SUMMARY The embryonic stem (ES) cell transcriptional and epigenetic networks are critical for the maintenance of ES cell self-renewal. However, it remains unclear whether components of these networks functionally interact and if so, what factors mediate such interactions. Here we show that WD-repeat protein-5 (Wdr5), a core member of the mammalian Trithorax (trxG) complex, positively correlates with the undifferentiated state and is a novel regulator of ES cell self-renewal. We demonstrate that Wdr5, an ‘effector’ of H3K4 methylation, interacts with the pluripotency transcription factor Oct4. Genome-wide protein localization and transcriptome analyses demonstrate overlapping gene regulatory functions between Oct4 and Wdr5. We show that the Oct4-Sox2-Nanog circuitry and trxG cooperate in activating transcription of key self-renewal regulators. Furthermore, Wdr5 expression is required for the efficient formation of induced pluripotent stem (iPS) cells. We propose an integrated model of transcriptional and epigenetic control, mediated by select trxG members, for maintenance of ES cell self-renewal and somatic cell reprogramming.

SUMMARY In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni Syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs.

Human embryonic stem cells (HESCs) are pluripotent cells derived from the ICM of blastocyst stage embryos. As the factors needed for their growth are largely undefined, they are propagated on feeder cells or with conditioned media from feeder cells. This is in contrast to mouse embryonic stem cells (MESCs) where addition of leukemia inhibitory factor (LIF) replaces the need for a feeder layer. Recently, the transcription factor Nanog was suggested to allow LIF and feeder-free growth of MESCs. Here, we show that NANOG overexpression in HESCs enables their propagation for multiple passages during which the cells remain pluripotent. NANOG overexpressing cells form colonies efficiently even at a very low density, an ability lost upon excision of the transgene. Cells overexpressing NANOG downregulate expression of markers specific to the ICM and acquire expression of a marker specific to the primitive ectoderm (the consecutive pluripotent population in the embryo). Examination of global transcriptional changes upon NANOG overexpression by DNA microarray analysis reveals new markers suggested to discriminate between these populations. These results are significant in the understanding of self-renewal and pluripotency pathways in HESCs, and of their use for modeling early development in humans.

SUMMARY Many signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to up-regulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming.

Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.

The present study provides a molecular basis for the identity of human ES cells and demonstrates that during their in vitro differentiation they express embryonic specific genes in a stage specific manner.

LIN-28 is a gene recently shown to be involved in the conversion of somatic cells to induced pluripotent stem cells. We have previously shown that LIN-28 is highly expressed in human embryonic stem cells (HESCs); however, its role in these cells has not been investigated. We now show that, like OCT4, SOX2, and NANOG, LIN-28 is downregulated during differentiation of HESCs into embryoid bodies. In addition, we investigate the role of LIN-28 in HESCs by manipulation of its expression levels. LIN-28 overexpression impairs the ability of cells to grow at clonal densities, due to increased differentiation and decreased cell division. Analysis of cell differentiation under these conditions revealed that it is mostly towards the extraembryonic endoderm lineage. Moreover, we show that, during early mouse development, high levels of Lin-28 are also observed in the extraembryonic endoderm, and therefore it seems that, both in vitro and in vivo, high levels of LIN-28 may specify an extraembryonic endoderm fate. However, LIN-28 seems dispensable for selfrenewal of HESCs; its downregulation neither impairs HESC proliferation nor leads to their differentiation. Thus, LIN-28 does not seem to be involved in the self-renewal of HESCs, but rather seems to be involved in their decision to switch from self-renewal to differentiation.

SummaryAnalyses of gene expression in single mouse embryonic stem cells (mESCs) cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM). In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN) reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP) data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB) suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.