Disruption of Cell-Adhesion Signaling Resolves Unwanted Progenitor Specification in Stem Cell-Derived α and β Cell Grafts

Directed differentiation protocols have recently been developed to produce stem cell-derived α (SC-α) cells as a potential component of a complete cell-based therapy for T1D, to complement the more widely studied stem cell-derived β (SC-β) cells. Differentiation protocols for SC-β cells produce off-target cell populations implicated in the development of outgrowths in SC-β cell grafts, but outgrowths from SC-α cells have not been explored. This study identifies that engrafted SC-α cells generate outgrowths of similar composition to SC-β cell outgrowths. Both cell types share outgrowth-driving populations marked by SOX9, CDX2, or SOX2. Single-cell RNA sequencing was used to reveal an enrichment in cell-adhesion signaling events in outgrowth-driving populations. Small-molecule inhibition of the Notch pathway was insufficient to disrupt all three outgrowth-driving populations. A comprehensive disruption of cell-adhesion signaling via single-cell dispersion and reaggregation is found to reduce the outgrowth propensity in engrafted SC-α and SC-β cells. Together, these results suggest that disrupting residual progenitor cells with SC-α and SC-β cell clusters can enhance the safety profile of these cell therapy products for T1D therapy.