Monothioglycerol

To induce hematopoietic differentiation from Rh-iPSCs, we slightly modified a previously described human iPSC method.² (link) In brief, small clumps (<100 cells) of Rh-iPSCs maintained on MEFs were collected and co-cultured on C3H10T1/2 feeder cells in EB medium (Iscove’s modified Dulbecco’s medium [Sigma] containing 20% fetal bovine serum [FBS], 1% l-glutamine-penicillin-streptomycin solution [Sigma], 100× insulin-transferrin-selenium solution [Thermo Fisher Scientific], 450 μM monothioglycerol [Nacalai], 50 μg/mL ascorbic acid 2-phosphate [Nacalai]), in addition to 20 ng/mL VEGF (R&D Systems). On day 0, 20 ng/mL BMP4 (R&D Systems), and on days 7, 10, and 12, 30 ng/mL stem cell factor (SCF) (R&D Systems) and 10 ng/mL FLT-3L (PeproTech) were added to EB medium. Hematopoietic cells generated in iPSC sacs were collected on day 14.

RPMI 1640 medium was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Opti-modified Eagle’s medium (Opti-MEM) and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Sodium chloride, sodium bicarbonate, potassium chloride, Tris-HCl, ethylenediaminetetraacetic acid (EDTA), ammonium chloride, and glucose were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Monothioglycerol, MEM non-essential amino acids, and penicillin-streptomycin-glutamine mixed solution were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The 20 bp ladder was purchased from Takara Bio (Otsu, Japan). Propidium iodide, R848, and ovalbumin were obtained from Merck KGaA (Darmstadt, Germany). Alexa Fluor 488 annexin V/ Dead Cell Apoptosis kit with Alexa Fluor 488 annexin V and PI for Flow Cytometry was purchased from Invitrogen (San Diego, CA, USA). All other chemicals were of the highest grade available and were used without further purification.