Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) into Hematopoietic Progenitors

Induced pluripotent stem cells are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells, and mature hematopoietic cells in a suitable culture system. Nowadays, HSCs transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Due to insufficient donors and limited cell numbers, pluripotent stem cell-induced hematopoietic cells have been considered as an alternative source of HSCs and mature hematopoietic cells for desired therapeutic goals.

Figure 1. Stages of hematopoietic development from hiPSCs.

Materials and Equipment

Reagents Preparation

1) Hematopoietic progenitor basal differentiation medium (SuperCult®, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 0.4 mM 1-thioglycerol, 50 µg/mL L-ascorbic acid)
2) Induction medium d0 (Hematopoietic progenitor basal differentiation medium, 8 ng/mL BMP4)
3) Induction medium d1 (Hematopoietic progenitor basal differentiation medium, 8 ng/mL BMP4, 10 ng/mL bFGF)
4) Induction medium d2 (Hematopoietic progenitor basal differentiation medium, 8 ng/mL BMP4, 10 ng/mL bFGF, 10 ng/mL VEGF, 10 ng/mL hyper-IL-6, 25 ng/mL IGF1, 100 ng/mL SCF)
5) Induction medium d4 (Hematopoietic progenitor basal differentiation medium, 10 ng/mL bFGF, 10 ng/mL VEGF, 10 ng/mL hyper-IL-6, 25 ng/mL IGF1, 100 ng/mL SCF, 10 ng/mL FLT3L, 20 ng/mL TPO, 30 ng/mL IL-3)
6) Induction medium d6 (Hematopoietic progenitor basal differentiation medium, 100 ng/mL SCF, 10 ng/mL FLT3L, 20 ng/mL TPO, 30 ng/mL IL-3)

Assay Procedure

1) Prepare induction medium d0 and prewarm at RT.
2) Harvest iPSC when they reached 70-80% confluence. Aspirate medium and add collagenase IV solution to the cells. The volume depends on the culture dish used. Make sure to completely cover the cells.
3) Incubate the cells with collagenase IV for 40-60 min at 37°C. Check if colony edges start to roll up. If necessary, incubate 10-15 min longer but do not exceed 80 min, as this will compromises cell viability.
4) Neutralize collagenase IV with DMEM in a ratio 1/3.
Note: If iPSCs were cultured on mouse embryonic fibroblasts (MEF), separate iPSCs clusters and single MEF by gravity for 10 min at RT. Large iPSCs clusters will settle to the bottom, while single MEF will remain in suspension. If iPSCs were cultured feeder-free, directly continue with step 5).
5) Collect iPSCs clusters by centrifugation at 200 g for 4 min. Aspirate supernatant and resuspend pellet in 1 mL induction medium d0. Pipette up and down with 1 mL pipette to break up clusters. Transfer a drop of the cell suspension onto a petri dish and check cluster size by microscopy.
6) If cluster size is appropriate, pass cell suspension through a 70 µm cell strainer to remove remaining larger clusters. Fill up flow-through to the final volume with induction medium d0 and transfer to petri dishes to allow embryoid bodies (EB) to form.
7) Place cells in a 37°C, 5% CO₂ incubator and set O₂ regulator of the automatic incubator to 5%.
8) On day 1 of differentiation (24 h after EB formation), many cells (approx. 40-50%) would have died. Prepare induction medium d1 and pre-warm at RT. Dilute old medium (induction medium d0) with fresh medium (induction medium d1) in a ratio of 1/2. Calculate volume of bFGF in induction medium d1 according to total volume (old medium + fresh medium).
9) On day 2 of differentiation, there will be a lot of cell debris visible. Rinse loosely adherent EB off the dishes twice with PBS and centrifuge at 150 g for 4 min. Aspirate supernatant, gently resuspend pellet in induction medium d2 and transfer to new petri dishes. Use the same volume as on day 0 (see step 6). Continue culture under hypoxic conditions (5% O₂, 5% CO₂).
10) On day 4 of differentiation, proceed as in step 9). Finally, resuspend EB in induction medium d4 and transfer to new petri dishes. Use the same volume as on day 0 (see step 6).
11) On day 6 of differentiation, proceed as in step 9). Finally, resuspend EB in induction medium d6 and transfer to tissue culture plastic dishes coated with 0.1% gelatin. Use the same volume as on day 0 (see step 6).
12) On day 8 of differentiation, most EB are adherent on plastic dishes. Collect medium and centrifuge at 150 g for 4 min. Aspirate supernatant, resuspend in induction medium d6 and transfer carefully back to the plastic dishes. Use the same volume as on day 0 (see step 6). From now on, avoid detaching EB from the dish. Transfer cells to 37°C, 5% CO₂ and normoxic incubator.
13) On day 10 and 12 of differentiation, medium change is performed as in step 12).
14) The hematopoietic progenitors will begin to bud off from adherent or loosely adherent EB on day 6-14.

Figure 2. Differentiation protocol used for hematopoietic induction of hiPSCs.

References

1. Choi K. D. et al. Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat Protoc, 2011, 6(3): 296-313.
2. Sontag S. et al. Differentiation of human induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) into dendritic cell (DC) subsets. Bio-protocol, 2017, 7(15): e2419.

For research use only. Not for any other purpose.