WNT/β-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3β inhibitor (GSKi) has been shown to facilitate in vitro endothelial cell differentiation from hESCs/hiPSCs. Since hematopoietic and endothelial cells are derived from common progenitors (hemogenic endothelial progenitors [HEPs]), we examined the effect of transient GSKi treatment on hematopoietic cell differentiation from hiPSCs. We found that transient GSKi treatment at the start of hiPSC differentiation induction altered the gene expression profile of the cells. Multiple CDX/HOX genes, which are expressed in the posterior mesoderm of developing embryos, were significantly upregulated by GSKi treatment. Further, inclusion of the GSKi in a serum- and stroma-free culture with chemically defined medium efficiently induced HEPs, and the HEPs gave rise to various lineages of hematopoietic and endothelial cells. Therefore, transient WNT/β-CATENIN signaling triggers the activation of the CDX/HOX pathway, which in turn confers hemogenic posterior mesoderm identity to differentiating hiPSCs. These data enhance our understanding of human embryonic hematopoietic/endothelial cell development and provide a novel in vitro system for inducing the differentiation of hematopoietic cells from hiPSCs.
LIM-homeobox transcription factor Lhx2 induces ex vivo amplification of adult hematopoietic stem cells (HSCs) in mice. We previously showed that engraftable HSC-like cells are generated from mouse embryonic stem cells (ESCs) and induced pluripotent stem cells by enforced expression of Lhx2. However, when these HSC-like cells were transplanted into irradiated congenic mice, donor-derived T cells were barely detectable, whereas other lineages of hematopoietic cells were continuously produced. Here we investigated T-cell differentiation potential of the Lhx2-induced HSC-like cells using ESCs carrying doxycycline (dox)-inducible Lhx2 expression cassette. Dox-mediated over-expression of Lhx2 conferred a self-renewing activity to ESC-derived cKit + CD41 + embryonic hematopoietic progenitor cells (HPCs), thereby converting them to HSClike cells. When these HSC-like cells were transplanted into irradiated immunodeficient mice and they were supplied with a dox-containing water, CD4/8 double negative T cells were detected in their thymi. Once the Lhx2 expression was terminated, differentiation of CD4/8 double positive and single positive T cells was initiated in the thymi of transplanted mice and mature T cells were released in the peripheral blood. These results showed that engraftable HSC-like cells with full hematopoietic potential can be obtained from ESCs by the conditional expression of Lhx2.
The generation of mouse hematopoietic stem cells from hemogenic endothelial cells (HECs) in the aorta/gonad/mesonephros region of developing embryos requires a zinc finger transcription factor Gata2. In the previous study, an enforced expression of Gata2 in vitro promoted the production of HECs from mesodermal cells differentiated from mouse embryonic stem cells (ESCs). Our research group has previously demonstrated that the enforced expression of Gata2 in ESC-derived HECs enhances erythroid and megakaryocyte differentiation and inhibits macrophage differentiation. However, the manner in which the multiple functions of Gata2 are regulated remains unclear. Mouse ESCs differentiate into various types of hematopoietic cells when cocultured with OP9 stromal cells (OP9 system). Using this system and the inducible gene cassette exchange system, which facilitates the establishment of ESCs carrying inducible transgenes under an identical gene expression regulatory unit, the domain-specific functions of Gata2 were systematically dissected in this study. We determined that the N-terminal (amino acid 1-110) region of Gata2 was an erythroid-inducing region, both the middle (amino acid 111-200) and C-terminal (amino acid 413-480) regions were megakaryocyte-inducing regions. Furthermore, the present data strongly suggest that intramolecular antagonistic interactions between each of these regions fine-tune the biological functions of Gata2.
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