Fibroblast to Pluripotent Stem Cell Reprogramming

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).

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Ahern D.T., Bansal P., Armillei M.K., Faustino I.V., Kondaveeti Y., Glatt-Deeley H.R., Banda E.C, & Pinter S.F. (2022). Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta. Proceedings of the National Academy of Sciences of the United States of America, 119(40), e2211073119.

Publication 2022

CytoTune iPSC 2.0 Sendai Reprogramming kit mTeSR media Geltrex

Centromere Ethylenediaminetetraacetic acid Extracellular matrix Fibroblasts Fish Hipscs Interphase Ipsc Mosaicism Sex chromosomal Stem cell

Corresponding Organization :

Other organizations : UConn Health, University of Connecticut

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Variable analysis

independent variables

Reprogramming of fibroblasts to hiPSCs using the CytoTune iPSC 2.0 Sendai Reprogramming kit

dependent variables

Confirmation of sex chromosomal mosaicism in starting fibroblasts by interphase DNA FISH using sex centromere probes
Expression of pluripotency markers and H3K27me3 in hiPSCs by standard IF
XIST expression in hiPSCs by FISH and qRT-PCR

control variables

Weekly mechanical passaging of hiPSCs
Transition of hiPSCs to mTeSR media and growth on extracellular matrix (Geltrex) prior to BAP differentiation
Weekly passaging of hiPSCs with ethylenediaminetetraacetic acid

controls

Positive control: Not specified
Negative control: Not specified

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