Stemmacs ipsc brew xf medium

In vitro differentiation of iPSCs from RTS individuals and unaffected family members to MSCs was performed by a defined optimized MSC differentiation protocol described previously [27 (link)]. Briefly, 80% confluent iPSCs were cultured in StemMACS iPSC-Brew XF medium (Miltenyi Biotec, 130-104-368) supplemented with 10 μM TGFβ inhibitor SB-431542 (MilliporeSigma, S4317) in Matrigel-coated plates at 37°C and 7.5% CO₂ and passaged at 80–90% confluence by Accutase cell detachment solution (StemCell Technology, 07922). After 25 days in culture, cells at the edge of differentiated cell clusters became spindle-shaped. These differentiated cells were trypsinized into single cells and maintained in gelatin-coated plates using modified human ESC-MSC medium (KnockOut DMEM/F-12 supplemented with 10% KnockOut Serum Replacement, L-glutamine, non-essential amino acids, β-mercaptoethanol, 20 ng/ml bFGF2, 10ng/ml EGF, 10 μM SB-431542, and penicillin/streptomycin). The medium was changed daily. When cells reached 80–90% confluence, cells were passaged at a 1 to 3 ratio for three weeks. The MSCs were characterized by staining for MSC surface markers CD44, CD73, and CD105.

Human iPSCs were generated from the HSE patient, family members as well as healthy controls by reprogramming erythroblasts via ectopic expression of a minimal set of transcription factors as previously described^{67 (link)}. Briefly, erythroblasts were amplified from PBMCs and nucleofected with episomal plasmids pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL (gifts from Shinya Yamanaka, Addgene #27077, #27080 and #27078)^{68 (link)}, plated on Matrigel-coated plates and cultured in a reprogramming medium (ReproTeSR, STEMCELL Technologies) until iPSC colonies started to appear. Human iPSC clones were then picked and lines expanded using StemMACS iPSC-Brew XF medium (Miltenyi Biotec, Bergisch Gladbach). Human iPSCs were characterized by immunofluorescence for pluripotency (expression of OCT4, TRA-1-60, and SOX2) and differentiation capacity (The three-germ layer differentiation capacity and expression of AFP, Pax6, SMA) as previously described^{67 (link)} and following standard quality controls for iPSCs. The genomic integrity of iPSCs was assessed by G-banding karyotype. The p.R841H mutation was confirmed by the Sanger sequencing of iPSCs’ genomic DNA. Two lines per donor were used in this study. Healthy control iPSC lines (n = 3, each from a different individual) were previously described^{36 (link)} and assessed to be WT for WWP2.