Type 1 diabetes is an autoimmune disease driven by T-cell mediated destruction of the pancreatic beta cells, resulting in blood glucose dysregulation. Although islet transplantation is currently used in clinical practice, there is a shortage of donor tissue. Pluripotent stem cells (PSCs) may potentially fill this shortfall by providing an unlimited supply of endocrine cells for transplantation.
We have generated a human embryonic stem cell line in which sequences encoding green fluorescent protein (GFP) and mCherry fluorescent protein (mCh) were placed under the regulatory control of the INSULIN and GLUCAGON genes respectively (INSGFP/w GCGmCh/w). This cell line enables endocrine cell specification to be monitored in real-time, as well as facilitating isolation of alpha and beta cells.
We utilized this INSGFP/w GCGmCh/w PSC line to identify novel cell surface markers expressed at intermediate stages of differentiation as well as on mature endocrine cells. RNAseq was performed on cells sorted based on their GFP and/or mCherry expression and compared to pancreatic progenitor cells. We discovered that a number of integrin subunits were dynamically regulated during the course of PSC differentiation. Specifically, we found that integrin αv marked both PSC-derived and adult human pancreatic endocrine cells.
We have used antibodies to integrin αv to isolate pancreatic endocrine cells from both adult cadaveric tissue and differentiated PSC cultures by flow cytometry. This method of endocrine cell enrichment permits the isolation and analysis of clinically-relevant pancreatic cell types, and is potentially of great value for cell replacement therapies for type 1 diabetes.
This work was supported by the Juvenile Diabetes Research Foundation and the National Health and Medical Research Council Australia.