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. CultrexTM) 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.



Stage1 Differentiation Medium Stage 2 Differentiation Medium Stage 3 Differentiation Medium Stage 3 Specific Medium
STAPEL medium   STAPEL medium   STAPEL medium   STAPEL medium  
CHIR 99021 (Cat.No. 4423) 0.5 μM BMP-4 (314-BP/CF) 20 ng/mL BMP-4 (314-BP/CF) 10 ng/mL SCF (7466-SC/CF) 100 ng/mL
BMP-4 (314-BP/CF) 20 ng/mL VEGF (293-VE/CF) 50 ng/mL SCF (7466-SC/CF) 100 ng/mL Flt3 (308-FK/CF) 100 ng/mL
VEGF (293-VE/CF) 25 ng/mL SCF (7466-SC/CF) 50 ng/mL Flt3 (308-FK/CF) 100 ng/mL TPO (288-TP/CF) 50 ng/mL
SCF (7466-SC/CF) 25 ng/mL IGF-2 (292-G2) 10 ng/mL TPO (288-TP/CF) 50 ng/mL IL-6 (7270-IL/CF) 25 ng/mL
Activin A (388-AC/CF) 7.5 ng/mL FGF-2 (3718-FB) 10 ng/mL VEGF (293-VE/CF) 50 ng/mL IL-3 (203-IL/CF) 25 ng/mL
FGF-2 (3718-FB) 10 ng/mL     IL-6 (7270-IL/CF) 25 ng/mL Penicillin/Streptomycin (B21210) Optional
From Day 1       IL-3 (203-IL/CF) 25 ng/mL    
CHIR 99021 (Cat.No. 4423) 3 μM     IGF-2 (292-G2) 10 ng/mL    
SB 431542 (Cat.No. 1614) 3.8 μM     FGF-2 (3718-FB) 10 ng/mL    
        Penicillin/Streptomycin (B21210) Optional    

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