Chunk #7 — Results — Reprogramming of 9p24.1-Carrier HFs Yields Isogenic Carrier and Non-carrier hiPSCs; 9p24.1-Carrier hiPSCs Show Variable Mosaicism in All Lines
We generated genetically unmanipulated and presumably clonal hiPSCs from the two 9p24.1 carriers (Figures 2 and 3) and the two non-carrier relatives (data not shown) using Sendai viral vectors to reprogram subject HFs, as described previously (Topol et al., 2016). All hiPSCs showed robust self-renewal as well as mRNA and protein expression of NANOG, OCT4, TRA-1-81, and TRA-1-60 (Figures 2A, 2B, S2A, and S2B). Validated hiPSCs were identical to their original fibroblasts by DNA fingerprinting (Figures S2F and S2G). At least three validated hiPSCs were generated per person; moreover, three 9p24.1-carrier and three isogenic 9p24.1-non-carrier hiPSCs were derived from both 9p24.1 carriers’ HFs and confirmed by PCR and/or aCGH (Figures 2C and S2C–S2E). Karyotyping of 9p24.1 hiPSCs detected a high frequency of mar elements in otherwise karyotypically normal lines (Figure 2D and Table S2). PCR of one of the CGR breakpoint junctions confirmed that mar-positive hiPSCs were carriers of the 9p24.1 rearrangement; in contrast, the CGR was not present in mar-negative hiPSCs; this mosaic pattern was maintained throughout hiPSC passaging from low passage (p3) to higher passage (p10) in both mar-positive and mar-negative lines (Figures S2C and S2D).
