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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|