Truncated CD19 as a selection marker for the isolation of stem cell derived β-cells

Abstract

Stem cell-derived {beta}-cells (SC{beta}-cell) are a renewable and scalable alternative to cadaveric islets as a cell replacement therapy for type 1 diabetes (T1D). However, heterogeneity within SC{beta}-cell cultures remains problematic for graft safety and function. Magnetic selection of SC{beta}-cells expressing a unique cell surface marker may help deplete undesirable cell types and facilitate functional maturation. Here, we explored CD19 as a potential cell surface marker for the enrichment of insulin-expressing SC{beta}-cells. Using CRISPR/Cas9 technology, we created a knock-in add-on of CD19-mScarlet downstream of the insulin coding sequence in human embryonic stem cells (hESCs). We established reproducible SC{beta}-cell surface expression of CD19-mScarlet. Importantly, we developed and optimized a magnetic sorting protocol for CD19-mScarlet-expressing cells, forming enriched SC{beta}-cell clusters with improved glucose-stimulated c-peptide secretion. This strategy holds promise to facilitate large-scale production of functional SC{beta}-cells for disease modeling and cell replacement therapy.