Stemspan serum free expansion medium 2

Primary human umbilical cord blood cells with the hematopoietic progenitor cell antigen CD34 (CD34⁺ cells) were purchased from STEMCELL™ Technologies (Vancouver, Canada). The stem cells were expanded once in StemSpan™ Serum-Free Expansion Medium II (SFEM II) supplemented with StemSpan™ CC100 solution (STEMCELL™ Technologies) containing both early- and late-acting recombinant human cytokines. The expanded CD34⁺ cells were cryopreserved in CryoStor®CS10 (BioLife Solutions, Bothell, WA). For testing the expansion and differentiation of CD34⁺ cells stimulated by the plant cell-derived SCF, the protocol developed by Kim et al. (2014) [25 (link)] was followed. Briefly, CD34⁺ cells (2×10⁴ cells/ml) were incubated in the SFEM II medium supplemented with 6 IU/ml EPO (STEMCELL™ Technologies), 10 ng/mL IL-3 (STEMCELL™ Technologies), and 100 ng/ml SCF equivalent of SCF-(SP)₂₀ or (SP)₂₀-SCF at 37 °C with 5% CO₂ for 7 days. The cells were then incubated in SFEM II supplemented with 3 IU/ml EPO, 10 ng/mL IL-3, and 50 ng/ml SCF equivalent of SCF-(SP)₂₀ or (SP)₂₀-SCF for another 7 days. As a comparison, the positive group was treated with all three commercial eryGFs (SCF, EPO and IL-3), and the negative group was treated with the commercial EPO and IL-3 lacking the SCF. The differentiation of the cultured CD34⁺ cells was analyzed by cell staining, flow cytometry, and hemoglobin development.

Human LSCs were cultured in StemSpan™ Serum-Free Expansion Medium II (STEMCELL, USA) with penicillin (100 U/mL), streptomycin (100 mg/mL), stem cell factor (SCF, 20 ng/ml), thrombopoietin (TPO, 20 ng/ml), Flt3-L (20 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml). Mouse LSCs were cultured in Iscove’s Modified Dulbecco’s Media (Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 mg/mL), SCF (20 ng/ml), TPO (20 ng/ml), Flt3-L (20 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml). Human and mouse ILC1s or NK cells were cultured in RPMI 1640 (Thermo Fisher Scientific, USA) with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), IL-12 (10 ng/ml), and IL-15 (100 ng/ml). The mouse AML cell line C1498 (American Type Culture Collection) was cultured in RPMI 1640 with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. All cytokines were from PeproTech. penicillin and streptomycin were from Thermo Fisher Scientific.