Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

Abstract

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a four-step workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions (mTeSR1 and Essential 8) and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic stability in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based stability test, and cell-line identity authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by q-PCR for progenitor, neuronal, cortical and glial markers. Taken together, we present a standardized quality control workflow to evaluate variability and reproducibility across and between iPSCs.HighlightsValidation of culture conditions is critical in the expansion and maintenance of an iPSC line.Characterization of pluripotency and genomic stability ensures each line is free of defects at the DNA level, while maintaining its ability to be directed into any of the three germ layers.Forebrain cortical neurons can be generated from all iPSC line tested; however, the morphology and expression pattern of these neurons can vary from line to line.

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“…The neural progenitor cells were dissociated, then purified by culturing in suspension for 48 hours in expansion medium: (DMEM/F12 with Glutamax supplemented with N2, B27 (ThermoFisher Scientific), NEAA (Gibco), laminin (1µg/ml) (Sigma) plus the growth factors EGF (20 ng/ml) and FGF (20 ng/ml) (Peprotech). The media used for cortical differentiation is described in the standard operating procedure published on the Early Drug Discovery Unit (EDDU) website (Chen et al, 2021) Briefly, purified NPCs were replated on Matrigel-coated dishes (Thermo-Fisher). Once cells attained 100% confluency, NPCs were passaged and seeded on a Poly-Ornithine-laminin coated dishes to be differentiated into neurons.…”

“…The other iPSC cells lines were reprogrammed with transducing retrovirus from PBMCs (AIW001-2; AIW002-2), fibroblasts (AJC001-5, KYOU-DRX0190B) or lymphocytes (522-2666-2). Quality control profiling for the iPSCs used was outlined in a previous study (Chen et al, 2021).…”

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“…For generation of hMOs, iPSCs were used only after a minimum of two passages following being thawed and were not passaged more than ten times. Details on iPSC passaging is found in our QC workflow for iPSC maintenance 29 . hMOs were generated according to the published method 30 .…”

“…For testing of hotspot mutations within the genome of the iPSCs used, genomic DNA was extracted with the Genomic DNA Mini Kit (Blood/Cultured Cell) (Geneaid). Genomic stability was then tested with the hPSC Genetic Analysis Kit (Stemcell, 07550) according to our earlier studies 29 .…”

“…S1B). Genomic stability was assessed by monitoring copy numbers in critical hotspot regions that are often commonly mutated during reprogramming or extensive cell passaging 29 . In these regions, we did not observe any of the recurrent abnormalities that have been reported [36][37][38] (Fig.…”