Enhanced CLIP Uncovers IMP Protein-RNA Targets in Human Pluripotent Stem Cells Important for Cell Adhesion and Survival.

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 levels, 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 increase in cell death. For cell adhesion, we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally, we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.

Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal cells.

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 codifferentiation 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). To better characterize the iPGCs, we performed Real-time polymerase chain reaction, 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.

Directed differentiation of human-induced pluripotent stem cells generates active motor neurons.

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 iPS-derived 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.

Histone h3 lysine 56 acetylation is linked to the core transcriptional network in human embryonic stem cells.

Lysine 56 acetylation in the helical core of histone H3 opens yeast chromatin and enables histone gene transcription, DNA replication, and 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 the K56Ac state more accurately reflects the epigenetic differences between hESCs and somatic cells than other active histone marks such as H3 K4 trimethylation and K9 acetylation. These results suggest that K56Ac is involved in the human core transcriptional network of pluripotency.

A self-renewal program controls the expansion of genetically unstable cancer stem cells in pluripotent stem cell-derived tumors.

Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer stem cells. To determine whether cancer stem cells can be identified in a transplantation model of testicular germ cell tumor, we transplanted murine embryonic germ cells (EGCs) into the testis of adult severe combined immunodeficient mice. Transplantation resulted in a locally invasive solid tumor, with a cellular component that generated secondary tumors upon serial transplantation. The secondary tumors were invariably metastatic, a feature not observed in the primary tumors derived from EGCs. To characterize the differences between EGCs and the tumor-derived stem cells, we performed karyotype and microarray analysis. Our results show that generation of cancer stem cells is associated with the acquisition of nonclonal genomic rearrangements not found in the originating population. Furthermore, pretreatment of EGCs with a potent inhibitor of self-renewal, retinoic acid, prevented tumor formation and the emergence of these genetically unstable cancer stem cells. Microarray analysis revealed that EGCs and first- and second-generation cancer stem cells were highly similar; however, approximately 1,000 differentially expressed transcripts could be identified corresponding to alterations in oncogenes and genes associated with motility and development. Combined, the data suggest that the activation of oncogenic pathways in a cellular background of genetic instability, coupled with an inherent ability to self-renew, is involved in the acquisition of metastatic behavior in the cancer stem cell population of tumors derived from pluripotent cells.