Generation of iPSCs from CB and PB

For generation of iPSCs from CB, we followed our previously published protocol. [40] (link), [54] (link) To generate PB iPSCs, human PBMNCs were cultured in HSC culture conditions. [75] (link), [76] (link) Iscove's modified Dulbecco's medium (IMDM)/10% FBS supplemented with TPO, SCF, FL and G-CSF each at 100 ng/ml, IL-3 at 10 ng/ml, and StemRegenin1 or SR1 (Cellagen Technology; San Diego, CA) [77] (link) at 1 uM. Cytokines were purchased from ProSpec (East Brunswick, NJ). After 6–8 days of culture, 1×10⁵ cells per well were seeded into non-TC treated 24-well plates that were pre-coated with fibronectin fragment RetroNectin or CH-296 (Takara Bio, Inc., Shiga, Japan). [78] (link) Lentiviral transduction was conducted for 5–6 hr with a multiplicity of infection (MOI) of 4. One day after transduction, cells were harvested and transferred to 6-well plates, which were pre-seeded with inactivated rat embryonic fibroblast (REF) feeder cells (Applied Biological Materials Inc. or ABM; Richmond, BC, Canada). Cells were maintained in the HSC culture condition for 2 more days before being gradually replaced with iPSC medium. The iPSC medium is composed of Knockout DMEM/F12 medium (Invitrogen) supplemented with 20% Knockout Serum Replacement (KSR) (Invitrogen; Carlsbad, CA), 1 mM GlutaMAX (Invitrogen), 2 mM nonessential amino acids (ABM), 1× penicillin/streptomycin (ABM), 0.1 mM β-mercaptoethanol (Sigma-Aldrich Corp; St. Louis, MO), 20 ng/ml FGF2 (ABM), and 50 µg/ml ascorbic acid. [63] (link), [79] (link) Culture medium was changed every 2 days. To increase reprogramming efficiency, an inhibitor of histone deacetylase sodium butyrate [80] (link), [81] (link) was added at 0.25 mM every 2 days from day 2 to 10, and cells were cultured under hypoxia throughout the experiment by placing culture plates in a hypoxia chamber (Stemcell Technologies, inc., Vancouver, BC, Canada) that was flushed with mixed air composed of 92%N₂/3%O₂/5%CO₂. [40] (link), [82] (link)
[54] (link). Starting from day 10, REF-conditioned medium was used.