Essential 8 medium e8

H9 hESCs (#WA09) were purchased from WiCell Research Institute. Fib-iPSCs were obtained from the Human Embryonic Stem Cell Core, Harvard Medical School. Fib-iPSCs were reprogrammed from fibroblasts, by George Q. Daley Lab (Children’s Hospital Boston, MA) and have been well characterized and described in the literature [43]. For 2D cell culture, H9s and iPSCs were cultured in 6-welll plate coated with Matrigel (BD Biosciences, #354277) in Essential 8™ medium (E8, Life Technologies, #A1517001). For differentiation, we followed our published protocols to prepare hPSCs-derived endothelial cells (ECs) and neural stem cells (NSCs) (Lin et al., 2018a (link); Lin et al., 2018b (link); Lin et al., 2018c (link)).

Dermal biopsies and blood samples were collected after informed consent and approval from the committee on Research Ethics of Northern Savo Hospital district (license no. 123/2016). Fibroblasts and peripheral blood mononuclear cells T cells were isolated and cultured as previously described (Korhonen et al., 2015 (link), Qu et al., 2013 (link)). Somatic cells were reprogrammed to iPSCs with either lentiviral vectors, CytoTune -iPS 1.0, or CytoTune -iPS 2.0 Sendai Reprogramming Kits (Invitrogen) as previously described (Holmqvist et al., 2016 (link)). iPSCs were grown on Matrigel-coated (Corning) plates in Essential 8 Medium (E8; Life Technologies) and passaged with 0.5 mM EDTA in the presence of 5 μM Y-27632 ROCK inhibitor (Selleckchem). Isogenic control lines were generated according to a previously published protocol (Chen et al., 2015 (link)). Further details are provided in Supplemental Experimental Procedures.

Fibroblasts were grown from skin biopsies under glass plates in DMEM + 20% fetal calf serum and antibiotics. Mononuclear cells were extracted freshly from blood by Ficoll-extraction method. PBMCs (1 × 10⁵ to 1 × 10⁶) and fibroblasts were transduced with SeVdp as previously described (Nishimura et al., 2011 (link), Trokovic et al., 2014 (link)). Cells were plated on mitomycin C-treated murine embryonic fibroblasts (3.75 × 10⁵ feeder cells/well) on a six-well plate in hES medium: DMEM/F12 with GlutaMAX, supplemented with 20% KO-serum replacement, 0.1  mM β-mercaptoethanol, 1% non-essential amino acids (all from Life Technologies), and 6 ng/ml basic fibroblast growth factor (bFGF; Sigma). For feeder-free cultures iPSC lines were grown on Matrigel (growth factor reduced, BD Biosciences) in StemPro (Life Technologies) or in Essential-8 medium (E8, Life Technologies). The donor identity of all iPSC lines was confirmed by microsatellite marker analyses.

BM2-3 hiPSCs from passages 16 to 20 were obtained from Dr. Sunita L. D'Souza. This BM2-3 iPSC line was derived from bone marrow of a human subject clinically normal and verified by fluorescent in situ hybridization test showing 46,XY. Cytogenetic analysis of cultured human stem cells revealed a normal female karyotype. This analysis does not show any evidence of a clinically significant numerical or structural chromosome abnormality. Cells were maintained under feeder-free conditions using Matrigel (BD Biosciences)-coated 6-well tissue culture plates in Essential 8 Medium (E8, Life Technologies), supplemented with 10 μM ROCK inhibitor Y27632 (Life Technologies) on passaging days. Cells were routinely passaged as small clumps using a previously described EDTA method (Beers et al., 2012 (link)).