan, Rajesh; Pettinato, Giuseppe; Beeston, John T; Lee, David D; Wen, Xuejun; Mangino, Martin J; Fisher, Robert A

    2015-08-01

    Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF. Intraperitoneal d-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF. A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen. Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. Elevated circulating endothelial cell-derived microparticle levels in patients with liver cirrhosis: a preliminary report

    PubMed Central

    Simon, Krzysztof Adam; Pazgan-Simon, Monika

    2015-01-01

    Aim of the study To determine plausible associations between liver cirrhosis and circulating endothelial cell-derived microparticles (EMPs), vascular endothelial growth factor (VEGF) levels and plasma nitric oxide (NO) metabolites. Material and methods Sixty patients with cirrhosis and 20 healthy control subjects were enrolled in the study. Circulating EMPs from platelet-poor plasma samples were examined by flow cytometry. These microparticles were categorized into endothelial cell-derived activated MPs (EMP-ac) (CD31+ CD42b– AN-V–) and endothelial cell-derived apoptotic MPs (EMP-ap) (CD31+ CD42b– AN-V+). Plasma VEGF levels were measured by enzyme-linked immunosorbent assay. Plasma NO metabolites (NOx–) levels were determined using a Greiss reaction method. Results Compared with the healthy control subjects, the patients with cirrhosis showed a significant increase in plasma levels of both phenotypes of EMPs. When the presence of ascites was considered, the plasma levels of EMP-ap were higher (p < 0.01), as well as NOx– (p < 0.05). EMP-ap positively correlated with VEGF level in all cirrhotic patients and this correlation was stronger in decompensated cirrhotic patients. In multivariate logistic regression analysis, the independent factors associated with the presence of ascites were high EMP-ap levels and elevated VEGF levels. Conclusions Elevated plasma levels of EMP-ap in addition to high levels of VEGF might be considered as valuable parameters for predicting the occurrence of ascites in cirrhotic patients. PMID:28856256

  18. 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.

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

    PubMed

    Lowenthal, Justin; Gerecht, Sharon

    2016-05-06

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. Differentiation and Application of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

    PubMed

    Maguire, Eithne Margaret; Xiao, Qingzhong; Xu, Qingbo

    2017-08-31

    Vascular smooth muscle cells (VSMCs) play a role in the development of vascular disease, for example, neointimal formation, arterial aneurysm, and Marfan syndrome caused by genetic mutations in VSMCs, but little is known about the mechanisms of the disease process. Advances in induced pluripotent stem cell technology have now made it possible to derive VSMCs from several different somatic cells using a selection of protocols. As such, researchers have set out to delineate key signaling processes involved in triggering VSMC gene expression to grasp the extent of gene regulatory networks involved in phenotype commitment. This technology has also paved the way for investigations into diseases affecting VSMC behavior and function, which may be treatable once an identifiable culprit molecule or gene has been repaired. Moreover, induced pluripotent stem cell-derived VSMCs are also being considered for their use in tissue-engineered blood vessels as they may prove more beneficial than using autologous vessels. Finally, while several issues remains to be clarified before induced pluripotent stem cell-derived VSMCs can become used in regenerative medicine, they do offer both clinicians and researchers hope for both treating and understanding vascular disease. In this review, we aim to update the recent progress on VSMC generation from stem cells and the underlying molecular mechanisms of VSMC differentiation. We will also explore how the use of induced pluripotent stem cell-derived VSMCs has changed the game for regenerative medicine by offering new therapeutic avenues to clinicians, as well as providing researchers with a new platform for modeling of vascular disease. © 2017 American Heart Association, Inc.

  5. Effector CD8(+) T cell-derived interleukin-10 enhances acute liver immunopathology.

    PubMed

    Fioravanti, Jessica; Di Lucia, Pietro; Magini, Diletta; Moalli, Federica; Boni, Carolina; Benechet, Alexandre Pierre; Fumagalli, Valeria; Inverso, Donato; Vecchi, Andrea; Fiocchi, Amleto; Wieland, Stefan; Purcell, Robert; Ferrari, Carlo; Chisari, Francis V; Guidotti, Luca G; Iannacone, Matteo

    2017-09-01

    Besides secreting pro-inflammatory cytokines, chemokines and effector molecules, effector CD8(+) T cells that arise upon acute infection with certain viruses have been shown to produce the regulatory cytokine interleukin (IL)-10 and, therefore, contain immunopathology. Whether the same occurs during acute hepatitis B virus (HBV) infection and role that IL-10 might play in liver disease is currently unknown. Mouse models of acute HBV pathogenesis, as well as chimpanzees and patients acutely infected with HBV, were used to analyse the role of CD8(+) T cell-derived IL-10 in liver immunopathology. Mouse HBV-specific effector CD8(+) T cells produce significant amounts of IL-10 upon in vivo antigen encounter. This is corroborated by longitudinal data in a chimpanzee acutely infected with HBV, where serum IL-10 was readily detectable and correlated with intrahepatic CD8(+) T cell infiltration and liver disease severity. Unexpectedly, mouse and human CD8(+) T cell-derived IL-10 was found to act in an autocrine/paracrine fashion to enhance IL-2 responsiveness, thus preventing antigen-induced HBV-specific effector CD8(+) T cell apoptosis. Accordingly, the use of mouse models of HBV pathogenesis revealed that the IL-10 produced by effector CD8(+) T cells promoted their own intrahepatic survival and, thus supported, rather than suppressed liver immunopathology. Effector CD8(+) T cell-derived IL-10 enhances acute liver immunopathology. Altogether, these results extend our understanding of the cell- and tissue-specific role that IL-10 exerts in immune regulation. Lay summary: Interleukin-10 is mostly regarded as an immunosuppressive cytokine. We show here that HBV-specific CD8(+) T cells produce IL-10 upon antigen recognition and that this cytokine enhances CD8(+) T cell survival. As such, IL-10 paradoxically promotes rather than suppresses liver disease. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  6. Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway.

    PubMed

    Lin, Jinghan; Xu, Yi; Zhao, Tingting; Sun, Lina; Yang, Meimei; Liu, Tingjiao; Sun, Hui; Zhang, Liming

    2015-08-07

    Genistein, as a protein tyrosine kinase inhibitor, has been shown to possess anti-atherosclerotic effects. Since the smooth muscle cell-derived foam cells are key components of atherosclerotic plaques. The aim of this study is to investigate the influence of genistein on foam cell transformation from vascular smooth muscle cells and possible mechanisms contributing to these effects. Vascular smooth muscle cells exposed to ox-LDL developed into foam cell, as demonstrated by Oil Red O staining and cholesterol content analysis. Ox-LDL induced phenotype transformation of smooth muscle cells, decreased expression of α-actin and increased expression of CD68 (a specific marker for monocytes, can also function as a subtype of scavenger receptors). The expression of scavenger receptors CD36 and LOX-1 was measured, and their role in foam cell formation in the presence of genistein, daidzein (a structurally similar analogue of genistein) and herbimycin A (a commonly tyrosine kinase inhibitor). The results showed that foam cell formation was markedly reduced by genistein and herbimycin A, as well as the expression of CD68, CD36 and LOX-1. However, daidzein had no such effect. In addition, genistein-induced down-regulation of CD68, CD36 and LOX-1 could be reversed by sodium orthovanadate (a membrane-permeable protein tyrosine phosphatase inhibitor). The results showed that ox-LDL induce smooth muscle cell-derived foam cell formation and transform the phenotype of smooth muscle cell. While tyrosine kinase inhibitor, genistein could suppress smooth muscle cell-derived foam cell formation through inhibiting the protein expressions of CD68, CD36 and LOX-1. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. Stem cell-derived cell-sheets for connective tissue engineering.

    PubMed

    Neo, Puay Yong; Teh, Thomas Kok Hiong; Tay, Alex Sheng Ru; Asuncion, Maria Christine Tankeh; Png, Si Ning; Toh, Siew Lok; Goh, James Cho-Hong

    2016-11-01

    Cell-sheet technology involves the recovery of cells with its secreted ECM and cell-cell junctions intact, and thereby harvesting them in a single contiguous layer. Temperature changes coupled with a thermoresponsive polymer grafted culture plate surface are typically used to induce detachment of this cell-matrix layer by controlling the hydrophobicity and hydrophilicity properties of the culture surface. This review article details the genesis and development of this technique as a critical tissue-engineering tool, with a comprehensive discussion on connective tissue applications. This includes applications in the myocardial, vascular, cartilage, bone, tendon/ligament, and periodontal areas among others discussed. In particular, further focus will be given to the use of stem cells-derived cell-sheets, such as those involving bone marrow-derived and adipose tissue-derived mesenchymal stem cells. In addition, some of the associated challenges faced by approaches using stem cells-derived cell-sheets will also be discussed. Finally, recent advances pertaining to technologies forming, detaching, and manipulating cell-sheets will be covered in view of the potential impact they will have on shaping the way cell-sheet technology will be utilized in the future as a tissue-engineering technique.

  16. 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.

  17. Human intestinal epithelial cell-derived molecule(s) increase enterohemorrhagic Escherichia coli virulence.

    PubMed

    Bansal, Tarun; Kim, Dae N; Slininger, Tim; Wood, Thomas K; Jayaraman, Arul

    2012-12-01

    To better understand the role of host cell-derived molecules on enterohemorrhagic Escherichia coli (EHEC) infection, we studied EHEC virulence gene expression when exposed to cell-free spent (conditioned) medium (CM) from HCT-8 intestinal epithelial cells. Exposure to HCT-8 CM for 1 h and 3 h increased the expression of 32 of 41 EHEC locus of enterocyte effacement (LEE) virulence genes compared with fresh medium (FM). Expression of the Shiga toxin 1 (stx1B) gene was up-regulated at 1 h of exposure. Seventeen genes encoded by prophage 933W, including those for Stx2, were also up-regulated at both time-points. The increase in 933W prophage expression was mirrored by a 2.7-fold increase in phage titers. Consistent with the increase in virulence gene expression, we observed a fivefold increase in EHEC attachment to epithelial cells when exposed to CM. The increase in EHEC attachment was abolished when CM was heated to 95 °C or treated with proteinase K to degrade the proteins. The host cell-derived molecule(s) were larger than 3 kDa, which suggests that the molecule(s) that increase EHEC virulence and attachment are protein-based. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. 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.

  19. 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.

  20. 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

  1. Glioma gene therapy using induced pluripotent stem cell derived neural stem cells.

    PubMed

    Lee, Esther Xingwei; Lam, Dang Hoang; Wu, Chunxiao; Yang, Jing; Tham, Chee Kian; Ng, Wai Hoe; Wang, Shu

    2011-10-03

    Using neural stem cells (NSCs) with tumor tropic migratory capacity to deliver therapeutic genes is an attractive strategy in eliminating metastatic or disseminated tumors. While different methods have been developed to isolate or generate NSCs, it has not been assessed whether induced pluripotent stem (iPS) cells, a type of pluripotent stem cells that hold great potential for regenerative medicine, can be used as a source for derivation of NSCs with tumor tropism. In this study, we used a conventional lentivirus transduction method to derive iPS cells from primary mouse embryonic fibroblasts and then generated NSCs from the iPS cells. To investigate whether the iPS cell derived NSCs can be used in the treatment of disseminated brain tumors, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected into the cerebral hemisphere contralateral to a tumor inoculation site in a mouse intracranial human glioma xenograft model. We observed that NSCs expressing the suicide gene were, in the presence of ganciclovir, effective in inhibiting the growth of the glioma xenografts and prolonging survival of tumor-bearing mice. Our findings provide evidence for the feasibility of using iPS cell derived NSCs as cellular vehicles for targeted anticancer gene therapy.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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).

  9. Induction of Protective Immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina Infections Using Dendritic Cell-Derived Exosomes

    PubMed Central

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

    2012-01-01

    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 Th1 cytokines, Th2 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 Th1 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 Th2 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. PMID:22354026

  10. 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.

  11. 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.

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

    PubMed

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

    2015-01-01

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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. Effect of purine alkaloids on the proliferation of lettuce cells derived from protoplasts.

    PubMed

    Sasamoto, Hamako; Fujii, Yoshiharu; Ashihara, Hiroshi

    2015-05-01

    To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 μM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with > 250 μM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

  2. 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.

  3. 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.

  4. Anatase-based implants nanocoating on stem cells derived from adipose tissue.

    PubMed

    Zollino, Ilaria; Girardi, Ambra; Palmieri, Annalisa; Cura, Francesca; Sollazzo, Vincenzo; Brunelli, Giorgio; Carinci, Francesco

    2012-04-01

    The aim of this study was to investigate the effect of a new anatase coating with antibacterial properties (Bactercline anatase coating [BAC]) on dental implants in the commitment of stem cells derived from adipose tissue to osteoblasts. Using real-time reverse transcription polymerase chain reaction, the quantitative expression of specific genes, such as transcriptional factors (runx2 and sp7), bone-related genes (spp1, col1a1, col3a1, alpl, and fosl1), and mesenchymal stem cells marker (eng), was examined. BAC caused induction of bone-related genes such as sp7, fosl1, alpl, and spp1. In contrast, the expression of runx2, col3a1, and col1a1 was decreased in stem cells treated with BAC with respect to untreated cells. The obtained results are relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

  5. Reversing drug resistance of soft tumor-repopulating cells by tumor cell-derived chemotherapeutic microparticles

    PubMed Central

    Ma, Jingwei; Zhang, Yi; Tang, Ke; Zhang, Huafeng; Yin, Xiaonan; Li, Yong; Xu, Pingwei; Sun, Yanling; Ma, Ruihua; Ji, Tiantian; Chen, Junwei; Zhang, Shuang; Zhang, Tianzhen; Luo, Shunqun; Jin, Yang; Luo, Xiuli; Li, Chengyin; Gong, Hongwei; Long, Zhixiong; Lu, Jinzhi; Hu, Zhuowei; Cao, Xuetao; Wang, Ning; Yang, Xiangliang; Huang, Bo

    2016-01-01

    Developing novel approaches to reverse the drug resistance of tumor-repopulating cells (TRCs) or stem cell-like cancer cells is an urgent clinical need to improve outcomes of cancer patients. Here we show an innovative approach that reverses drug resistance of TRCs using tumor cell-derived microparticles (T-MPs) containing anti-tumor drugs. TRCs, by virtue of being more deformable than differentiated cancer cells, preferentially take up T-MPs that release anti-tumor drugs after entering cells, which in turn lead to death of TRCs. The underlying mechanisms include interfering with drug efflux and promoting nuclear entry of the drugs. Our findings demonstrate the importance of tumor cell softness in uptake of T-MPs and effectiveness of a novel approach in reversing drug resistance of TRCs with promising clinical applications. PMID:27167569

  6. 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

  7. 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

  8. 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

  9. N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm.

    PubMed

    Mallanna, Sunil K; Waas, Matthew; Duncan, Stephen A; Gundry, Rebekah L

    2017-03-01

    Using cell surface capture technology, the cell surface N-glycoproteome of human-induced pluripotent stem cell derived hepatic endoderm cells was assessed. Altogether, 395 cell surface N-glycoproteins were identified, represented by 1273 N-glycopeptides. This study identified N-glycoproteins that are not predicted to be localized to the cell surface and provides experimental data that assist in resolving ambiguous or incorrectly annotated transmembrane topology annotations. In a proof-of-concept analysis, combining these data with other cell surface proteome datasets is useful for identifying potentially cell type and lineage restricted markers and drug targets to advance the use of stem cell technologies for mechanistic developmental studies, disease modeling, drug discovery, and regenerative medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. Aquaporin expression and function in human pluripotent stem cell-derived retinal pigmented epithelial cells.

    PubMed

    Juuti-Uusitalo, Kati; Delporte, Christine; Grégoire, Francoise; Perret, Jason; Huhtala, Heini; Savolainen, Virpi; Nymark, Soile; Hyttinen, Jari; Uusitalo, Hannu; Willermain, Francois; Skottman, Heli

    2013-05-01

    Aquaporins (AQPs), a family of transmembrane water channel proteins, are essential for allowing passive water transport through retinal pigmented epithelial (RPE) cells. Even though human native RPE cells and immortalized human RPEs have been shown to express AQPs, the expression of AQPs during the differentiation in stem cell-derived RPE remains to be elucidated. In human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs)-derived RPE cells, the expression of several AQPs was determined by quantitative real-time PCR and the localization of AQP1 was assessed with confocal microscopy. The functionality of AQP water channels was determined by cell volume assay in hESC-derived RPE cells. AQP1, AQP3, AQP4, AQP5, AQP6, AQP7, AQP10, AQP11, and AQP12 were expressed in hESC- and hiPSC-derived RPE cells. Furthermore, the expression of AQP1 and AQP11 genes were significantly upregulated during the maturation of both hESC and iPSC into RPE. Confocal microscopy shows the expression of AQP1 at the apical plasma membrane of polarized cobblestone hESC- and hiPSC-derived RPE cells. Lastly, aquaporin inhibitors significantly reduced AQP functionality in hESC-RPE cells. hESC-RPE and hiPSC-RPE cells express several AQP genes, which are functional in mature hESC-derived RPE cells. The localization of AQP1 on the apical plasma membrane in mature RPE cells derived from both hESC and hiPSC suggests its functionality. These data propose that hESC- and hiPSC-derived RPE cells, grown and differentiated under serum-free conditions, resemble their native counterpart in the human eye.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. A non-invasive platform for functional characterization of stem-cell-derived cardiomyocytes with applications in cardiotoxicity testing.

    PubMed

    Maddah, Mahnaz; Heidmann, Julia D; Mandegar, Mohammad A; Walker, Chase D; Bolouki, Sara; Conklin, Bruce R; Loewke, Kevin E

    2015-04-14

    We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy

    PubMed Central

    2013-01-01

    Background Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis. Methods To identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells. Results Using SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search. Conclusions We

  7. 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

  8. Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect the Fetal Brain After Hypoxia-Ischemia.

    PubMed

    Ophelders, Daan R M G; Wolfs, Tim G A M; Jellema, Reint K; Zwanenburg, Alex; Andriessen, Peter; Delhaas, Tammo; Ludwig, Anna-Kristin; Radtke, Stefan; Peters, Vera; Janssen, Leon; Giebel, Bernd; Kramer, Boris W

    2016-06-01

    Preterm neonates are susceptible to perinatal hypoxic-ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic-ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in a preclinical model of preterm hypoxic-ischemic brain injury. Ovine fetuses were subjected to global hypoxia-ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC-EVs. The therapeutic effects of MSC-EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC-EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC-EV treatment. Our data demonstrate that MSC-EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic-ischemic injury of the preterm brain. Our study results suggest that a cell-free preparation comprising neuroprotective MSC-EVs could substitute MSCs in the treatment of preterm neonates with hypoxic-ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells. Bone marrow-derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic-ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration

  9. 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

  10. 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

  11. Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy.

    PubMed

    Hara, Yasuhiro; Kawasaki, Naoko; Hirano, Ken-ichi; Hashimoto, Yuuki; Adachi, Jun; Watanabe, Shio; Tomonaga, Takeshi

    2013-12-21

    Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis. To identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells. Using SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search. We performed the SILAC- and SRM

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Mesenchymal Stem Cell-Derived Factors Restore Function to Human Frataxin-Deficient Cells.

    PubMed

    Kemp, Kevin; Dey, Rimi; Cook, Amelia; Scolding, Neil; Wilkins, Alastair

    2017-08-01

    Friedreich's ataxia is an inherited neurological disorder characterised by mitochondrial dysfunction and increased susceptibility to oxidative stress. At present, no therapy has been shown to reduce disease progression. Strategies being trialled to treat Friedreich's ataxia include drugs that improve mitochondrial function and reduce oxidative injury. In addition, stem cells have been investigated as a potential therapeutic approach. We have used siRNA-induced knockdown of frataxin in SH-SY5Y cells as an in vitro cellular model for Friedreich's ataxia. Knockdown of frataxin protein expression to levels detected in patients with the disorder was achieved, leading to decreased cellular viability, increased susceptibility to hydrogen peroxide-induced oxidative stress, dysregulation of key anti-oxidant molecules and deficiencies in both cell proliferation and differentiation. Bone marrow stem cells are being investigated extensively as potential treatments for a wide range of neurological disorders, including Friedreich's ataxia. The potential neuroprotective effects of bone marrow-derived mesenchymal stem cells were therefore studied using our frataxin-deficient cell model. Soluble factors secreted by mesenchymal stem cells protected against cellular changes induced by frataxin deficiency, leading to restoration in frataxin levels and anti-oxidant defences, improved survival against oxidative stress and stimulated both cell proliferation and differentiation down the Schwann cell lineage. The demonstration that mesenchymal stem cell-derived factors can restore cellular homeostasis and function to frataxin-deficient cells further suggests that they may have potential therapeutic benefits for patients with Friedreich's ataxia.

  1. 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.

  2. 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

  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. 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.

  7. 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

  8. 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.

  9. 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.

  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. © 2012 by the Wound Healing Society.

  11. 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.

  12. 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

  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. Epithelial Cell-Derived Cytokines Contribute to the Pathophysiology of Eosinophilic Chronic Rhinosinusitis.

    PubMed

    Kouzaki, Hideaki; Matsumoto, Koji; Kato, Tomohisa; Tojima, Ichiro; Shimizu, Shino; Shimizu, Takeshi

    2016-03-01

    The epithelial cell-derived cytokines, thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33 induce T helper 2 type immune responses. In the present study, we investigate the role of these cytokines in the pathophysiology of eosinophilic chronic rhinosinusitis (ECRS). Nasal tissue specimens from chronic rhinosinusitis patients were assayed for the expression of TSLP, IL-25, IL-33, protease-activated receptor (PAR)-2, and P2Y2 receptor (P2Y2R). Cytokine production in cultured nasal epithelial cells (PNECs) was also examined. The mRNA levels of TSLP and IL-25 and the concentrations of IL-25 and IL-33 increased in PNECs from ECRS patients. Immunohistological staining demonstrated that TSLP, IL-25, and IL-33 were localized in the epithelial cells of nasal polyps, and that their expression was increased in ECRS. The mRNA levels of TSLP and IL-25 correlated with the clinical severity of ECRS, as indicated by the computed tomography score. The TSLP mRNA levels and IL-33 protein concentration correlated with the number of eosinophils in the nasal polyps of patients with ECRS. Airborne allergen-induced cytokine production increased in PNECs of these patients. Expression levels of the PAR-2 and P2Y2R increased in cultured PNECs and nasal polyps from patients with ECRS. The results indicate that increased induction and expression of TSLP, IL-25, and IL-33 from nasal epithelial cells contribute to the pathophysiology of ECRS.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26392547

  17. 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

  18. 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

  19. 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.

  20. Using induced pluripotent stem cells derived neurons to model brain diseases.

    PubMed

    McKinney, Cindy E

    2017-07-01

    The ability to use induced pluripotent stem cells (iPSC) to model brain diseases is a powerful tool for unraveling mechanistic alterations in these disorders. Rodent models of brain diseases have spurred understanding of pathology but the concern arises that they may not recapitulate the full spectrum of neuron disruptions associated with human neuropathology. iPSC derived neurons, or other neural cell types, provide the ability to access pathology in cells derived directly from a patient's blood sample or skin biopsy where availability of brain tissue is limiting. Thus, utilization of iPSC to study brain diseases provides an unlimited resource for disease modelling but may also be used for drug screening for effective therapies and may potentially be used to regenerate aged or damaged cells in the future. Many brain diseases across the spectrum of neurodevelopment, neurodegenerative and neuropsychiatric are being approached by iPSC models. The goal of an iPSC based disease model is to identify a cellular phenotype that discriminates the disease-bearing cells from the control cells. In this mini-review, the importance of iPSC cell models validated for pluripotency, germline competency and function assessments is discussed. Selected examples for the variety of brain diseases that are being approached by iPSC technology to discover or establish the molecular basis of the neuropathology are discussed.

  1. Schwann cell-derived factors modulate synaptic activities at developing neuromuscular synapses.

    PubMed

    Cao, Guan; Ko, Chien-Ping

    2007-06-20

    Glial cells are active participants in the function, formation, and maintenance of the chemical synapse. To investigate the molecular basis of neuron-glia interactions at the peripheral synapse, we examined whether and how Schwann cell-derived factors modulate synaptic function at developing neuromuscular junctions (NMJs). Schwann cell-conditioned medium (SC-CM) from Xenopus Schwann cell cultures was collected and applied to Xenopus nerve-muscle cocultures. We found that SC-CM increased the frequency of spontaneous synaptic currents (SSCs) within 3-15 min by an average of approximately 150-fold at developing neuromuscular synapses. The increase in SSC frequency by SC-CM is a presynaptic effect independent of neuronal excitability and requires the influx of Ca2+. In contrast to its potentiating effect on spontaneous transmitter release, SC-CM suppressed the evoked transmitter release. The SC-CM effect required the presence of motoneuron soma but not protein synthesis. Using molecular weight cutoff filters and dialysis membranes, we found that the molecular weight of functional factor(s) in SC-CM was within 500 and 5000 Da. The SC-CM effect was not attributable to currently known factors that modulate synaptic efficacy, including neurotrophins, glutamate, and ATP. SC-CM also enhanced spontaneous synaptic release at developing NMJs in Xenopus tadpoles in situ. Our results suggest that Schwann cells release small molecules that enhance spontaneous synaptic activities acutely and potently at developing neuromuscular synapses, and the glial cell-enhanced spontaneous neurotransmission may contribute to synaptogenesis.

  2. Simultaneous Measurement of Contraction and Calcium Transients in Stem Cell Derived Cardiomyocytes.

    PubMed

    Ahola, A; Pölönen, R-P; Aalto-Setälä, K; Hyttinen, J

    2017-10-03

    Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) provide a powerful platform for disease modeling and drug development in vitro. Traditionally, electrophysiological methods or fluorescent dyes (e.g. calcium) have been used in their functional characterization. Recently, video microscopy has enabled non-invasive analysis of CM contractile motion. Simultaneous assessments of motion and calcium transients have not been generally conducted, as motion detection methods are affected by changing pixel intensities in calcium imaging. Here, we present for the first time a protocol for simultaneous video-based measurement of contraction and calcium with fluorescent dye Fluo-4 videos without corrections, providing data on both ionic and mechanic activity. The method and its accuracy are assessed by measuring the effect of fluorescence and background light on transient widths and contraction velocity amplitudes. We demonstrate the method by showing the contraction-calcium relation and measuring the transient time intervals in catecholaminergic polymorphic ventricular tachycardia patient specific iPSC-CMs and healthy controls. Our validation shows that the simultaneous method provides comparable data to combined individual measurements, providing a new tool for measuring CM biomechanics and calcium simultaneously. Our results with calcium sensitive dyes suggest the method could be expanded to use with other fluorescent reporters as well.

  3. 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.

  4. 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.

  5. Induced Pluripotent HD Monkey Stem Cells Derived Neural Cells for Drug Discovery.

    PubMed

    Kunkanjanawan, Tanut; Carter, Richard; Ahn, Kwan-Sung; Yang, Jinjing; Parnpai, Rangsun; Chan, Anthony W S

    2017-07-01

    Huntington's disease (HD) is a neurodegenerative disease caused by an expansion of CAG trinucleotide repeat (polyglutamine [polyQ]) in the huntingtin ( HTT) gene, which leads to the formation of mutant HTT (mHTT) protein aggregates. In the nervous system, an accumulation of mHTT protein results in glutamate-mediated excitotoxicity, proteosome instability, and apoptosis. Although HD pathogenesis has been extensively studied, effective treatment of HD has yet to be developed. Therapeutic discovery research in HD has been reported using yeast, cells derived from transgenic animal models and HD patients, and induced pluripotent stem cells from patients. A transgenic nonhuman primate model of HD (HD monkey) shows neuropathological, behavioral, and molecular changes similar to an HD patient. In addition, neural progenitor cells (NPCs) derived from HD monkeys can be maintained in culture and differentiated to neural cells with distinct HD cellular phenotypes including the formation of mHTT aggregates, intranuclear inclusions, and increased susceptibility to oxidative stress. Here, we evaluated the potential application of HD monkey NPCs and neural cells as an in vitro model for HD drug discovery research.

  6. 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.

  7. 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

  8. 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

  9. Myocardial regeneration strategies using human embryonic stem cell-derived cardiomyocytes.

    PubMed

    Capi, Oren; Gepstein, Lior

    2006-11-28

    Regenerative medicine is a new biomedicine discipline that takes advantage of the recent advancements in the fields of stem cell biology, molecular biology, and tissue engineering to derive tissue substitutes, in an attempt to replace or modify the function of diseased organs. The heart represents an attractive candidate for these emerging technologies since adult cardiac tissue has limited regenerative capacity. Consequentially, myocardial cell replacement therapy has emerged as a novel therapeutic paradigm for restoration of the myocardial electromechanical function. This innovative strategy has been significantly hampered, however, by the paucity of cell sources for human cardiomyocytes. The recent establishment of the human embryonic stem cell (hESC) lines may provide a possible solution for this cell-sourcing problem. These unique pluripotent cell lines can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. This review will describe the hESC system, their differentiation into cardiomyocytes, and the structural and functional characterization of these cardiac lineage derivatives. The potential applications of this unique differentiating system in several research areas will be discussed with special emphasis on the steps required to fully harness their unique potential in the emerging field of cardiovascular regenerative medicine.

  10. 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.

  11. 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

  12. Malignant cell-derived extracellular vesicles express different chromogranin epitopes compared to prostasomes.

    PubMed

    Dubois, Louise; Stridsberg, Mats; Kharaziha, Pedram; Chioureas, Dimitris; Meersman, Niels; Panaretakis, Theocharis; Ronquist, K Göran

    2015-07-01

    Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers. © 2015 Wiley Periodicals, Inc.

  13. Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model

    PubMed Central

    Thompson, William R.; Uzer, Gunes; Brobst, Kaitlyn E.; Xie, Zhihui; Sen, Buer; Yen, Sherwin S.; Styner, Maya; Rubin, Janet

    2015-01-01

    Studying osteocyte behavior in culture has proven difficult because these embedded cells require spatially coordinated interactions with the matrix and surrounding cells to achieve the osteocyte phenotype. Using an easily attainable source of bone marrow mesenchymal stem cells, we generated cells with the osteocyte phenotype within two weeks. These “stem cell derived-osteocytes” (SCD-O) displayed stellate morphology and lacunocanalicular ultrastructure. Osteocytic genes Sost, Dmp1, E11, and Fgf23 were maximally expressed at 15 days and responded to PTH and 1,25(OH)2D3. Production of sclerostin mRNA and protein, within 15 days of culture makes the SCD-O model ideal for elucidating regulatory mechanisms. We found sclerostin to be regulated by mechanical factors, where low intensity vibration significantly reduced Sost expression. Additionally, this model recapitulates sclerostin production in response to osteoactive hormones, as PTH or LIV repressed secretion of sclerostin, significantly impacting Wnt-mediated Axin2 expression, via β-catenin signaling. In summary, SCD-O cells produce abundant matrix, rapidly attain the osteocyte phenotype, and secrete functional factors including sclerostin under non-immortalized conditions. This culture model enables ex vivo observations of osteocyte behavior while preserving an organ-like environment. Furthermore, as marrow-derived mesenchymal stem cells can be obtained from transgenic animals; our model enables study of genetic control of osteocyte behaviors. PMID:26056071

  14. 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

  15. Glial cell-derived neurotrophic factor gene polymorpisms affect severity and functionality of bipolar disorder.

    PubMed

    Safari, Roghaiyeh; Tunca, Zeliha; Özerdem, Ayşegül; Ceylan, Deniz; Yalçın, Yaprak; Sakizli, Meral

    2017-01-01

    Glial cell-derived neurotrophic factor and other neurotrophins have important role in the development of mental disorders. Here, we aimed to assess the effects of Single nucleotide polymorphisms at potentially regulated regions of GDNF on severity and functionality of bipolar disorder and GDNF serum levels in bipolar disorder patients and healthy volunteers. Severity and functionality of bipolar disorder were evaluated using the Clinical Global Impression and Global Assessment of Functioning scales in sixty-six bipolar disorder patients. The GDNF serum levels obtained from bipolar disorder patients and healthy volunteers who had been already reported SNPs information by our group. GAF scales were lower and GDNF serum levels were higher in Bipolar disorder patients with T/A genotype at 5:37812784 and 5:37812782 compared to patients with T/T genotype. There were significant difference in severity and functionality scores, but not in GDNF serum levels, between patients with G/G and G/A genotype of rs62360370 G > A SNP.rs2075680 C > A and rs79669773 T > C SNPs had no effect on bipolar disorder severity and functionality scores and GDNF serum levels. The results suggest that some SNPs of GDNF have potential association with severity and functionality of bipolar disorder. In addition, except two SNPs, none of GDNF SNPs had association with GDNF serum levels.

  16. 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.

  17. Autologous Pluripotent Stem Cell-Derived β-Like Cells for Diabetes Cellular Therapy.

    PubMed

    Millman, Jeffrey R; Pagliuca, Felicia W

    2017-05-01

    Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into β-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach. © 2017 by the American Diabetes Association.

  18. 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

  19. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    PubMed

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-03-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  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. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Mistletoe lectin modulates intestinal epithelial cell-derived cytokines and B cell IgA secretion.

    PubMed

    Lyu, Su-Yun; Park, Won-Bong

    2009-03-01

    A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. var. coloratum agglutinin, VCA), which is known for its anti-cancer activity, was isolated from Korean mistletoe. In this study, IEC-6 rat intestinal epithelial cells and IM-9 human B-cells were cultured to determine the effect of VCA on cytokine and immunoglobulin (Ig) secretion. In lipopolysaccharide (LPS)-stimulated IEC-6 cells, VCA significantly shifted the interleukin (IL)-2, IL-5, IL-6, and tumor necrosis factor-alpha (TNF-alpha) secretion toward a more immunostimulatory response. Since intestinal epithelial cell-derived secretions may be capable of affecting local B cell Ig production in a variety of ways, we mimicked this condition by deriving a 2-day culture supernatant from IEC-6 cell line which was treated VCA in the presence or absence of LPS, and adding these supernatants to cultures of IM-9 human B cells. As a result, IgA secretion was significantly enhanced at in the presence of VCA at 10(-8)-10(-4) microg/mL. This study suggests that cytokines derived from IEC by VCA may create an environment which may contribute to the enhancement of IgA secretion seen in mucosal tissues. Overall, the induction of cytokines in intestinal epithelial cells, and IgA in B cells by Korean mistletoe lectin could indicate an enhanced immunosurveillance to prevent intestinal infections or other intestinal pathologies.

  8. 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.

  9. Cell-derived matrices for tissue engineering and regenerative medicine applications1

    PubMed Central

    Fitzpatrick, Lindsay E.; McDevitt, Todd C.

    2014-01-01

    The development and application of decellularized extracellular matrices (ECM) has grown rapidly in the fields of cell biology, tissue engineering and regenerative medicine in recent years. Similar to decellularized tissues and whole organs, cell-derived matrices (CDMs) represent bioactive, biocompatible materials consisting of a complex assembly of fibrillar proteins, matrix macromolecules and associated growth factors that often recapitulate, at least to some extent, the composition and organization of native ECM microenvironments. The unique ability to engineer CDMs de novo based on cell source and culture methods makes them an attractive alternative to conventional allogeneic and xenogeneic tissue-derived matrices that are currently harvested from cadaveric sources, suffer from inherent heterogeneity, and have limited ability for customization. Although CDMs have been investigated for a number of biomedical applications, including adhesive cell culture substrates, synthetic scaffold coatings, and tissue engineered products, such as heart valves and vascular grafts, the state of the field is still at a relatively nascent stage of development. In this review, we provide an overview of the various applications of CDM and discuss successes to date, current limitations and future directions. PMID:25530850

  10. 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

  11. 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.

  12. 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...

  13. 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...

  14. Long noncoding RNA BDNF-AS regulates ketamine-induced neurotoxicity in neural stem cell derived neurons.

    PubMed

    Zheng, Xiaozhu; Lin, Chunshui; Li, Yuhong; Ye, Jing; Zhou, Jiali; Guo, Peipei

    2016-08-01

    Ketamine is an anesthetic commonly used in both humans and animals. Emerging evidence has demonstrated that ketamine may induce neurotoxicity in neural stem cell-derived neurons. In this work, we investigated whether long noncoding RNA (lncRNA) Brain derived neurotrophic factor antisense (BDNF-AS) was involved in ketamine-induced neurotoxicity in differentiation of mouse embryonic neural stem cells. Mouse embryonic neural stem cells were differentiated in vitro, and treated with ketamine. The corresponding change in gene expression levels of BDNF and BDNF-AS were assessed by qRT-PCR. BDNF-AS was subsequently downregulated by siRNA. And its effect on protecting neuronal apoptosis, promoting neurite regrowth, and activating TrkB signaling pathways were assessed by TUNEL assay, neurite outgrowth assay, and western blot assay, respectively. In ketamine-injured mouse embryonic neural stem cell-derived neurons, BDNF was downregulated, whereas BDNF-AS was upregulated in dose-dependent manner. SiRNA-mediated BDNF-AS downregulation ameliorated neuronal apoptosis, induced neurite outgrowth, and phosphorylated TrkB signaling pathway after ketamine-induce neurotoxicity in mouse embryonic neural stem cell-derived neurons. Inhibition of BDNF-AS is a novel method to protect ketamine-induced neurotoxicity in mouse embryonic neural stem cell-derived neurons, very likely through the activation of TrkB signaling pathway. Copyright © 2016. Published by Elsevier Masson SAS.

  15. 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

  16. 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.

  17. [Analysis of factors related to the number of mesenchymal stem cells derived from synovial fluid of the temporomandibular joint].

    PubMed

    Sun, Y P; Zheng, Y H; Zhang, Z G

    2017-06-09

    Objective: To analyze related factors on the number of mesenchymal stem cells in the synovial fluid of the temporomandibular joint (TMJ) and provide an research basis for understanding of the source and biological role of mesenchymal stem cells derived from synovial fluid in TMJ. Methods: One hundred and twenty-two synovial fluid samples from 91 temporomandibular disorders (TMD) patients who visited in Department of TMJ Center, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University from March 2013 to December 2013 were collected in this study, and 6 TMJ synovial fluid samples from 6 normal volunteers who were studying in the North Campus of Sun Yat-sen University were also collected, so did their clinical information. Then the relation between the number of mesenchymal stem cells derived from synovial fluid and the health status of the joints, age of donor, disc perforation, condylar bony destruction, blood containing and visual analogue scale score of pain were investigated using Mann-Whitney U test and Spearman rank correlation test. Results: The number of mesenchymal stem cells derived from synovial fluid had no significant relation with visual analogue scale score of pain (r=0.041, P=0.672), blood containing (P=0.063), condylar bony destruction (P= 0.371). Linear correlation between the number of mesenchymal stem cells derived from synovial fluid and age of donor was very week (r=0.186, P=0.043). The number of mesenchymal stem cells up-regulated when the joint was in a disease state (P=0.001). The disc perforation group had more mesenchymal stem cells in synovial fluid than without disc perforation group (P=0.042). Conclusions: The number of mesenchymal stem cells derived from synovial fluid in TMJ has no correlation with peripheral blood circulation and condylar bony destruction, while has close relation with soft tissue structure damage of the joint.

  18. Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro.

    PubMed

    Stary, Martina; Pasteiner, Waltraud; Summer, Alexandra; Hrdina, Astrid; Eger, Andreas; Weitzer, Georg

    2005-11-01

    Cardiomyogenesis proceeds in the presence of signals emanating from extra-embryonic lineages emerging before and during early eutherian gastrulation. In embryonic stem cell derived embryoid bodies, primitive endoderm gives rise to visceral and parietal endoderm. Parietal endoderm undergoes an epithelial to mesenchymal transition shortly before first cardiomyocytes start to contract rhythmically. Here, we demonstrate that Secreted Protein, Acidic, Rich in Cysteine, SPARC, predominantly secreted by mesenchymal parietal endoderm specifically promotes early myocardial cell differentiation in embryoid bodies. SPARC enhanced the expression of bmp2 and nkx2.5 in embryoid bodies and fetal cardiomyocytes. Inhibition of either SPARC or Bmp2 attenuated in both cases cardiomyogenesis and downregulated nkx2.5 expression. Thus, SPARC directly affects cardiomyogenesis, modulates Bmp2 signaling, and contributes to a positive autoregulatory loop of Bmp2 and Nkx2.5 in cardiomyocytes.

  19. 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.

  20. 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

  1. 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.

  2. Differential expression of cell-cycle regulators in human beta-cells derived from insulinoma tissue.

    PubMed

    Ueberberg, Sandra; Tannapfel, Andrea; Schenker, Peter; Viebahn, Richard; Uhl, Waldemar; Schneider, Stephan; Meier, Juris J

    2016-05-01

    The low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. To identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. The frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (p<0.0001), Cyclin C (p<0.0001), and p57 (p=0.018) expression levels were lower. The abundance of Cyclin D3 (p=0.62) and p27 (p=0.68) was not different between the groups. The reduced expression of p16 (p<0.0001) and p57 (p=0.012) in insulinomas and the unchanged expression of Cyclin D3 (p=0.77) and p27 (p=0.55) were confirmed using qRT-PCR. The expression of certain cell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Characterizing human pluripotent-stem-cell-derived vascular cells for tissue engineering applications.

    PubMed

    Kusuma, Sravanti; Facklam, Amanda; Gerecht, Sharon

    2015-02-15

    Tissue-engineered constructs are rendered useless without a functional vasculature owing to a lack of nutrients and oxygen. Cell-based approaches to reconstruct blood vessels can yield structures that mimic native vasculature and aid transplantation. Vascular derivatives of human induced pluripotent stem cells (hiPSCs) offer opportunities to generate patient-specific therapies and potentially provide unlimited amounts of vascular cells. To be used in engineered vascular constructs and confer therapeutic benefit, vascular derivatives must exhibit additional key properties, including extracellular matrix (ECM) production to confer structural integrity and growth factor production to facilitate integration. In this study, we examine the hypothesis that vascular cells derived from hiPSCs exhibit these critical properties to facilitate their use in engineered tissues. hiPSCs were codifferentiated toward early vascular cells (EVCs), a bicellular population of endothelial cells (ECs) and pericytes, under varying low-oxygen differentiation conditions; subsequently, ECs were isolated and passaged. We found that EVCs differentiated under low-oxygen conditions produced copious amounts of collagen IV and fibronectin as well as vascular endothelial growth factor and angiopoietin 2. EVCs differentiated under atmospheric conditions did not demonstrate such abundant ECM expression, but exhibited greater expression of angiopoietin 1. Isolated ECs could proliferate up to three passages while maintaining the EC marker vascular endothelial cadherin. Isolated ECs demonstrated an increased propensity to produce ECM compared with their EVC correlates and took on an arterial-like fate. These findings illustrate that hiPSC vascular derivates hold great potential for therapeutic use and should continue to be a preferred cell source for vascular construction.

  4. 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.

  5. MicroRNA-4443 regulates mast cell activation by T cell-derived microvesicles.

    PubMed

    Shefler, Irit; Salamon, Pazit; Levi-Schaffer, Francesca; Mor, Adam; Hershko, Alon Y; Mekori, Yoseph A

    2017-08-16

    The mechanism by which mast cells (MCs) are activated in T cell-mediated inflammatory processes remains elusive. Recently, we have shown that microvesicles derived from activated T cells (mvT*s) can stimulate MCs to degranulate and release several cytokines. The aim of this study was to characterize the contribution of microRNAs (miRs) delivered by microvesicles to MC activation. miR profiling was performed with NanoString technology and validated by using real-time PCR. The biological role of mvT* miR was verified by overexpression of miRs in MCs using mimic or inhibitory molecules and analyzing the effect on their predicted targets. mvT*s were found to downregulate the expression of the tyrosine phosphatase protein tyrosine phosphatase receptor type J (PTPRJ), a known extracellular signal-regulated kinase inhibitor. Bioinformatics analysis predicted that miR-4443 regulates the PTPRJ gene expression. Indeed, miR-4443, which was present in mvT*s, was also found to be overexpressed in human MCs stimulated with these MVs. α-Amanitin insensitivity confirmed that overexpression of miR-4443 was not due to transcriptional activation. The luciferase reporter assay indicated that the 3' untranslated region of PTPRJ was targeted by this miR. Transfection of MCs with mimic or inhibitor of miR-4443 resulted in decreased or enhanced PTPRJ expression, respectively. Furthermore, miR-4443 regulated extracellular signal-regulated kinase phosphorylation and IL-8 release in MCs activated by mvT*s. These results support a scenario by which T cell-derived microvesicles act as intercellular carriers of functional miR-4443, which might exert heterotypic regulation of PTPRJ gene expression in MCs, leading to their activation in the context of T cell-mediated inflammatory processes. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. 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

  7. 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. ©AlphaMed Press.

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

    PubMed Central

    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-μm 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. PMID:19900180

  9. 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.

  10. 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.

  11. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

    PubMed

    Ayers, Lisa; Kohler, Malcolm; Harrison, Paul; Sargent, Ian; Dragovic, Rebecca; Schaap, Marianne; Nieuwland, Rienk; Brooks, Susan A; Ferry, Berne

    2011-04-01

    Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  12. Clinical safety of intrathecal administration of mesenchymal stromal cell-derived neural progenitors in multiple sclerosis.

    PubMed

    Harris, Violaine K; Vyshkina, Tamara; Sadiq, Saud A

    2016-12-01

    There is a critical unmet need to develop regenerative therapies for the demyelinating disease multiple sclerosis (MS). We previously characterized the immunoregulatory and trophic properties of neural progenitors derived from bone marrow mesenchymal stromal cells (MSC-NPs) and established that cells derived from MS and non-MS patients alike were therapeutically viable. In an experimental model of MS, intrathecal MSC-NP injection resulted in disease amelioration with decreased T-cell infiltration, and less severe lesion pathology associated with recruitment of resident progenitors to inflammatory sites. In this pilot feasibility study, we investigated safety and dosing of intrathecal MSC-NP therapy in six patients with MS. Patients with progressive MS and advanced disability who were refractory to all other conventional MS treatments were enrolled in the study. For each dose, MSC-NP cells were cultured from autologous MSCs and tested for quality control before intrathecal administration. Patients were evaluated for adverse events and neurological status to assess safety of the treatment. Six patients with progressive MS were treated with between 2 and 5 intrathecal injections of escalating doses of autologous MSC-NPs and were followed an average of 7.4 years after initial injection. There were no safety concerns noted, no serious adverse events, and the multiple dosing regimen was well tolerated. Four of the six patients showed a measurable clinical improvement following MSC-NP treatment. This pilot study supports preliminary first-in-human safety and tolerability of autologous MSC-NP treatment for MS. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. Stromal Cell-Derived Factor-1 Alpha Is Decreased in Women With Migraine With Aura.

    PubMed

    Liman, Thomas G; Neeb, Lars; Rosinski, Jana; Reuter, Uwe; Endres, Matthias

    2016-09-01

    Endothelial dysfunction may contribute to the pathophysiology of migraine with aura. Stromal cell-derived factor-1 alpha (SDF-1α) is involved in the maintenance of endothelial integrity via mobilization of vascular stem cells. We sought to determine whether SDF-1α levels are decreased in women with MA. In this post hoc analysis of a case-cohort study, levels of SDF-1α were determined by enzyme-linked immunosorbent assay. Endothelial function was assessed using peripheral arterial tonometry. Arterial stiffness was assessed by fingertip tonometry derived and heart-rate-adjusted augmentation index (AI). Twenty-eight women with MA and 27 age-matched healthy women were included in this study. Levels of SDF-1α were significantly lower in women with MA compared to age- and risk factor-matched healthy women (1763 ± 281 vs 2013 ± 263 pg/mL, P = 0.006). SDF-1α levels were positively correlated with AI in healthy women (r = 0.49, P = 0.009), but not in women with MA (r = 0.05, P = 0.78). SDF-1α levels were negatively correlated with CD144-positive endothelial microparticles (EMP; r = -0.31, P = .02), and activated CD62E-positive EMP (r = -0.35, P = .01). Levels of SDF-1α are decreased in women with MA and are associated with EMPs as a surrogate marker of endothelial dysfunction. This might contribute to the pathophysiology and vascular risk in MA, but evidence from larger prospective studies is warranted. © 2016 American Headache Society.

  14. Using stem cell-derived gametes for same-sex reproduction: an alternative scenario.

    PubMed

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

    2017-10-01

    It has been suggested that future application of stem-cell derived gametes (SCD-gametes) might lead to the possibility for same-sex couples to have genetically related children. Still, for this to become possible, the technique of gamete derivation and techniques of reprogramming somatic cells to a pluripotent state (directly or via somatic cell nuclear transfer) would have to be perfected. Moreover, egg cells would have to be derived from male cells and sperm cells from female cells, which is believed to be particularly difficult, if not impossible. We suggest a more plausible scenario to provide same-sex couples with the possibility to parent a child who is genetically related to both parents. Although technical feasibility is an advantage (also in terms of safety), disadvantages are that cooperation of a donor of the opposite sex is still required and that the partners are genetically linked to the resulting child in a different degree. However, since in our scenario the donor's genetic contribution would not outweigh any of the parents' genetic contribution, this alternative route may ease the fear for a possible parental claim by the donor. Like many other applications in the field of infertility treatment, the goal to create SCD-gametes for reproductive purposes is largely based on the high value attributed to genetic parenthood. Although we believe that genetic relatedness is neither a necessary nor a sufficient condition for 'good' parenthood, we do believe that many people may consider our scenario a welcome alternative. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  15. Notochordal cell-derived conditioned medium protects human nucleus pulposus cells from stress-induced apoptosis.

    PubMed

    Mehrkens, Arne; Matta, Ajay; Karim, Muhammad Zia; Kim, Sarah; Fehlings, Michael G; Schaeren, Stefan; Mark Erwin, William

    2017-04-01

    Degenerative disc disease (DDD) remains without an effective therapy and presents a costly burden to society. Based upon prior reports concerning the effects of notochordal cell-conditioned medium (NCCM) on disc cells, we performed a proof of principle study to determine whether NCCM could reduce cytotoxic stress-induced apoptosis in human disc nucleus pulposus (NP) cells. This is an "in vitro" fundamental or basic science study. Nucleus pulpous cells derived from 15 patients undergoing spinal surgery were treated with interleukin (IL)-1β and Fas ligand or etoposide in the presence of NCCM. We determined pro- or antiapoptotic events using activated caspase assays and determined genomic regulation of apoptosis using polymerase chain reaction arrays validated using Western blotting methods. We interrogated cellular apoptotic regulation using JC-1 dye and flow cytometry and performed enzyme-linked immunosorbent assays to evaluate NP inflammatory cytokine secretion. Notochordal cell-conditioned medium inhibits cytotoxic stress-induced caspase-9 and -3/7 activities and maintains the mitochondrial membrane potential in human NP cells, thereby suppressing the intrinsic apoptotic pathway. Gene expression analysis revealed the X-linked inhibitor of apoptosis protein as a key player responsible for evading etoposide-induced apoptosis in the presence of NCCM, and we verified these data using Western blotting. Enzyme-linked immunosorbent assay results revealed distinct differences in IL-6 and IL-8 secretions by NP cells in response to etoposide in the presence of NCCM. Here we demonstrate for the first time that NCCM reduces cytotoxic stress-induced apoptosis in human NP cells. Soluble factors present in NCCM could be harnessed for the development of novel therapeutics for the treatment of DDD. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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. Copyright © 2016 the American Physiological Society.

  1. 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

  2. Xenopus laevis Stromal cell-derived factor 1: conservation of structure and function during vertebrate development.

    PubMed

    Braun, Mike; Wunderlin, Markus; Spieth, Kathrin; Knöchel, Walter; Gierschik, Peter; Moepps, Barbara

    2002-03-01

    Transmembrane signaling of the CXC chemokine stromal cell-derived factor-1 (SDF-1) is mediated by CXCR4, a G protein-coupled receptor initially identified in leukocytes and shown to serve as a coreceptor for the entry of HIV into lymphocytes. Characterization of SDF-1- and CXCR4-deficient mice has revealed that SDF-1 and CXCR4 are of vital developmental importance. To study the role of the SDF-1/CXCR4-chemokine/receptor system as a regulator of vertebrate development, we isolated and characterized a cDNA encoding SDF-1 of the lower vertebrate Xenopus laevis (xSDF-1). Recombinant xSDF-1 was produced in insect cells, purified, and functionally characterized. Although xSDF-1 is only 64-66% identical with its mammalian counterparts, it is indistinguishable from human (h)SDF-1alpha in terms of activating both X. laevis CXCR4 and hCXCR4. Thus, both xSDF-1 and hSDF-1alpha promoted CXCR4-mediated activation of heterotrimeric G(i2) in a cell-free system and induced release of intracellular calcium ions in and chemotaxis of intact lymphoblastic cells. Analysis of the time course of xSDF-1 mRNA expression during Xenopus embryogenesis revealed a tightly coordinated regulation of xSDF-1 and X. laevis CXCR4. xSDF-1 mRNA was specifically detected in the developing CNS, incipient sensory organs, and the embryonic heart. In Xenopus, CXCR4 mRNA appears to be absent from the heart anlage, but present in neural crest cells. This observation suggests that xSDF-1 expressed in the heart anlage may attract cardiac neural crest cells expressing CXCR4 to migrate to the primordial heart to regulate both septation of the cardiac outflow tract and differentiation of the myocardium during early heart development.

  3. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    PubMed Central

    Kucera, Jan P.; Prudat, Yann; Marcu, Irene C.; Azzarito, Michela; Ullrich, Nina D.

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias. PMID:26442264

  4. 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

  5. Flow rate calibration to determine cell-derived microparticles and homogeneity of blood components.

    PubMed

    Noulsri, Egarit; Lerdwana, Surada; Kittisares, Kulvara; Palasuwan, Attakorn; Palasuwan, Duangdao

    2017-07-18

    Cell-derived microparticles (MPs) are currently of great interest to screening transfusion donors and blood components. However, the current approach to counting MPs is not affordable for routine laboratory use due to its high cost. The current study aimed to investigate the potential use of flow-rate calibration for counting MPs in whole blood, packed red blood cells (PRBCs), and platelet concentrates (PCs). The accuracy of flow-rate calibration was investigated by comparing the platelet counts of an automated counter and a flow-rate calibrator. The concentration of MPs and their origins in whole blood (n=100), PRBCs (n=100), and PCs (n=92) were determined using a FACSCalibur. The MPs' fold-changes were calculated to assess the homogeneity of the blood components. Comparing the platelet counts conducted by automated counting and flow-rate calibration showed an r(2) of 0.6 (y=0.69x+97,620). The CVs of the within-run and between-run variations of flow-rate calibration were 8.2% and 12.1%, respectively. The Bland-Altman plot showed a mean bias of -31,142platelets/μl. MP enumeration revealed both the difference in MP levels and their origins in whole blood, PRBCs, and PCs. Screening the blood components demonstrated high heterogeneity of the MP levels in PCs when compared to whole blood and PRBCs. The results of the present study suggest the accuracy and precision of flow-rate calibration for enumerating MPs. This flow-rate approach is affordable for assessing the homogeneity of MPs in blood components in routine laboratory practice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Postacute stromal cell-derived factor-1α expression promotes neurovascular recovery in ischemic mice.

    PubMed

    Li, Yaning; Huang, Jun; He, Xiaosong; Tang, Guanghui; Tang, Yao-Hui; Liu, Yanqun; Lin, Xiaojie; Lu, Yifan; Yang, Guo-Yuan; Wang, Yongting

    2014-06-01

    Acute interventions of stroke are often challenged by a narrow treatment window. In this study, we explore treatments in the postacute phase of stroke with wider windows of opportunity. We investigated the effects of stromal cell-derived factor (SDF-1α) in neurovascular recovery during the postacute phase and downstream signaling pathways, underlying SDF-1α-mediated neurovascular recovery. Adult male Institute of Cancer Research (ICR) mice underwent middle cerebral artery occlusion. One week after middle cerebral artery occlusion, the animals received stereotactic injection of adenoassociated virus (AAV) carrying SDF-1α gene as treatment or AAV-green fluorescent protein as control and were monitored for 5 weeks. Neurobehavioral outcomes were evaluated, and brain atrophy was measured. Neurogenesis and angiogenesis were examined. The proliferation and migration of neural progenitor cells were evaluated. Downstream pathways of SDF-1α were investigated. Inflammatory response was monitored. Neurobehavioral outcomes were improved, and brain atrophy was greatly reduced for ≤5 weeks in AAV-SDF-1α groups when compared with the control. SDF-1 receptor CXCR4 was upregulated and colocalized with neural and endothelial progenitor cells. The number of nestin(+) and doublecortin(+)/bromodeoxyuridine(+) cells in the subventricular zone, doublecortin(+) and neuron(+)/bromodeoxyuridine(+) cells in the perifocal region, and cluster of differentiation (CD)31(+) and bromodeoxyuridine(+)/CD31(+) microvessels are also significantly increased in AAV-SDF-1α groups. Administration of CXCR4 antagonist AMD3100 eliminated the beneficial effects of SDF-1α. SDF-1α/CXCR4 interaction activated AKT, extracellular signal-regulated kinases (ERK), and P38 mitogen-activated protein kinase (MAPK) signaling pathways but not the c-Jun N-terminal kinase (JNK) pathway. SDF-1α promoted neurogenesis and angiogenesis during the postacute phase of ischemia without eliciting an inflammatory response

  7. Objective evaluation of the degree of pigmentation in human induced pluripotent stem cell-derived RPE.

    PubMed

    Kamao, Hiroyuki; Mandai, Michiko; Wakamiya, Shunji; Ishida, Junko; Goto, Katsutoshi; Ono, Takaaki; Suda, Taiji; Takahashi, Masayo; Kiryu, Junichi

    2014-11-11

    For the transplantation of human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE), determination of the maturation status of these cells is essential, and the degree of pigmentation (dPG) can serve as a good indicator of this status. The aim of this study was to establish a method of objectively and quantitatively evaluating the dPG of hiPSC-RPE. Two observers determined the dPG subjectively by observing recorded images of hiPSC-RPE as follows: the dPG of a single cell was classified into three different pigmentation stages, and the overall dPG was compared between two cell groups to identify the group with the higher dPG. The κ statistic was applied to assess interobserver reproducibility. Next, the dPG of single cells and cell groups was objectively determined by the lightness of the hue, saturation, and value (HSL) color space, and the correlation between the subjective evaluation and time-dependent change in the objective dPG of hiPSC-RPE was investigated. The κ statistic was 0.88 and 0.81 in the single-cell and cell-group observations, respectively. The objective dPG of single cells and cell groups was highly correlated with the subjective dPG. However, the observers were occasionally unable to subjectively determine the group with the higher dPG. The objective dPG increased in a time-dependent manner. The lightness of the HSL color space can be used to objectively and quantitatively evaluate the dPG of hiPSC-RPE in culture. The objective evaluation was consistent and was able to better identify small differences than subjective evaluation. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  8. 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

    2017-09-01

    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.

  9. 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

  10. 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.

  11. Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function.

    PubMed

    Liau, Brian; Christoforou, Nicolas; Leong, Kam W; Bursac, Nenad

    2011-12-01

    Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell-derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25 cm/s, and 3) significant contractile forces of up to 2 mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease. Copyright © 2011

  12. Stromal cell-derived factor 2 is critical for Hsp90-dependent eNOS activation.

    PubMed

    Siragusa, Mauro; Fröhlich, Florian; Park, Eon Joo; Schleicher, Michael; Walther, Tobias C; Sessa, William C

    2015-08-18

    Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser(1177), a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser(1177) in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells. Copyright © 2015, American Association for the Advancement of Science.

  13. 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

  14. 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

  15. 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

  16. Vascular Morphogenesis of Human Umbilical Vein Endothelial Cells on Cell-Derived Macromolecular Matrix Microenvironment

    PubMed Central

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

    2014-01-01

    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

  17. 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.

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

    PubMed

    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

    2015-09-01

    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

  19. Comparison between fibroblasts and mesenchymal stem cells derived from dermal and adipose tissue.

    PubMed

    Brohem, C A; de Carvalho, C M; Radoski, C L; Santi, F C; Baptista, M C; Swinka, B B; de A Urban, C; de Araujo, L R R; Graf, R M; Feferman, I H S; Lorencini, M

    2013-10-01

    Stem cells have the ability to renew themselves and differentiate into various cell types. For this reason, numerous research groups have been studying these cells for their therapeutic potential. Some of the therapies, however, are not producing the expected results because of contamination by other cell types, especially by fibroblasts. In the cosmetic industry, stem cells are used to test the efficacy of anti-ageing and rejuvenation products. The purpose of this work was to gain a better understanding of the differences in phenotype, in gene expression associated with stem cells, in the pattern of cell surface proteins and in the differentiation capacity of adipose-derived stem cells, of skin-derived stem cells and of commercially available fibroblasts. In this study, we compared fibroblasts with mesenchymal stem cells derived from bone marrow, skin (dermis) and adipose tissue, to assess the differentiation potential of fibroblasts. Dermal and adipose stem cells were isolated from aesthetic surgery patients, and fibroblasts were obtained from a commercial source. The following parameters were used in this study: immunophenotypic profile (positive: CD29, CD73, CD90 and CD105; negative: CD14, CD45 and HLA-DR); differentiation into osteoblastic, chondrogenic and adipogenic cell types; and PCR array to analyse the gene expression of cells isolated from different culture passages. Fibroblasts express the same cell immunophenotypic markers, as well as the genes that are known to be expressed in stem cells, and were shown to be expressed also in adipose and dermis stem cells. Fibroblasts are also able to differentiate into the three cell lineages mentioned above, that is, adipocytes, osteocytes and chondrocytes. Human dermal fibroblasts have a potential to adhere to plastic surfaces and differentiate into other cell types. However, for stem cells intended to be used in cosmetics, experiments conducted with contaminated fibroblasts may produce poor or even falsely

  20. Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.

    PubMed

    Lee, Junmin; Abdeen, Amr A; Tang, Xin; Saif, Taher A; Kilian, Kristopher A

    2016-09-15

    Mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical and biochemical properties of the extracellular matrix. In this work we conduct a combinatorial study of matrix properties that influence adipogenesis and neurogenesis including: adhesion proteins, stiffness, and cell geometry, for mesenchymal stem cells derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). We uncover distinct differences in integrin expression, the magnitude of traction stress, and lineage specification to adipocytes and neuron-like cells between cell sources. In the absence of media supplements, adipogenesis in AT-MSCs is not significantly influenced by matrix properties, while the converse is true in BM-MSCs. Both cell types show changes in the expression of neurogenesis markers as matrix cues are varied. When cultured on laminin conjugated microislands of the same adhesive area, BM-MSCs display elevated adipogenesis markers, while AT-MSCs display elevated neurogenesis markers; integrin analysis suggests neurogenesis in AT-MSCs is guided by adhesion through integrin αvβ3. Overall, the properties of the extracellular matrix guides MSC adhesion and lineage specification to different degrees and outcomes, in spite of their similarities in general characteristics. This work will help guide the selection of MSCs and matrix components for applications where high fidelity of differentiation outcome is desired. Mesenchymal stem cells (MSCs) are an attractive cell type for stem cell therapies; however, in order for these cells to be useful in medicine, we need to understand how they respond to the physical and chemical environments of tissue. Here, we explore how two promising sources of MSCs-those derived from bone marrow and from adipose tissue-respond to the compliance and composition of tissue using model extracellular matrices. Our results demonstrate a source-specific propensity to undergo adipogenesis and neurogenesis, and

  1. 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

  2. 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

  3. 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

  4. 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

  5. Label-free identification and characterization of human pluripotent stem cell-derived cardiomyocytes using second harmonic generation (SHG) microscopy

    PubMed Central

    Awasthi, Samir; Matthews, Dennis L.; Li, Ronald A.; Chiamvimonvat, Nipavan; Lieu, Deborah K.; Chan, James W

    2013-01-01

    Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSCCM populations is important for this application, but is hampered by a lack of CM-specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non-specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC-CMs that is attributable to sarcomeric myosin, dependent on PSC-CM maturity, and retained while PSCCMs are in suspension. Our study demonstrates the feasibility of developing a SHG-activated flow cytometer for the noninvasive purification of PSC-CMs. Second harmonic generation (SHG) from sarcomeric myosin can be used to identify human plutipotent stem cell-derived cardiomyocytes (PSC-CMs) in suspension and discriminate groups of PSC-CMs according to maturity. PMID:22083829

  6. 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

  7. 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

  8. Microvesicles released from human embryonic stem cell derived-mesenchymal stem cells inhibit proliferation of leukemia cells.

    PubMed

    Ji, Yuan; Ma, Yongbin; Chen, Xiang; Ji, Xianyan; Gao, Jianyi; Zhang, Lei; Ye, Kai; Qiao, Fuhao; Dai, Yao; Wang, Hui; Wen, Xiangmei; Lin, Jiang; Hu, Jiabo

    2017-08-01

    Human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSCs) are able to inhibit proliferation of leukemia cells. Microvesicles released from human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSC‑MVs) might play an important part in antitumor activity. Microvesicles were isolated by ultracentrifugation and identified under a scanning electron microscopy and transmission electron microscope separately. After 48-h cocultured with hESC‑MSCs and hESC‑MSC‑MVs, the number of K562 and HL60 was counted and tumor cell viability was measured by CCK8 assay. The expression of proteins Bcl-2 and Bax were estimated by western blotting. Transmission electron microscope and western blot analysis were adopted to evaluate the autophagy level. Results showed that both hESC‑MSCs and hESC‑MSC‑MVs inhibited proliferation of leukemia cells in a concentration-dependent manner. hESC‑MSC‑MVs reduced the ratio of Bcl/Bax, enhanced the protein level of Beclin-1 and LC3-II conversion, thus upregulating autophagy and apoptosis. In conclusion, microvesicles released from human embryonic stem cell derived-mesenchymal stem cells inhibited tumor growth and stimulated autophagy and excessive autophagy might induce apoptosis.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

    PubMed

    Tønnesen, Jan; Parish, Clare L; Sørensen, Andreas T; Andersson, Angelica; Lundberg, Cecilia; Deisseroth, Karl; Arenas, Ernest; Lindvall, Olle; Kokaia, Merab

    2011-03-04

    Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

  14. Finding the rhythm of sudden cardiac death: new opportunities using induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Sallam, Karim; Li, Yingxin; Sager, Philip T; Houser, Steven R; Wu, Joseph C

    2015-06-05

    Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death. © 2015 American Heart Association, Inc.

  15. 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

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

    PubMed

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

    2016-07-01

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

  17. 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.

  18. 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 ...

  19. 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.

  20. Virtual karyotyping reveals greater chromosomal stability in neural cells derived by transdifferentiation than those from stem cells.

    PubMed

    Weissbein, Uri; Ben-David, Uri; Benvenisty, Nissim

    2014-12-04

    Neural cells can be derived either from pluripotent or adult stem cells via differentiation or by transdifferentiation from other cell types with the aid of tissue regulators. We compared the chromosomal stability of over 500 neural cell samples from human and mouse with virtual karyotyping (e-karyotyping). We detected notable genomic instability in cells derived from pluripotent or adult stem cells, but surprisingly, transdifferentiated cells seemed more chromosomally stable, except if they were reprogrammed using pluripotency factors. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. 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.

  2. Occurrence of Donor Cell-derived Lymphoid Blast Crisis 24 Years Following Related Bone Marrow Transplantation for Chronic Myeloid Leukemia.

    PubMed

    Kurosawa, Shuhei; Doki, Noriko; Hino, Yutaro; Sakaguchi, Masahiro; Fukushima, Kazuaki; Shingai, Naoki; Hattori, Keiichiro; Watanabe, Ken; Hagino, Takeshi; Igarashi, Aiko; Najima, Yuho; Kobayashi, Takeshi; Kakihana, Kazuhiko; Sakamaki, Hisashi; Ohashi, Kazuteru

    2016-01-01

    We herein report a unique case of donor cell leukemia (DCL), as donor cell-derived lymphoid blast crisis of chronic myeloid leukemia (CML) was observed 24 years after related bone marrow transplantation for CML in the chronic phase. Short tandem repeat testing of the leukemic blast sample revealed full donor chimerism, strongly indicative of DCL. The original donor is healthy with a normal complete blood cell count for the past 24 years. This rare case may provide a precious opportunity to consider not only the underlying mechanism of DCL, but also the pathogenesis of CML.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

    SciTech Connect

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; Meer, Berend van; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G.J.; Mummery, Christine L.; Casini, Simona

    2015-11-27

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation. - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. Akt Suppression of TGFβ Signaling Contributes to the Maintenance of Vascular Identity in Embryonic Stem Cell-Derived Endothelial Cells

    PubMed Central

    Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

    2016-01-01

    The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (EC) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells, and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs, and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs, and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. PMID:23963623

  19. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors.

    PubMed

    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte; Kozubenko, Nataliya; Itier, Valerie; Tine, Samir; Tritschler, Laurent; Brenot, Marion; Guidou, Emmanuelle; Blondeau, Johanna; Lhuillier, Mickael; Bugi, Aurore; Aubry, Laetitia; Jendelova, Pavla; Sykova, Eva; Perrier, Anselme L; Finsen, Bente; Onteniente, Brigitte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors. Four differentiation stages were identified on the basis of quantitative polymerase chain reaction expression of pluripotency, proliferation, and differentiation markers. Neural progenitors were transplanted at these 4 stages into rats with no, small, or large middle cerebral artery occlusion lesions. The fate of each transplant was compared with their pretransplantation status 1 to 4 months posttransplantation. The influence of the postischemic environment was limited to graft survival and occurrence of nonneuroectodermal structures after transplantation of very immature neural progenitors. Both effects were lost with differentiation. We identified a particular stage of differentiation characterized in vitro by a rebound of proliferative activity that produced highly proliferative grafts susceptible to threaten surrounding host tissues. The effects of the ischemic environment on the formation of teratoma by transplanted human embryonic stem cell-derived neural progenitors are limited to early differentiation stages that will likely not be used for stem cell therapy. In contrast, hyperproliferation observed at later stages of differentiation corresponds to an intrinsic activity that should be monitored to avoid tumorigenesis.

  20. 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

  1. 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

  2. Prediction of drug-induced immune-mediated hepatotoxicity using hepatocyte-like cells derived from human embryonic stem cells.

    PubMed

    Kim, Dong Eon; Jang, Mi-Jin; Kim, Young Ran; Lee, Joo-Young; Cho, Eun Byul; Kim, Eunha; Kim, Yeji; Kim, Mi Young; Jeong, Won-Il; Kim, Seyun; Han, Yong-Mahn; Lee, Seung-Hyo

    2017-07-15

    Drug-induced liver injury (DILI) is a leading cause of liver disease and a key safety factor during drug development. In addition to the initiation events of drug-specific hepatotoxicity, dysregulated immune responses have been proposed as major pathological events of DILI. Thus, there is a need for a reliable cell culture model with which to assess drug-induced immune reactions to predict hepatotoxicity for drug development. To this end, stem cell-derived hepatocytes have shown great potentials. Here we report that hepatocyte-like cells derived from human embryonic stem cells (hES-HLCs) can be used to evaluate drug-induced hepatotoxic immunological events. Treatment with acetaminophen significantly elevated the levels of inflammatory cytokines by hES-HLCs. Moreover, three human immune cell lines, Jurkat, THP-1, and NK92MI, were activated when cultured in conditioned medium obtained from acetaminophen-treated hES-HLCs. To further validate, we tested thiazolidinedione (TZD) class, antidiabetic drugs, including troglitazone withdrawn from the market because of severe idiosyncratic drug hepatotoxicity. We found that TZD drug treatment to hES-HLCs resulted in the production of pro-inflammatory cytokines and eventually associated immune cell activation. In summary, our study demonstrates for the first time the potential of hES-HLCs as an in vitro model system for assessment of drug-induced as well as immune-mediated hepatotoxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-Derived Myocardial Precursors

    PubMed Central

    Mihardja, Shirley S.; Liszewski, Walter; Erle, David J.; Lee, Randall J.; Bernstein, Harold S.

    2011-01-01

    Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease. PMID:21245908

  8. Human Mesenchymal Stem Cell-Derived Conditioned Media for Hair Regeneration Applications.

    PubMed

    Ramdasi, Sushilkumar; Tiwari, Shashi Kant

    Hair loss can have major psychological impact on affected population belonging to varied ethnic background. Hair is a mini organ in itself and serves many distinguishing functions ranging from maintaining body temperature to promoting social interactions. Major cause of hair loss is androgenic alopecia. Hair follicles possess receptor for androgen. However, DHT (Dihydrotestosterone) in excess results into shrinkage of hair follicle affecting hair growth adversely. The present review is focused on etiology of hair loss, traditional treatment approach and their limitations with side effects with special emphasis on unique properties of stem cells, favourable growth factors secreted by stem cells and strategies to enhance favourable growth factor/cytokine production for hair loss therapeutics. We discussed in details the present available treatment options for hair loss like drugs (Finasteride and Minoxidil), follicular hair transplant, laser therapy and serum therapy. These treatment options have their own disadvantages and side effects with appropriate alerts from regulatory authorities. The side effects of these modalities cannot be ignored and demands alternate therapy approach with less or no side effects. We feel that the stem cell therapy is advancing and is a promising modality in near future owing to its advantages and promising outcomes. This review article discusses possible stem cell therapy for hair regrowth and its advantages. We focused on use of conditioned media derived from stem cells instead of using stem cells directly for the therapy.

  9. 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.

  10. 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.

  11. Prothrombin Loading of Vascular Smooth Muscle Cell-Derived Exosomes Regulates Coagulation and Calcification.

    PubMed

    Kapustin, Alexander N; Schoppet, Michael; Schurgers, Leon J; Reynolds, Joanne L; McNair, Rosamund; Heiss, Alexander; Jahnen-Dechent, Willi; Hackeng, Tilman M; Schlieper, Georg; Harrison, Paul; Shanahan, Catherine M

    2017-03-01

    The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative co