Taqman hpsc scorecard assay

To confirm the iPSC status of reprogrammed donor fibroblasts, we performed a TaqMan iPSC Scorecard Assay^{44 (link)}, which also confirmed the cells’ trilineage potential (Fig. 2b). We followed the protocol described by the manufacturer of the TaqMan hPSC Scorecard Assay (Thermo Fisher Scientific).
Stem cells were cultured on Geltrex matrix (Gibco) in mTeSR^™1 media (StemCell Technologies) under standard incubator conditions of 5% CO2 and humidity. On the day of analysis, the cells were dissociated using Accutase and pelleted by centrifugation. RNA was extracted using a Qiagen extraction kit and cDNA was synthesized as per Scorecard kit instructions. Embryonic bodies were generated as per Scorecard kit instructions, RNA was extracted and cDNA synthesized in the same way as for iPSC pellets. The TaqMan hPSC Scorecard Kit 384w plate was amplified using Lightcycler 480 (Roche Diagnostics) and the data were uploaded to the hPSC Scorecard analysis software available online from Thermo Fisher Scientific. The resulting graphs were downloaded and included in Fig. 2.

iPSCs were subjected to spontaneous differentiation mediated by the formation of embryoid bodies (EBs) and subsequently analyzed for three-lineage differentiation using the ScoreCard methodology (Thermo Fisher Scientific). On the day of EB formation, 60 to 80% confluent iPSCs were washed with PBS and incubated with 0.5 mM EDTA (Invitrogen) for 1 to 3 min to dissociate the colonies. Cells were harvested in Essential 8 flex medium (Gibco) and counted before gently spinning down at 300g for 5 min. Cells were plated in a 24-well Corning Ultra-Low Attachment Surface plates (Corning) at a density of 1.5 × 10⁶ cells per well. The plates were incubated overnight at 37°C. The following day, half of the Essential 8 flex medium was replaced with Essential 6 medium (Gibco). For the spontaneous differentiation, EBs were kept for 14 days in E6 medium, which was changed every 2 days. After 14 days in culture, EBs were collected for RNA extraction with the GenElute Mammalian Total RNA Kit (Sigma-Aldrich). Complementary DNA (cDNA) synthesis was performed with SuperScript III (Thermo Fisher Scientific), and qPCR was performed according to the manufacturer’s protocol with TaqMan hPSC Scorecard assay (Thermo Fisher Scientific). Data were analyzed with the online Scorecard software (Thermo Fisher Scientific).

The TaqMan hPSC Scorecard Assay (Thermo Fisher) was used to assess the pluripotency of cultured Gibco hiPSCs and the XCL-1 hiPSC clones as described^{32 (link)}. The assay outputs scores for self-renewal, ectoderm, endoderm, and mesoderm genes.

Patient-derived and control iPSCs were analysed for pluripotency using the Taqman hPSC Scorecard Assay (Thermo Fisher Scientific). Undifferentiated cells and randomly differentiated cells through embryoid (EB) formation were prepared for the plate according to the manufacturer’s instructions. Cells were collected in TRIzol reagent on Day 8. RNA was extracted, and cDNA prepared from iPSC and differentiated cells according to the manufacturer’s protocol for hPSC Scorecard analysis. The qRT-PCR was performed on a StepOne Real-Time PCR system using the manufacturer’s ScoreCard experimental template file. Gene expression data was assessed using hPSC Scorecard Analysis Software (Thermo Fisher Scientific).

All hiPSC lines were assessed for chromosomal abnormalities by performing karyotyping (WiCell). Their identity was confirmed using short-tandem repeat (STR) analysis (WiCell) and comparison with the donor’s blood DNA. The potential for self-renewal and pluripotency of the hiPSC lines was assessed by utilizing hiPSC RNA with the Taqman hPSC Scorecard Assay (Thermo Fisher, A15870). For pluripotency, RNA was isolated after random differentiation of hiPSCs into embryoid bodies [43 (link)] to generate the three primary germ layers and assessed utilizing EB RNA with the Taqman hPSC Scorecard Assay. Full elimination of the Sendai virus vectors was confirmed by immunostaining and with the Taqman hPSC Scorecard Assay. The e-Myco Mycoplasma PCR Detection Kit (Bulldog Bio, 25233) and the MycoAlert Mycoplasma Detection Kit (Lonza, LT07-118) were used to ensure that all the cells used in this study were mycoplasma-free.