(84b) Scalable Generation of Functional Pancreatic β Cells from Human Pluripotent Stem Cells for Tissue Engineering and Drug Screening Applications
The generation of functional, insulin-secreting pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for cell replacement therapy and drug screening in diabetes. However, insulin-producing cells generated in vitro from human pluripotent stem cells, including embryonic stem (ES) and induced pluripotent stem (iPS) cells, lack many characteristics of bona fide β cells, including glucose-stimulated insulin secretion, appropriate marker expression, and rapid function after transplantation. Furthermore, production at a scale for practical use is highly desirable and technically challenging.
We have developed an in vitro, suspension-based protocol that from both ES and iPS cells produces β cells, termed stem cell-β cells (SC-β), in a 300-mL spinner flask using defined factors. SC-β cells increased intracellular [Ca2+] and secreted insulin in response to multiple sequential high glucose challenges at amounts comparable to cadaveric β cells. Insulin was packaged into secretary granules in a manner that was indistinguishable from cadaveric β cells when visualized by electron microscopy. In addition to insulin, SC-β cells expressed the β cell markers PDX1, MAFA, and NKX6-1 and did not express other non-β cell markers, including glucagon, somatostatin, ghrelin, and pancreatic polypeptide. This scalable protocol produced approximately 3x108 cells, of which 30-50% are SC-β cells.
As soon as two weeks after transplantation into SCID/Beige mice, high amounts of human insulin was detected in the serum of mice that received SC-β cells, similar to that found in mice transplanted with cadaveric β cells. The amount of human insulin increased after intraperitoneal injection of glucose, demonstrating the cells were functional in vivo. No human insulin was detected in mice transplanted with hESC-derived pancreatic progenitors and non-functional insulin-producing endocrine, consistent with previous findings. When transplanted into diabetic NRG-Akita mice, SC-β cells rapidly reversed hyperglycemia and maintained normoglycemia for 4 months.
To further demonstrate the utility of these SC-β cells, we treated SC-β cells with different categories of known anti-diabetic drugs that increase insulin secretion and with prolactin, which increases β cell proliferation. Increasing β cell function and mass are highly sought after drug targets for treating diabetes. Treatment with anti-diabetic drugs increased insulin release from SC-β cells. In addition, treatment with prolactin increased proliferation of SC-β cells, as indicated by an increase in Ki67 staining.
We have developed a scalable process to generate functional SC-β cells in vitro that are virtually indistinguishable from cadaveric β cells. This protocol is able to produce >108 SC-β cells per batch, meaning it is the first scalable human source of functional β cells for therapy and drug discovery. Given that the only alternative is the very limited and unreliable supply of cadaveric β cells, this discovery represents a major advance in stem cell engineering for treating diabetes.