The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPSderived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases.

SUMMARY Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3′UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.

The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that co-differentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). In order to better characterize the iPGCs we performed Real time PCR, microarray and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure.

Summary Lysine 56 acetylation in the helical core of histone H3 opens yeast chromatin and enables histone gene transcription, DNA replication, DNA repair, and prevents epigenetic silencing. While K56Ac is globally abundant in yeast and flies its presence has been uncertain in mammals. We show here using mass spectrometry and genome wide analyses that K56Ac is present in human embryonic stem cells (hESCs) overlapping strongly at active and inactive promoters with the binding of the key regulators of pluripotency NANOG, SOX2 and OCT4. This includes also the canonical histone gene promoters and those for the hESC-specific microRNAs. K56Ac then relocates to developmental genes upon cellular differentiation. Thus K56Ac state more accurately reflects the epigenetic differences between hESCs and somatic cells than other active histone marks such as H3 K4 tri-methylation and K9 acetylation. These results suggest that K56Ac is involved in the human core transcriptional network of pluripotency.