Differentiation of Hematopoietic Progenitors from hPSCs

This is intended as a guide only; for full experimental details please read the reference provided.

Nafria et al. describe a 3-stage procedure for the differentiation of human pluripotent stem cells to multipotent hematopoietic progenitors in 14+ days using a cocktail of small molecules and growth factors.

Briefly, human pluripotent stem cells (hPSCs) are co-cultured  with feeder cells for 1 week, after which the hPSCs are harvested, dissociated and seeded in non-adherent round-bottom 96 well plates in stage 1 differentiation medium. On day 0 plates are centrifuged and cells aggregate forming embryoid bodies (EB; 1 per well).  On day 1, SB 431542 and CHIR 99021 are added to the Stage 1 differentiation medium to direct the mesoderm toward the definitive hematopoietic program. On day 4 the medium is exchanged for Stage 2 medium without small molecule inhibitors. At day 8 of differentiation EBs are transferred to Basement Membrane Extract (BME, e.g. Cultrex^TM) coated 6-well plates containing stage 2 medium. The next day (Day 9) the medium is removed and replaced with stage 3 medium. Then around day 13-15 Stage 3 medium is replaced with Stage 3 Specific medium to promote proliferation and differentiation of hematopoietic lineage cells.

Vascular structures and hemogenic endothelium expressing the SOX17 marker are generated from day 8 to 14 and from day 14 onward, SOX17- CD34+ RUNX1C-/+ hematopoietic progenitors are generated within the SOX17+ endothelium displaying similarities to human aorta-gonad mesonephros (AGM) and adult hematopoietic stem cells.