(84b) Scalable Generation of Functional Pancreatic β Cells from Human Pluripotent Stem Cells for Tissue Engineering and Drug Screening Applications

Millman, J. R., Harvard University
Pagliuca, F. W., Harvard University
Gurtler, M., Harvard University
Segel, M., Harvard University
Van Dervort, A., Harvard University
Ryu, J. H., Harvard University
Peterson, Q., Harvard University
Melton, D. A., Harvard University

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.