Mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. We determined global gene expression profiles for mouse and human hematopoietic stem cells and other stages of the hematopoietic hierarchy. Murine and human hematopoietic stem cells share a number of expressed gene products, which define key conserved regulatory pathways in this developmental system. Moreover, in the mouse, a portion of the genetic program of hematopoietic stem cells is shared with embryonic and neural stem cells. This overlapping set of gene products represents a molecular signature of stem cells.

Dendritic cells (DC) migrate into inflamed peripheral tissues where they capture antigens and, following maturation, to lymph nodes where they stimulate T cells. To gain insight into this process we compared chemokine receptor expression in immature and mature DC. Immature DC expressed CCR1, CCR2, CCR5 and CXCR1 and responded to their respective ligands, which are chemokines produced at inflammatory sites. Following stimulation with LPS or TNF-§ maturing DC expressed high levels of CCR7 mRNA and acquired responsiveness to the CCR7 ligand EBI1 ligand chemokine (ELC), a chemokine produced in lymphoid organs. Maturation also resulted in up-regulation of CXCR4 and down-regulation of CXCR1 mRNA, while CCR1 and CCR5 mRNA were only marginally affected for up to 40 h. However, CCR1 and CCR5 were lost from the cell surface within 3 h, due to receptor down-regulation mediated by chemokines produced by maturing DC. A complete down-regulation of CCR1 and CCR5 mRNA was observed only after stimulation with CD40 ligand of DC induced to mature by LPS treatment. These different patterns of chemokine receptors are consistent with "inflammatory" and "primary response" phases of DC function.

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.

Generation of reprogrammed induced pluripotent stem cells (iPSC) from patients with defined genetic disorders promises important avenues to understand the etiologies of complex diseases, and the development of novel therapeutic interventions. We have generated iPSC from patients with LEOPARD syndrome (LS; acronym of its main features: Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary valve stenosis, Abnormal genitalia, Retardation of growth and Deafness), an autosomal dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-MAPK signaling diseases, which also includes Noonan syndrome (NS), with pleiomorphic effects on several tissues and organ systems1,2. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSC have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LS-iPSC are larger, have a higher degree of sarcomeric organization and preferential localization of NFATc4 in the nucleus when compared to cardiomyocytes derived from human embryonic stem cells (HESC) or wild type (wt) iPSC derived from a healthy brother of one of the LS patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signaling pathways that may promote the disease phenotype.

Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells1–5. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-β and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.

Summary Definitive hematopoiesis emerges during embryogenesis via an endothelial-to-hematopoietic transition. We attempted to induce this process in mouse fibroblasts by screening a panel of factors for hemogenic activity. We identified a combination of four transcription factors, Gata2, Gfi1b, cFos, and Etv6 that efficiently induces endothelial-like precursor cells with the subsequent appearance of hematopoietic cells. The precursor cells express a human CD34 reporter, Sca1 and Prominin1 within a global endothelial transcription program. Emergent hematopoietic cells possess nascent/specifying hematopoietic stem cell gene expression profiles and cell surface phenotypes. After transgene silencing and reaggreagtion culture the specified cells generate hematopoietic colonies in vitro. Thus, we have shown that a simple combination of transcription factors is sufficient to induce a complex, dynamic and multi-step developmental program in vitro. These findings provide insights into the specification of definitive hemogenesis and a platform for future development of patient-specific stem/progenitor cells as well as more differentiated blood products.

The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Here, using fibroblasts derived from macroH2A double knockout mice we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild type fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A double knockout induced pluripotent cells are able to differentiate properly in vitro and in vivo, such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation ‘lock’ at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram.

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.