SUMMARY Self-renewal of human embryonic stem (ES) cells is promoted by FGF and TGFβ/Activin signaling, and differentiation is promoted by BMP signaling, but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium, we show here that both TGFβ and FGF signals synergize to inhibit BMP signaling, sustain expression of pluripotency-associated genes such as NANOG, OCT4, and SOX2, and promote long-term undifferentiated proliferation of human ES cells. We also show that both TGFβ- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFβ/Activin and FGF signaling, and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels, and makes NANOG unresponsive to BMP and TGFβ signaling. These results suggest that direct binding of TGFβ/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ES cell self-renewal.

X chromosome inactivation (XCI) is an essential mechanism for dosage compensation of X-linked genes in female cells. We report that subcultures from lines of female human embryonic stem cells (hESCs) exhibit variation (0 -100%) for XCI markers, including XIST RNA expression and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on the inactive X chromosome (Xi). Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a monoallelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have already completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already not random. Moreover, XIST gene expression in subsets of cultured female hESCs is unstable and subject to stable epigenetic silencing by DNA methylation. In the absence of XIST expression, Ϸ12% of X-linked promoter CpG islands become hypomethylated and a portion of X-linked alleles on the Xi are reactivated. Because alterations in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine.culture variation ͉ DNA methylation ͉ gene regulation H uman embryonic stem cells (hESCs) are regarded as one of the most promising stem cells for regenerative medicine because of their unusual capacity of self-renewal and pluripotency (1). However, given the variations in the derivation and propagation of hESCs in different laboratories, it is imperative to establish a common set of criteria for the quality control of hESCs. Efforts have been devoted to characterizing whether established lines of hESCs carry inherent differences in gene expression and epigenetic modifications such as DNA methylation (2). Although different lines of hESCs can exhibit a common set of stem cell markers, differences in gene expression are observed including allelic expression of several imprinted genes and XIST, a crucial gene for X-inactivation (2). Several studies also demonstrated that in vitro cultures or differentiation of hESCs can contribute to changes in CpG methylation patterns and genome stability in different lines of hESCs (2-4). Thus, routine and thorough characterization of genetic and epigenetic stability in hESCs is a necessary step to ensure the quality of hESCs for regenerative medicine.X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in female cells (5). So far, only a few studies have examined XCI in female hESCs and conflicting data exist regarding the nature of XCI. It has been reported that Ϸ50% of all established female hESC lines exhibit XCI markers such as XIST expression and/or punctate histone H3 lysine 27 trimethylation (H3K27me3) staining on the inactive X chromosome (Xi), whereas other lines do not (2, 6-9). Moreover, discrepan...

Dendritic cells (DCs) are the most commonly studied source of the cytokine interleukin-15 (IL-15). Using an IL-15 reporter transgenic mouse, we have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8+ and CD8-). Here we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (Lin-Sca-1+c-kit+; LSKs) expressed high levels of IL-15 suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was Notch-dependent and occurred in a step-wise pattern coincident with increasing maturation and commitment to a T cell fate. Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory activity of IL-15 production occurring at the pre-DC branch point leading to the generation of both IL-15+ CD8+ and IL-15-/low CD8-DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells.

Nuclear receptors (NRs) regulate gene expression in essential biological processes including differentiation and development. Here we report the systematic profiling of NRs in human and mouse embryonic stem cell (ESC) lines and during their early differentiation into embryoid bodies. Expression of the 48 human and mouse NRs was assessed by quantitative real-time PCR. In general, expression of NRs between the two human cell lines was highly concordant, whereas in contrast, expression of NRs between human and mouse ESCs differed significantly. In particular, a number of NRs that have been implicated previously as crucial regulators of mouse ESC biology, including ERRbeta, DAX-1, and LRH-1, exhibited diametric patterns of expression, suggesting they may have distinct species-specific functions. Taken together, these results highlight the complexity of the transcriptional hierarchy that exists between species and governs early development. These data should provide a unique resource for further exploration of the species-specific roles of NRs in ESC self-renewal and differentiation.

Bone marrow (BM) fibrosis is a feature of severe hyperparathyroidism. Consistent with this observation, mice expressing constitutively active parathyroid hormone (PTH)/PTH-related peptide receptors (PPR) in osteoblasts (PPR*Tg) display BM fibrosis. To obtain insight into the nature of BM fibrosis in such a model, a double-mutant mouse expressing constitutively active PPR and green fluorescent protein (GFP) under the control of the type I collagen promoter (PPR*Tg/GFP) was generated. Confocal microscopy and flow cytometry revealed the presence of a cell population expressing GFP (GFP(+)) that was also positive for the hematopoietic marker CD45 in the BM of both PPR*Tg/GFP and control animals. This cell population was expanded in PPR*Tg/GFP. The existence of cells expressing both type I collagen and CD45 in the adult BM was confirmed by IHC and fluorescence-activated cell sorting. An analysis of total RNA extracted from sorted GFP(+)CD45(+) cells showed that these cells produced type I collagen and PTH/PTH-related peptide receptor and receptor activator for NF-κB mRNAs, further supporting their features of being both mesenchymal and hematopoietic lineages. Similar cells, known as fibrocytes, are also present in pathological fibroses. Our findings, thus, indicate that the BM is a permissive microenvironment for the differentiation of fibrocyte-like cells and raise the possibility that these cells could contribute to the pathogenesis of BM fibrosis.