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Abstract

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