We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem cells (ESCs) by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ESC line harboring an inducible Lhx2 gene cassette. When ESCs are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/cofactors are involved in the Lhx2-mediated expansion of HSC-like cells from ESCs. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells.
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.
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