SteMClass: A Novel DNA Methylation-Based Classifier for iPSC In Vitro Differentiation States

Human induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modelling, and drug discovery, but most downstream applications require differentiation into specialised cell types not covered by current quality control assays. Here, we present “SteMClass”, a proof-of-concept DNA methylation-based classifier that standardises iPSC differentiation state identification across protocols with one test. We curated a reference cohort of 15 iPSC lines differentiated into seven distinct states (n = 97), performed array-based DNA methylation profiling, and trained a random forest model to classify the eight distinct differentiation states. In nested cross-validation, SteMClass achieved a Brier score of 0.0264, and on an independent cohort (n = 58) attained 96.5% accuracy (Cohen’s K = 0.959) with a 3% rejection rate. Applied to external data (n = 241), performance was 76.5% accuracy (Cohen’s K = 0.593) with a 16.6% rejection rate and enabled the detection of potentially inefficient differentiations. SteMClass is compatible with all Illumina methylation array versions, and accessible via an interactive web interface that supports classification and exploration of DNA methylation profiles. By providing a harmonised, single-assay framework for iPSC-derived differentiation state characterisation, SteMClass improves reproducibility and comparability across studies, paving the way for robust quality control standards and accelerating clinical translation. iPSCs were differentiated into seven distinct cell states, and DNA was extracted from harvested pellets using the Maxwell® RSC system (Promega) with kits appropriate for fresh, frozen, or PFA-fixed samples, yielding comparable methylation quality. DNA quantity was assessed with the Quantus/QuantiFluor® ONE dsDNA system (Promega), followed by bisulfite conversion using the EpiTect Fast Bisulfite Kit (Qiagen). Converted DNA was analysed on Infinium MethylationEPIC BeadChip arrays (v1.0 or v2.0) and scanned with the Illumina iScan System.