Multiomics analysis of keratinocyte differentiation identifies secreted markers for non-destructives cell therapy in-process controls

Pluripotent stem cell-based therapy holds great promise for regenerative medicine, but industrial-scale bioproduction is still in its infancy. A particular challenge for cells that need to adhere to a substrate to differentiate properly is currently the absence of non-destructive cellular controls during the process. This renders biomanufacturing essentially blind until the final stage of differentiation -several weeks away for some cell types- thus preventing potentially necessary corrective measures or premature, money-saving termination. We have overcome this problem by creating a methodology that allows the selection of appropriate markers in the culture medium, based on single-cell RNA sequencing analysis and quantitative proteomics at several stages of cell differentiation, enabling the identification of informative secreted markers. As a proof of concept, we applied this approach to the differentiation of basal keratinocytes from pluripotent stem cells and identified IGFBP6 and TNC as two secreted proteins. These biomarkers can be directly measured in the culture supernatant, providing informative and non-invasive indicators of the differentiation process.