Serum- and Stromal Cell-Free Hypoxic Generation of Embryonic Stem Cell-Derived Hematopoietic Cells In Vitro, Capable of Multilineage Repopulation of Immunocompetent Mice

Inducedpluripotentstemcells(iPSCs)maybecomeapromisingsourceforthegenerationofpatient- 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 strategyforgeneratingHSCsfrompluripotentcellsinvitrohasbeenestablishedinthemousemodel involving ectopic expression of the human transcription factor HOXB4. However, most differentia- tion 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 repopu- latingHSCscan,inprinciple,begeneratedfromembryonicstemcellswithoutstromacellsorserum. Here, we showed that long-term multilineage engraftment could be accomplished in immunocom- petent mice when HSCs were generated in serum-free medium without stroma cell support and when hypoxic conditions were used. Under those conditions, HOXB4embryonic stem cell-derived hematopoietic stem and progenitor cells were immunophenotypically similar to definitive bone marrow resident E-SLAM (CD150CD48 − CD45CD201) 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