Expression Of Pluripotency-associated Factors Research Articles

Snap29 Is Dispensable for Self-Renewal Maintenance but Required for Proper Differentiation of Mouse Embryonic Stem Cells

Pluripotent embryonic stem cells (ESCs) can self-renew indefinitely and are able to differentiate into all three embryonic germ layers. Synaptosomal-associated protein 29 (Snap29) is implicated in numerous intracellular membrane trafficking pathways, including autophagy, which is involved in the maintenance of ESC pluripotency. However, the function of Snap29 in the self-renewal and differentiation of ESCs remains elusive. Here, we show that Snap29 depletion via CRISPR/Cas does not impair the self-renewal and expression of pluripotency-associated factors in mouse ESCs. However, Snap29 deficiency enhances the differentiation of ESCs into cardiomyocytes, as indicated by heart-like beating cells. Furthermore, transcriptome analysis reveals that Snap29 depletion significantly decreased the expression of numerous genes required for germ layer differentiation. Interestingly, Snap29 deficiency does not cause autophagy blockage in ESCs, which might be rescued by the SNAP family member Snap47. Our data show that Snap29 is dispensable for self-renewal maintenance, but required for the proper differentiation of mouse ESCs.

GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients.

GENYOi004-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with autism-related ADNP syndrome carrying a pTyr719* mutation

ADNP syndrome is an intellectual disability associated with Autism spectrum disorder caused by mutations in ADNP. We generated an iPSC line from an ADNP syndrome pediatric patient harboring the mutation p.Trp719* (GENYOi004-A). Peripheral blood mononuclear cells were reprogrammed using a non-transmissible form of Sendai viruses expressing the four Yamanaka factors (Oct3/4, SOX2, KLF4 and c-MYC). Characterization of GENYOi004-A included mutation analysis of ADNP by allele-specific PCR, genetic identity by Short Tandem Repeats polymorphism profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and pluripotency studies in vivo. GENYOi004-A will be useful to evaluate ADNP syndrome alterations at early developmental stages.

Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst.

The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst-stage embryo. The EPI lineage isdistinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur invivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages invivo. A period of apoptosis occurred concomitantly with ICM cell-fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early mammalian embryonic development prevents fluctuations in cell fate.

Hypoxia Supports Reprogramming of Mesenchymal Stromal Cells Via Induction of Embryonic Stem Cell–Specific microRNA-302 Cluster and Pluripotency-Associated Genes

Pluripotency is characterized by specific transcription factors such as OCT4, NANOG, and SOX2, but also by pluripotency-associated microRNAs (miRs). Somatic cells can be reprogrammed by forced expression of these factors leading to induced pluripotent stem cells (iPSCs) with characteristics similar to embryonic stem cells (ESCs). However, current reprogramming strategies are commonly based on viral delivery of the pluripotency-associated factors, which affects the integrity of the genome and impedes the use of such cells in any clinical application. In an effort to establish nonviral, nonintegrating reprogramming strategies, we examined the influence of hypoxia on the expression of pluripotency-associated factors and the ESC-specific miR-302 cluster in primary and immortalized mesenchymal stromal cells (MSCs). The combination of hypoxia and fibroblast growth factor 2 (FGF2) treatments led to the induction of OCT4 and NANOG in an immortalized cell line L87 and primary MSCs, accompanied with increased doubling rates and decreased senescence. Most importantly, the endogenous ECS-specific cluster miR-302 was induced upon hypoxic culture and FGF2 supplementation. Hypoxia also improved reprogramming of MSCs via episomal expression of pluripotency factors. Thus, our data illustrate that hypoxia in combination with FGF2 supplementation efficiently facilitates reprogramming of MSCs.