hematopoiesis ͉ HOX genes ͉ microarray ͉ self-renewal ͉ embryoid bodies

Induced pluripotent stem cells (iPSCs) may become a promising source for the generation of patientspecific 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. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:581-591

Ectopic expression of the homeodomain transcription factor HOXB4 has been shown to enhance the in vivo repopulation ability of in vitro differentiated mouse ES‐cells. How it promotes the conversion of ES‐cells to HSCs is not yet known. Here we demonstrate that its expression enforces the development of the developmentally earliest known hematopoietic cell during ES‐cell differentiation and promotes its expansion in stroma‐cell free suspension cultures. As these cultures lead to long‐term engraftment after transplantation into Rag2(‐/‐)γ(‐/‐) mice, the ability to engraft may correlate with the presence of these earliest, CD41hi cells. To test whether the CD41+ subpopulation depends on expression levels of HOXB4, we designed new tetracycline regulatable cassettes in which all necessary components were embedded in a single retroviral vector.The presence of a CD41hi / c‐Kit+/− / CD45− subpopulation correlated with HOXB4 expression levels in clonal ES‐HC cultures. This subpopulation was capable of reconstituting the entire heterogeneity of the primary suspension culture and differentiated towards all lineages in colony assays.In summary, we show that HOXB4 expression enhances the in vitro generation and expansion of the earliest known HSCs from mouse ES‐cells. Its tight and fine‐tuned regulation may also support the generation and safe expansion of patient‐specific pluripotent stem cell derived HSCs.