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

Stem cell-derived β-cells (SCβ-cell) are a renewable and scalable alternative to cadaveric islets 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. Here, we explored the transmembrane glycoprotein CD19 as a potential cell-surface marker for the enrichment of insulin-expressing SCβ-cells. Using CRISPR/Cas9 technology, we created a knock-in add-on of CD19-mScarlet downstream of insulin (INS) coding sequence exon 2 in human embryonic stem cells (hESCs). We developed and optimized a magnetic-activated cell 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 disease modeling and cell-replacement therapy.