In Vivo Repopulating Activity Emerges at the Onset of Hematopoietic Specification during Embryonic Stem Cell Differentiation

The generation of in vivo repopulating hematopoietic cells from in vitro differentiating embryonic stem cells has remained a long-standing challenge. To date, hematopoietic engraftment has mostly been achieved through the enforced expression of ectopic transcription factors. Here, we describe serum-free culture conditions that allow the generation of in vivo repopulating hematopoietic cells in the absence of ectopically expressed factors. We show that repopulating activity arises immediately upon the commitment of mesodermal precursors to the blood program, within the first wave of hematopoietic specification. We establish that the formation of these progenitors is extremely transient and exquisitely sensitive to the cytokine milieu. Our findings define the precise differentiating stage at which hematopoietic repopulating activity first appears in vitro, and suggest that during embryonic stem cell differentiation, all hematopoietic programs are unraveled simultaneously from the mesoderm in the absence of cues that restrict the coordinated emergence of each lineage as is normally observed during embryogenesis.