Cytotune ipsc 2.0 sendai reprogramming kit

PBMCs were isolated from whole blood samples using Percoll density gradient medium (17089109, GE Healthcare). Cells were purified with multiple rounds of DPBS wash (14190144, ThermoFisher Scientific). Cells were cultured in complete PBMC medium composed of StemPro^®−34 SFM medium (10639011, ThermoFisher Scientific) containing 100 ng/mL SCF (300–07, Peprotech), 100 ng/mL FLT3 (PHC9414, ThermoFisher Scientific), 20 ng/mL IL-3 (200–3, Peprotech), 20 ng/mL IL-6 (PHC0063, ThermoFisher Scientific), and 20 ng/mL EPO (PHC9631, ThermoFisher Scientific). The medium was refreshed daily until the cell count remained stable for a few days. PBMC transduction was performed using the Sendai virus reprogramming cocktail according to the manufacturer’s instructions (CytoTune^™-iPSC 2.0 Sendai Reprogramming Kit, A16517, ThermoFisher Scientific). The transduced cells were resuspended and plated in a Matrigel-coated plate (356231, Corning) and cultured in StemPro^™−34 medium (Thermo Fisher). Media was replaced daily until day 7 post transduction. On Day 7, the medium was switched to StemMACS^™ iPS-Brew XF medium (130–104–368, Miltenyi Biotec) and was replaced every other day until Day 10–15 post transduction until the colonies appeared. Selected colonies were expanded and frozen down until experimental usage.

Sex chromosomal mosaicism in starting fibroblasts (partial 45,X recorded in Coriell Biorepository) was confirmed by interphase DNA FISH using sex centromere (DXZ1/DYZ3) probes (WiCell). Fibroblasts were reprogrammed to hiPSCs using the CytoTune iPSC 2.0 Sendai Reprogramming kit (Thermo Fisher Scientific) and maintained by weekly mechanical passaging, as described (26 (link)). Prior to BAP differentiation, hiPSCs were transitioned to mTeSR media (Stem Cell Technologies), grown on extracellular matrix (Geltrex, Thermo Fisher), and passaged weekly with ethylenediaminetetraacetic acid. Standard IF was performed for pluripotency markers and H3K27me3, as well as FISH and qRT-PCR for XIST (SI Appendix, SI Methods include details).

To obtain PBMCs, the patient’s whole blood was subjected to isolation using Percoll^® density gradient separation medium (GE Healthcare #17089109), followed by purification through multiple washes with DPBS (Thermo Fisher Scientific #14190144). Subsequently, the isolated PBMCs were cultured in StemPro^®-34 SFM medium (Thermo Fisher Scientific #10639011), supplemented with 100 ng/mL SCF (Peprotech #300–07), 100 ng/mL FLT3 (Thermo Fisher Scientific #PHC9414), 20 ng/mL IL-3 (Peprotech #200–3), 20 ng/mL IL-6 (Thermo Fisher Scientific #PHC0063), and 20 ng/mL EPO (Thermo Fisher Scientific #PHC9631). The medium was refreshed every two days until the cell culture reached a stable state.
For cell reprogramming, the CytoTune^™-iPSC 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific #A16517) was employed following the manufacturer’s instructions. The transduced cells were plated on Matrigel-coated plates and cultured in StemPro^®-34 medium. The medium was replaced every two days until day 7, at which point it was switched to supplemented StemMACS^™ iPS-Brew XF medium (Miltenyi Biotec #130–104-368) until day 10–15 post-transduction. Colonies then became visible and ready for clonal expansion. Selected colonies were expanded and cryopreserved for future experimental use.

PBMCs were isolated from blood using Percoll density gradient medium (#17089109, GE Healthcare), purified using DPBS, and plated in a 24-well plate as previously described (Belbachir et al., 2021 (link)). Cells were cultured in StemPro®-34 SFM medium (#10639011, Thermo Fisher Scientific) and nourished with specific supplements: SCF (100 ng/mL, #300–07, Peprotech), FLT3 (100 ng/mL, #PHC9414, Thermo Fisher Scientific), IL-3 (20 ng/mL, #200–3, Peprotech), IL-6 (20 ng/mL, #PHC0063, ThermoFisher Scientific), and EPO (20 ng/mL, #PHC9631, Thermo Fisher Scientific). PBMCs were reprogramed according to the instructions provided with CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (#A16517, Thermo Fisher Scientific). Transduced PBMCs were resuspended and plated on a Matrigel-coated plate. Cells were cultured in StemPro™-34 medium (Thermo Fisher Scientific). On day 7, the medium was redirected to StemMACS™ iPS-Brew XF medium (#130–104–368, Miltenyi Biotec), and cells were maintained until days 10–15 post-transduction. Cell colonies were picked, and clones expanded as previously described (Belbachir et al., 2021 (link)).