Human Embryonic Stem Cell Fate Research Articles

Generation of an RNF1-deficient human pluripotent stem cell line using CRISPR/Cas9 technology

RNF1 (RING1A) is a catalytic component of the polycomb repressive complex 1 (PRC1) involved in regulation of, among others, embryonic development and disease progression. However, the exact role of RNF1 in self-renewal and differentiation of human embryonic stem cells (ESCs) remains unknown. Here, we derive one RNF1 knockout human ESC line using CRISPR/Cas9 system. The cell line retains the canonical stem cell morphology and normal karyotype. Moreover, the cell line highly expresses pluripotency genes and has three germ-layer differentiation potential. The RNF1 -/- cell line will be useful for studies on the function and role of RNF1 in human embryonic stem cell fate decisions.

β-catenin coordinates with Jup and the TCF1/GATA6 axis to regulate human embryonic stem cell fate

β-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of human embryonic stem cells (hESCs). However, the results are controversial and the mechanism by which β-catenin regulates the hESC fate remains unclear. Here, we report that β-catenin and γ-catenin are functionally redundant in mediating hESC adhesion and are required for embryoid body formation, but both genes are dispensable for hESC maintenance, as the undifferentiated state of β-catenin and γ-catenin double deficient hESCs can be maintained. Overexpression of β-catenin induces rapid hESC differentiation. Functional assays revealed that TCF1 plays a crucial role in hESC differentiation mediated by β-catenin. Forced expression of TCF1, but not other LEF1/TCF family members, resulted in hESC differentiation towards the definitive endoderm. Conversely, knockdown of TCF1 or inhibition of the interaction between TCF1 and β-catenin delayed hESC exit from pluripotency. Furthermore, we demonstrated that GATA6 plays a predominant role in TCF1-mediated hESC differentiation. Knockdown of GATA6 completely eliminated the effect of TCF1, while forced expression of GATA6 induced hESC differentiation. Our data thus reveal more detailed mechanisms for β-catenin in regulating hESC fate decisions and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.

Activin A Modulates CRIPTO-1/HNF4α+Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes.

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.

SummaryNonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

Computational Analysis of Transcriptional Circuitries in Human Embryonic Stem Cells Reveals Multiple and Independent Networks

It has been known that three core transcription factors (TFs), NANOG, OCT4, and SOX2, collaborate to form a transcriptional circuitry to regulate pluripotency and self-renewal of human embryonic stem (ES) cells. Similarly, MYC also plays an important role in regulating pluripotency and self-renewal of human ES cells. However, the precise mechanism by which the transcriptional regulatory networks control the activity of ES cells remains unclear. In this study, we reanalyzed an extended core network, which includes the set of genes that are cobound by the three core TFs and additional TFs that also bind to these cobound genes. Our results show that beyond the core transcriptional network, additional transcriptional networks are potentially important in the regulation of the fate of human ES cells. Several gene families that encode TFs play a key role in the transcriptional circuitry of ES cells. We also demonstrate that MYC acts independently of the core module in the regulation of the fate of human ES cells, consistent with the established argument. We find that TP53 is a key connecting molecule between the core-centered and MYC-centered modules. This study provides additional insights into the underlying regulatory mechanisms involved in the fate determination of human ES cells.

6 Differentiation of human embryonic stem cells towards the endothelial lineage involves microRNAs

There is evidence demonstrating the regulation of microRNAs in a myriad of vascular biology events such as cardiogenesis, but the role of miRNA in controlling human embryonic stem cell fate...

13-P041 The balancing action between activin A/TGFβ, FGF, Wnt and BMP signalling instructs human embryonic stem cell fate decisions

13-P041 The balancing action between activin A/TGFβ, FGF, Wnt and BMP signalling instructs human embryonic stem cell fate decisions