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

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 multistep 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 and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic integrity in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based test for the detection of hotspot mutations test. Cell-line identity was 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 qPCR for progenitor, neuronal, cortical, and glial markers. Taken together, we present a multistep quality control workflow to evaluate variability and reproducibility across and between iPSCs. ### Competing Interest Statement The authors have declared no competing interest. * 5-FAM : 5-carboxyfluorescein CV : crystal violet E8 : Essential 8 media EB : embryoid body ESC : embryonic stem cell iPSC : induced pluripotent stem cell Ho342 : Hoechst 33342 ICC : immunocytochemistry NE : neuroepithelial NEAA : non-essential amino acids NPC : neural progenitor cell PBMC : peripheral blood mononuclear cells PFA : paraformaldehyde RT : room temperature