Differentiation Of hESCs Research Articles

Abstract 16005: Highly Efficient Derivation of Human Pacemaker Cells From Pluripotent Stem Cells in Chemically Defined Conditions

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. Dysfunction of the cardiac conduction system plays a central role in the pathogenesis of arrhythmia. While much progress has been made understanding cardiomyocyte differentiation, the molecular mechanisms regulating the specification and patterning of cells that form this conductive network is largely unknown. The LIM-homeodomain transcription factor ISL1 is highly expressed in the secondary heart field (SHF) progenitor population that makes a substantial contribution to the developing heart, comprising most cells in the right ventricle, both atria and pacemaker cells. Pacemaker cells comprise the most proximal component of the cardiac conduction system, which have been proposed as the source of most arrhythmogenic events. Their dominance on other spontaneous beating cell types makes them a suitable target for pharmacologic compounds, making access to this cell lineage necessary for the study of new therapeutic agents. To identify the signaling pathways that control the differentiation of human embryonic stem cell (hESC)-derived SHF cells into pacemaker cells, we performed RNA sequencing to compare the hESC-derived ISL1 + population, non-enriched population and undifferentiated hESCs. Furthermore, using a small molecule screen we identified compounds that can improve differentiation of hESCs toward pacemaker cells. Pathway analysis identified the Wnt pathway as the most significant regulator of SHF specification. Further differentiation of human pluripotent stem cells by stage-specific activation of BMP and WNT signaling pathways resulted in phenotypic pacemaker cells, which display morphological characteristics. More than 80% of these cells stained positively for HCN4, Contactin2(CNTN2) and GATA6, key markers of pacemaker cells. The differentiated cells express pacemaker markers, including CNTN2, TBX2, TBX3, HCN4, TBX18, GATA6 indicated by qRT-PCR. They show inward potassium currents through HCN channels in patch clamp experiments. Our data provides a new strategy to obtain human cardiac conduction cells in large scale for disease modeling, drug screening and cell therapy.

Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.

ABSTRACTShort bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue.

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.

Transcriptional Activation of Human CDCA8 Gene Regulated by Transcription Factor NF-Y in Embryonic Stem Cells and Cancer Cells

The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

MicroRNA dynamics during human embryonic stem cell differentiation to pancreatic endoderm

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as critical regulators of human embryonic stem cell (hESC) pluripotency and differentiation. Despite the wealth of information about the role individual that miRNAs play in these two processes, there has yet to be a large-scale temporal analysis of the dynamics of miRNA expression as hESCs move from pluripotency into defined lineages. In this report, we used Next Generation Sequencing (NGS) to map temporal expression of miRNAs over ten 24-hour intervals as pluripotent cells were differentiated into pancreatic endoderm. Of the 2042 known human miRNAs, 694 had non-zero expression on all 11days. Of these 694 miRNAs, 494 showed statistically significant changes in expression during differentiation. Clusters of miRNAs were identified, each displaying unique expression profiles distributed over multiple days. Selected miRNAs associated with pluripotency/differentiation (miR-302/367 and miR-371/372/373) and development/growth (miR-21, miR-25, miR-103, miR-9, and miR-92a) were found to have distinct expression profiles correlated with changes in media used to drive the differentiation process. Taken together, the clustering of miRNAs to identify expression dynamics that occur over longer periods of time (days vs. hours) provides unique insight into specific stages of differentiation. Major shifts in defined stages of hESC differentiation appear to be heavily dependent upon changes in external environmental factors, rather than intrinsic conditions in the cells.

Gene Manipulation of Human Embryonic Stem Cells by In Vitro-Synthesized mRNA for Gene Therapy.

The difficulty in producing genetically modified human embryonic stem cells (hESCs) limits research on their applications. Virus-based gene transfer is not safe for clinical use, whereas DNAbased non-viral methods are not efficient or safe, and mRNA-based methods are useful for genetic manipulation. In this study, we easily obtained multiple types and large amounts of in vitro-synthesized mRNA by PCR. The efficiency of different transfection methods was studied by flow cytometry. The effect of different mRNA modifications on protein translation efficiency and dynamics of luciferase mRNA expression in hESCs were studied using a bioluminescence imaging system. The pluripotency of hESCs after transfection was studied by immunofluorescence. In vitro-synthesized pancreatic-duodenal homeobox 1 (PDX1) mRNA was used to induce the differentiation of hESCs into insulin-producing cells. We found that electroporation is the most efficient transfection method, and it produces more than 95% transgene expression in multiple hESC lines. Synthesized mRNA with a combination of a polyA tail, cap and base analogues is more efficiently translated into protein in hESCs compared with single-modified mRNA. Transfection of mRNA into hESCs by trypsinizing the cells into single-cell suspensions did not affect their pluripotency, and multiple types of mRNAs can be transfected into hESCs efficiently. We found that PDX-1 mRNA transfection significantly improved the expression level of genes related to beta cells and differentiated cells that express insulin and C-peptide. ELISA analysis validate the insulin secretion of islet-like cell clusters in response to glucose stimulation. Our results indicate that electroporation of in vitro-synthesized mRNA is useful for genetic manipulation of hESCs and differentiation of hESCs into particular cell types, and this finding will pave the way for clinical applications of this method.

Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

Human embryonic stem cells (hESCs) undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency “signature” would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI) methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5) induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells.

RING1B O-GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells

O-linked-N-acetylglucosamine (O-GlcNAc) post-translationally modifies and regulates thousands of proteins involved in various cellular mechanisms. Recently, O-GlcNAc has been linked to human embryonic stem cells (hESC) differentiation, however the identity and function of O-GlcNAc proteins regulating hESC remain unknown. Here, we firstly identified O-GlcNAc modified human stem cell regulators such as hnRNP K, HP1γ, and especially RING1B/RNF2. Thereafter, we focused our work on RING1B which is the catalytic subunit of the polycomb repressive complex 1 (PRC1) a major epigenetic repressor essential for pluripotency maintenance and differentiation. By point-mutation, we show that T250/S251 and S278 RING1B residues are bearing O-GlcNAc, and that T250/S251 O-GlcNAcylation decreases during differentiation. O-GlcNAc seems to regulate RING1B-DNA binding as suggested by our ChIP-sequencing results. Non-O-GlcNAcylated RING1B is found to be enriched near cell cycle genes whereas O-GlcNAcylated RING1B seems preferentially enriched near neuronal genes. Our data suggest that during hESC differentiation, the decrease of RING1B O-GlcNAcylation might enable PRC1 to switch its target to induce neuron differentiation. Overall, we demonstrate that O-GlcNAc modifies and regulates an essential epigenetic tool, RING1B, which may contribute to hESC pluripotency maintenance and differentiation.

Efficient in vitro generation of functional thymic epithelial progenitors from human embryonic stem cells.

Thymic epithelial cells (TECs) are the major components of the thymic microenvironment for T cell development. TECs are derived from thymic epithelial progenitors (TEPs). It has been reported that human ESCs (hESCs) can be directed to differentiate into TEPs in vitro. However, the efficiency for the differentiation is low. Furthermore, transplantation of hESC-TEPs in mice only resulted in a very low level of human T cell development from co-transplanted human hematopoietic precursors. We show here that we have developed a novel protocol to efficiently induce the differentiation of hESCs into TEPs in vitro. When transplanted into mice, hESC-TEPs develop into TECs and form a thymic architecture. Most importantly, the hESC-TECs support the long-term development of functional mouse T cells or a higher level of human T cell development from co-transplanted human hematopoietic precursors. The hESC-TEPs may provide a new approach to prevent or treat patients with T cell immunodeficiency.

Wnt/β-Catenin and MEK-ERK Signaling are Required for Fibroblast-Derived Extracellular Matrix-Mediated Endoderm Differentiation of Embryonic Stem Cells.

Human embryonic stem cells (hESCs) have the potential to differentiate into all cells of the three germ layers, thus making them an attractive source of cells for use in regenerative medicine. The greatest challenge lies in regulating the differentiation of hESCs into specific cell lineages by both intrinsic and extrinsic factors. In this study we determined the effect of a fibroblast-derived extracellular matrix (fd-ECM) on hESCs differentiation. We demonstrate that growth of hESCs on fd-ECM results in hESCs losing their stemness and proliferation potential. As the stem cells differentiate they attain gene expression profiles similar to the primitive streak of the in vivo embryo. The activation of both the MEK-ERK and Wnt/β-catenin signaling pathways is required for the fd-ECM-mediated differentiation of hESCs towards the endoderm and involves integrins α1, α2, α3 and β1. This study illustrates the importance of the cellular microenvironment in directing stem cell fate and that the nature and composition of the extracellular matrix is a crucial determining factor.

Variability of miRNA expression during the differentiation of human embryonic stem cells into retinal pigment epithelial cells

Embryonic stem cells (ESCs) and induced pluripotent stem cells can be induced to differentiate into retinal pigment epithelium (RPE). MiRNAs have been characterized and found playing important roles in the differentiation process of ESCs, but their length and sequence heterogeneity (isomiRs), and their non-canonical forms of miRNAs are underestimated or ignored. In this report, we found some non-canonical miRNAs (dominant isomiRs) in all differentiation stages, and 27 statistically significant editing sites were identified in 24 different miRNAs. Moreover, we found marked major-to-minor arm-switching events in 14 pre-miRNAs during the hESC to RPE cell differentiation phases. Our study for the first time reports exploring the variability of miRNA expression during the differentiation of hESCs into RPE cells and the results show that miRNA variability is a ubiquitous phenomenon in the ESC differentiation.

A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells

SummaryThe development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs), including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy.

Nanotopographical control of human embryonic stem cell differentiation into definitive endoderm.

Derivation of definitive endoderm (DE) from human embryonic stem cells (hESCs) can address the needs of regenerative medicine for endoderm-derived organs such as the pancreas and liver. Fibrous substrates which topographically recapitulate native extracellular matrix have been known to promote the stem cell differentiation. However, the optimal fiber diameter remains to be determined for the desired differentiation. Here, we have developed a simple method to precisely fabricate electrospun poly(ε-caprolactone) fibers with four distinct average diameters at nano- and microscale levels (200, 500, 800, and 1300 nm). Human ESCs were cultured as clumps or single cells and induced into DE differentiation to determine the optimal topography leading to the promoted differentiation compared with planar culture plates. Gene expression analysis of the DE-induced cells showed significant upregulation of DE-specific genes exclusively on the 200-nm fibers. By Western blot analysis, significant expression of DE-specific proteins was found when hESCs were cultured on the 200 nm substrate as single cells rather than clumps, probably due to more efficient cell-matrix interaction realized by morphological observations of the cell colonies. The results indicated that nanofibrillar substrates, only at ultrathin fiber diameters, provided a better environment for DE differentiation of hESC, which holds great promise in prospective tissue engineering applications.

Derivation of Engrafting Skeletal Muscle Precursors from Human Embryonic Stem Cells Using Serum-Free Methods

Degenerative muscle diseases affect muscle tissue integrity and function. Human embryonic stem cells (hESC) are an attractive source of cells to use in regenerative therapies due to their unlimited capacity to divide and ability to specialize into a wide variety of cell types. A practical way to derive therapeutic myogenic stem cells from hESC is lacking. In this study, we demonstrate the development of two serumfree conditions to direct the differentiation of hESC towards a myogenic precursor state. Using TGFs and PI3Kinase inhibitors in combination with bFGF we showed that one week of differentiation is sufficient for hESC to specialize into PAX3 + /PAX7 + myogenic precursor cells. These cells also possess the capacity to further differentiate in vitro into more specialized myogenic cells that express MYOD, Myogenin, Desmin and MYHC, and showed engraftment in vivo upon transplantation in immunodeficient mice. Ex vivo myomechanical studies of dystrophic mouse hindlimb muscle showed functional improvement one month post-transplantation. In summary, this study describes a promising system to derive engrafting muscle precursor cells solely using chemical substances in serum-free conditions and without genetic manipulation.

HESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature

SummaryTo gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.

Cardiac Development in Zebrafish and Human Embryonic Stem Cells Is Inhibited by Exposure to Tobacco Cigarettes and E-Cigarettes

BackgroundMaternal smoking is a risk factor for low birth weight and other adverse developmental outcomes.ObjectiveWe sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo.MethodsZebrafish (Danio rerio) were used to assess developmental effects in vivo and cardiac differentiation of human embryonic stem cells (hESCs) was used as a model for in vitro cardiac development.ResultsIn zebrafish, exposure to both types of cigarettes results in broad, dose-dependent developmental defects coupled with severe heart malformation, pericardial edema and reduced heart function. Tobacco cigarettes are more toxic than e-cigarettes at comparable nicotine concentrations. During cardiac differentiation of hESCs, tobacco smoke exposure results in a delayed transition through mesoderm. Both types of cigarettes decrease expression of cardiac transcription factors in cardiac progenitor cells, suggesting a persistent delay in differentiation. In definitive human cardiomyocytes, both e-cigarette- and tobacco cigarette-treated samples showed reduced expression of sarcomeric genes such as MLC2v and MYL6. Furthermore, tobacco cigarette-treated samples had delayed onset of beating and showed low levels and aberrant localization of N-cadherin, reduced myofilament content with significantly reduced sarcomere length, and increased expression of the immature cardiac marker smooth muscle alpha-actin.ConclusionThese data indicate a negative effect of both tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Tobacco cigarettes are more toxic than E-cigarettes and exhibit a broader spectrum of cardiac developmental defects.

Co-culture with endometrial stromal cells enhances the differentiation of human embryonic stem cells into endometrium-like cells.

In vitro differentiation of human embryonic stem cells (hESCs) into endometrium-like cells may provide a useful tool for clinical treatment. The aim of the present study was to investigate the differentiation potential of hESCs into endometrium-like cells using three methods, which included induction by feeder cells, co-culture with endometrial stromal cells and induction with embryoid bodies. Following differentiation, the majority of cells positively expressed cytokeratin and epithelial cell adhesion molecule (EPCAM). Factors associated with endometrium cell function, namely the estrogen and progesterone receptors (ER and PR), were also detected. At day 21 following the induction of differentiation, the expression levels of cytokeratin, EPCAM, ER and PR were significantly increased in the co-culture method group, as compared with the other two methods. Furthermore, these cells became decidualized in response to progesterone and prolactin. In addition, the number of cytokeratin-positive or EPCAM-positive cells significantly increased following the induction of differentiation using the co-culture method, as compared with the other two methods. The mRNA expression levels of Wnt members that are associated with endometrial development were subsequently examined, and Wnt5a was found to be significantly upregulated in the differentiated cells induced by feeder cells and co-culture with endometrial stromal cells; however, Wnt4 and Wnt7a expression levels were unaffected. Additionally, the mRNA expression levels of Wnt5a in the differentiated cells co-cultured with endometrial stromal cells were higher when compared with those induced by feeder cells. In conclusion, the present findings indicated that the co-culture system is the optimal protocol for the induction of hESC differentiation into endometrium-like cells, and Wnt5a signaling may be involved in this process.

A ground state of PPARγ activity and expression is required for appropriate neural differentiation of hESCs.

Several evidences indicate stimulation of peroxisome proliferator activated receptor γ (PPARg), promotes neuronal differentiation. This study was conducted to testify the prominence of PPARγ during neural differentiation of human embryonic stem cells (hESCs). PPARγ expression level was assessed during neural differentiation of hESCs. Meanwhile, the level of endogenous miRNAs, which could be engaged in regulation of PPARγ expression, was measured. Next, natural and synthetic components of PPARγ agonists and antagonist were implemented on neural progenitor formation during neural differentiation of hESCs. Data showed an increasing wave of PPARγ expression level when human neural progenitors (NPs) were formed upon retinoic acid treatment. Interestingly, there was no significant difference in the amount of PPARγ proteins during the differentiation of hESCs that is inconsistent with what we observed for RNA level. Our results indicated that miRNAs are not involved in the regulation of PPARγ expression, while proteasome-mediated degradation may to some degree be involved in this process. Among numerous treatments, PPARγ inactivation during NPs formation significantly decreased expression of NP markers. We conclude that a ground state of PPARγ activity is required for NP formation of hESCs during early neural differentiation. However, high expression and activity of PPARγ could not enhance the required neural differentiation, whereas the PPARγ inactivation could negatively influence NP formation from hESCs by antagonist.

661. Assay Development and Qualification for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Studies

Human embryonic stem cell (hESC)-derived cellular products have been considered as promising candidates for cell replacement therapy for multiple degenerative diseases. At the Center of Biomedicine and Genetics (CBG), we have manufactured under cGMP numerous hESC-differentiated products including neuron stem cells (NSC), neuronal progenitor cells (NPC), retinal pigment epithelium (RPE) cells, dopaminergic neurons and cardiomyocytes intended for pre-clinical and clinical studies. Specifically, cardiomyocytes are produced through hESC differentiation procedure utilizing adherent and notably suspension culture systems. Prior to their release for clinical application, cardiomyocytes must be thoroughly tested for their identity and purity. We have developed a systematic approach to characterize hESC-derived cardiomyocytes using assays such as real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. The development and qualification of each assay involve the selection and banking of positive and negative controls, selection and banking of critical reagents, determination of assay conditions, qualification of assay, and generation of assay qualification report and standard operating procedure (SOP). We will also examine a number of critical factors including assay specificity, sensitivity, assay controls, and establishment of assay specifications.