In our first reprogramming experiments, fibroblasts from “Angalifu” (laboratory no. KB 9947), passage 10, were reprogrammed using the CytoTune™ 2.0 Kit (Thermo Fisher Scientific). Cells were thawed 2 days before the start of reprogramming, plated on 0.1% gelatin in supplemented DMEM at an approximate cell density of 13,000 cells/cm2 (∼70% confluent on day 0), and transduced with the recommended multiplicity of infection (MOI) for each vector for human cell reprogramming (ie, KOS-5, MYC-5, and KLF4-3).

The medium was changed according to the protocol. In brief, fibroblast medium (supplemented DMEM) was changed within 24 h postinfection and every other day until replating. The cells were replated 5 days after transduction using 0.05% Trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco), 2 days earlier than recommended due to overgrowth of the cells. Cells were plated at a density of 180 cells/cm2 onto irradiated (mitotically inactivated) CF-1 mouse embryonic fibroblast (MEF) feeder layers or 5,300 cells/cm2 onto Geltrex™ (Gibco). All cultures were changed to iPSC medium 24 h later and subsequently fed every day.

Seven colonies, across both conditions, were picked using a needle between days 14 and 16 post-transduction and plated onto MEFs. Plates became severely overgrown after day 16, resulting in high cell death; therefore, we did not maintain the plates for the full 4 weeks recommended by the protocol. We successfully obtained one clone from this reprogramming attempt, which originated from a Geltrex coated plate. The remaining colonies were either partially reprogrammed or outcompeted by the carried over fibroblasts.

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