Generation of monohormonal INSULIN+ cells from human embryonic stem cells

Human Embryonic Stem Cells (hESCs) are immortal, pluripotent cells derived from the inner cell mass of the pre implantation embryo. These cells have the potential to differentiate into all cell types including insulin producing beta cells, which could provide an alternative to cadaver‐derived islets for the treatment of type 1 diabetes. We have explored the formation of pancreatic progenitor cells and mature β‐cells using hESC reporter lines which express GFP under the control of regulatory sequences from the PDX1 and INSULIN genes respectively. Using serum‐free media, we have found that specific combinations of factors applied in a sequential fashion result in the formation of a pancreatic precursor population, which can be further differentiated to hormone expressing cells. These endocrine cells have been shown to be a heterogeneous population that contains a substantial population of INSULIN expressing cells that do not express either glucagon or somatostatin, of which a subpopulation expresses NKX6.1. An INSULINGFP/w hESC reporter line was further modified by the addition of a luciferase transgene under the control of an EF1α promoter, which allowed for in vivo imaging of transplanted INSULIN expressing cells. Differentiated Tg‐EF1αLuc.INSULINGFP/w cells were sorted based on INSULIN‐GFP expression, and transplanted under the kidney capsule of immunocompromised mice and assessed for the retention of INSULIN‐GFP+ cells. It was found that the transplanted INSULIN+ cells had the ability to form monohormonal endocrine cells of three different lineages. Additionally it was seen that persisting INSULIN+ cells co‐expressed transcription factors associated with beta‐cell maturity, such as NKX6.1 and MAFA.