Modifications in chromatin, including DNA methylation and histone modification, are known to be important epigenetic determinants of gene transcription. DNA methylation levels fluctuate markedly in early mouse development. In preimplantation development, the mouse embryo undergoes active and passive genomic demethylation (3,15). This is restored at the time of implantation by the combined action of de novo and maintenance DNA methyltransferases (Dnmts). Studies of DNA methyltransferase 1-deficient (Dnmt1 Ϫ/Ϫ ) and Dnmt3a/ Dnmt3b-deficient (Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ]) mouse embryos have demonstrated that restoring DNA methylation is essential for development (13,19). Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryos exhibit an early-lethal phenotype. At day 9.5 postcoitus, the embryos appear to have gastrulated but exhibit marked growth delay, having failed to turn or develop somites. In the presence of Dnmt1 deficiency, development is thought to fail because of cell death. Dnmt1-deficient embryoid bodies (EBs) aberrantly express Xist, down-regulate X-linked genes, and apoptose when induced to differentiate (20). Late-passage hypomethylated Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryonic stem (ES) cells are unable to form teratomas in vivo, but the cause of their differentiation failure has not been studied (4).Early embryonic development is characterized by high levels of Dnmt3a and Dnmt3b expression. These enzymes clearly have roles in initiating remethylation of the genome following preimplantation demethylation, but it is not known whether continued de novo methyltransferase activity is required for development once global remethylation has taken place. This was our reason for studying the differentiation of Dnmt[3a Ϫ/Ϫ , 3b Ϫ/Ϫ ] ES cells in vitro. These mutant ES cells were derived from fully methylated wild-type ES cells and would have been predicted to have retained most of their methylation because of the continued presence of the maintenance methyltransferase Dnmt1. In fact, while early-passage Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells are well methylated, DNA methylation levels fall progressively in culture (4). However, the rate of loss and the precise levels of methylation remaining have not been quantified. We have used a quantitative assay of DNA methylation to examine the effects of progressively decreasing genomic methylation levels on differentiation in vitro. Our studies reveal a clear but unexpected difference between the behaviors of hypomethylated Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells in in vitro assays of differentiation. At very low levels of DNA methylation, Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells demonstrate an inability to initiate differentiation upon leukemia inhibitory factor (LIF) withdrawal, remaining viable and retaining markers characteristic of undifferentiated ES cells. MATERIALS AND METHODSES cell culture. ES cells were maintained on gelatin in a Glasgow modification of Eagle medium (Invitrogen) supplemented with 10% fetal calf serum, 100 M 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 ...

Hematopoietic stem cells derived from human embryonic stem cells (hESCs) could provide a therapeutic alternative to bone marrow transplants, but the efficiency of currently available derivation protocols is low. In this study, we investigated whether coculture with monolayers of cells derived from mouse AGM and fetal liver, or with stromal cell lines derived from these tissues, can enhance hESC hematopoietic differentiation. We found that under such conditions hESC-derived differentiating cells formed early hematopoietic progenitors, with a peak at day 18-21 of differentiation that corresponded to the highest CD34 expression. These hESC-derived hematopoietic cells were capable of primary and secondary hematopoietic engraftment into immunocompromised mice at substantially higher levels than described previously. Transcriptional and functional analysis identified TGF-beta1 and TGF-beta3 as positive enhancers of hESC hematopoietic differentiation that can further stimulate this process when added to the culture. Overall, our findings represent significant progress toward the goal of deriving functional hematopoietic stem cells from hESCs.

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