Interleukin 3 (il 3)

The construction of PUER-derived reporter cell lines is described elsewhere [33 (link)]. PUER cells were cultured and differentiated as described previously [12 (link), 34 (link)]. Briefly, PUER cells were grown in complete Iscove’s Modified Dulbecco’s Glutamax medium (IMDM; Gibco, 12440061) supplemented with 10% FBS, 50μM β-mercaptoethanol, 5ng/ml IL3 (Peprotech, 213–13). PUER cells were differentiated into macrophages by adding 200nM 4-hydroxy-tamoxifen (OHT; Sigma, H7904–5MG). Cells were differentiated into neutrophils by replacing IL3 with 10ng/ml Granulocyte Colony Stimulating Factor (GCSF; Peprotech, 300–23) and inducing with 100nM 4-hydroxy-tamoxifen (OHT; Sigma, H7904–5MG) after 48 hours.

PUER cells were cultured and differentiated as described previously [18 (link), 28 (link)]. Briefly, PUER cells were routinely maintained in complete Iscove’s Modified Dulbecco’s Glutamax medium (IMDM; Gibco, 12440061) supplemented with 10% FBS, 50μM β-mercaptoethanol, 5ng/ml IL3 (Peprotech, 213–13). PUER cells were differentiated into macrophages by adding 200nM 4-hydroxy-tamoxifen (OHT; Sigma, H7904–5MG). Cells were differentiated into neutrophils by replacing IL3 with 10ng/ml Granulocyte Colony Stimulating Factor (GCSF; Peprotech, 300–23) and inducing with 100nM OHT after 48 hours.

The hiPSCs were differentiated into HPCs using the embryoid bodied (EB)-based HPC differentiation method [32 (link),33 (link)]. hiPSCs were briefly treated with the indicated flavonoids for 3 days before differentiation into EBs. For EB formation, cells were transferred onto Corning Ultra-Low Attachment Surface dishes with mTesR1 supplemented with 10 μM ROCK inhibitor for 6 days. On day 7, the formed EBs were transferred onto Matrigel-coated plate and incubated with the HPC differentiation medium (Iscove’s Modified Dulbecco′s Medium (Thermo Fisher Scientific) containing 20% FBS, with 100 ng/mL SCF (PeproTech), 10 ng/mL IL-3 (PeproTech), 10 ng/mL IL-6 (PeproTech), 20 ng/mL FLT3L (PeproTech), and 20 ng/mL BMP4 (PeproTech) with media exchange every 2 days until day 21. To obtain NKCs, hiPSC-derived HPCs were differentiated for 4 weeks in the presence of 10 ng/mL IL-15 (PeproTech), 5 ng/mL IL-3, 20 ng/mL IL-7 (PeproTech), 20 ng/mL SCF, and 10 ng/mL FLT3L. Medium containing cytokines was changed weekly with the exception of IL-3, which was only included for the first week of differentiation.

PB MNCs were expanded for 4–10 days in a serum-free medium supplemented with a mixture of cytokines. The two main culture media (erythroid culture medium (ECM) and granulocyte culture medium (GCM)) were tested. ECM included IMDM (50%; Invitrogen) and Ham’s F12 (50%; Invitrogen) with ITS-X (100×; Invitrogen), chemically defined lipid concentrate (100×; Invitrogen), l-glutamine (100×; Invitrogen), ascorbic acid (0.05 mg/ml; Sigma), BSA (5 mg/ml; Sigma), l-thioglycerol (200 μM; Sigma), SCF (100 ng/ml; PeproTech), IL-3 (10 ng/ml; PeproTech), erythropoietin (2 U/ml; PeproTech), IGF-1 (40 ng/ml; PeproTech), dexamethasone (1 μM; Sigma), and holo-transferrin (100 μg/ml; R&D). GCM was supplemented with IMDM (50%) and Ham’s F12 (50%), ITS-X (100×), chemically defined lipid concentrate (100×), l-glutamine (100×), ascorbic acid (0.05 mg/ml), BSA (5 mg/ml), 1-thioglycerol (200 μM), Thrombopoietin (100 ng/ml; PeproTech), SCF (100 ng/ml), Flt3 ligand (100 ng/ml; PeproTech), granulocyte-colony stimulating factor (G-CSF) (100 ng/ml; PeproTech), and IL-3 (10 ng/ml). During culture, we quantified the living cells by FACS staining and counted them automatically using a cell number counting machine (Bio-Rad).

PUER cells were cultured and differentiated as described previously (Dahl et al. 2003 (link); Repele et al. 2019 (link)). Briefly, PUER cells were routinely maintained in complete Iscove’s Modified Dulbecco’s Glutamax medium (IMDM; Gibco, 12440061) supplemented with 10% FBS, 50 $\mu$ M β-mercaptoethanol, 5 ng/mL IL3 (Peprotech, 213-13). PUER cells were differentiated into macrophages by adding 200 nM 4-hydroxy-tamoxifen (OHT; Sigma, H7904-5MG). Cells were differentiated into neutrophils by replacing IL3 with 10 ng/mL granulocyte colony stimulating factor (GCSF; Peprotech, 300-23) and inducing with 100 nM OHT after 48 h.

We used a modified version of the previously described EB-based hematopoietic differentiation protocol^{92 (link),106} to differentiate human induced pluripotent stem cells (iPSCs) into hematopoietic cells. To initiate differentiation, we generated embryoid bodies (EBs) from detached iPSC colonies (#MHHi015-A, hPSCreg) using 2 mg/mL collagenase IV (#17104019, Invitrogen) and cultivated them on an orbital shaker in iPSC-medium without bFGF but supplemented with 10 μM Rock inhibitor (Y-27632; Tocris). After 2–3 days, the medium was changed, and after 5 days, mature EBs were manually selected and transferred to adherent plates in differentiation medium I supplemented with either 50 ng/ml IL-3 or a combination of 25 ng/ml IL-3 (Peprotech) and 50 ng/ml M-CSF (Peprotech). We acquired samples for single-cell RNA transcriptome analysis at the mature EB stage and also 8 or 16 days after initiation of hematopoietic differentiation medium I for both cytokine conditions. EB and adherent myeloid-cell-forming-complexes were dissociated with TrypLE Express (#12605028, Thermo Fischer Scientific) for 10–30 minutes at 37°C and filtered through 70 μm mesh. Subsequently, the single-cell suspension was stained for CD34 expression (#11–0349, 1:50, eBioscience) and with DAPI (Sigma) and was further enriched by sorting (FACS Aria, BD) for live and CD34⁺ cells.