Serum- and stromal cell-free hypoxic generation of embryonic stem cell-derived hematopoietic cells in vitro, capable of multilineage repopulation of immunocompetent mice.

Induced pluripotent stem cells (iPSCs) may become a promising source for the generation of patient-specific hematopoietic stem cells (HSCs) in vitro. A crucial prerequisite will be the availability of reliable protocols for the directed and efficient differentiation toward HSCs. So far, the most robust strategy for generating HSCs from pluripotent cells in vitro has been established in the mouse model involving ectopic expression of the human transcription factor HOXB4. However, most differentiation protocols include coculture on a xenogenic stroma cell line and the use of animal serum. Involvement of any of both would pose a major barrier to the translation of those protocols to human autologous iPSCs intended for clinical use. Therefore, we asked whether long-term repopulating HSCs can, in principle, be generated from embryonic stem cells without stroma cells or serum. Here, we showed that long-term multilineage engraftment could be accomplished in immunocompetent mice when HSCs were generated in serum-free medium without stroma cell support and when hypoxic conditions were used. Under those conditions, HOXB4(+) embryonic stem cell-derived hematopoietic stem and progenitor cells were immunophenotypically similar to definitive bone marrow resident E-SLAM(+) (CD150(+)CD48(-)CD45(+)CD201(+)) HSCs. Thus, our findings may ease the development of definitive, adult-type HSCs from pluripotent stem cells, entirely in vitro.

HOXB4's road map to stem cell expansion.

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent "universal" targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-alpha inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.