Human stem cell factor

The derivation of iMAC from iPSCs has been previously described^{10 (link)}. Briefly, 8000 iPSCs were seeded in 96-well round-bottom plates with APEL2 medium (05271, STEMCELL Technologies) containing 100 ng/mL human Stem Cell Factor (SCF), 50 ng/mL human Vascular Endothelial Growth Factor (VEGF), 10 ng/mL recombinant human Bone Morphogenetic Protein 4 (BMP-4), 5 ng/mL human FGF-basic (154 a.a.), and 10 mM Rho kinase inhibitor (ROCK inhibitor, Y27632, Sigma). After eight days of hematopoietic differentiation, spin embryoid bodies (EB) were transferred into Matrigel-coated 6-well plates under macrophage differentiation conditions. Macrophage differentiation medium is StemSpan-XF (100-0073, STEMCELL Technologies) containing 10 ng/mL human FGF-basic (154 a.a.), 50 ng/mL human Vascular Endothelial Growth Factor (VEGF), 50 ng/mL human Stem Cell Factor (SCF), 10 ng/mL recombinant human Insulin-like Growth Factor-1 (IGF1), 20 ng/mL IL-3, 50 ng/mL recombinant human M-CSF, and 50 ng/mL recombinant human GM-CSF. The floating cells were collected from the supernatant and directly transferred into uncoated 6-well plates in macrophage culture medium. The macrophage culture medium is StemSpan-XF containing 50 ng/mL recombinant human M-CSF and 50 ng/mL recombinant human GM-CSF.

The cells were purified as follows:

bPGCs: ACK treatment or Percoll

gPGCs: trypsin or trypsin and Percoll

After purification, the cells were transfected using two methods: electroporation at 200 V and 900 μF for 32 ms or lipofection with Xtreme (Roche) and 20 μg pEGFP-N1. Next, they were seeded into 4-well plates with a density of 5 × 10⁵ cells and over a period of 24 h, the PGCs were grown in OptiMEM I supplemented with antibiotics at 37 °C and 5 % CO₂ in air. After 24 h, the medium was replaced with OptiMEM I C [C: supplemented with 2 % chicken serum (Gibco), 10 % FBS (Sigma), 20 ng/ml bFGF, basic fibroblast growth factor (Sigma), 9 ng/ml mLIF, murine leukaemia inhibitory factor (Sigma), 5 ng/ml hSCF, human stem cell factor (Sigma), antibiotics and G418 (50 μg/ml, Sigma)]. Every 3 days, the medium was changed.

Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, U.S.A.). Human stem cell factor, basic fibroblast growth factor, retinoic acid (RA), and murine leukemia inhibitory factor were acquired from Sigma-Aldrich (Saint Louis, MO, U.S.A.). TRNzol, FastQuant-RT kit, and SuperReal premix color kit were obtained from TIANGEN (Beijing, China). Fugen was from Promega (Madison, WI, U.S.A.). Antibodies specific to the following proteins were integrinα6 (Millipore, temecula, CA, U.S.A.; dilution ratio 1:100), integrin β1 (Millipore, temecula, CA, U.S.A.; no. MAB1378; dilution ratio 1:100), goat anti-Rat IgG (Proteintech, Chicago, Illinois U.S.A.; no. SA00003-11; [FITC] labeled; dilution ratio, 1:100), and goat anti-rabbit IgG (Proteintech, Chicago, Illinois, U.S.A.; no. SA00008-9; [PE] labeled; dilution ratio, 1:100).