379286 Scalable Generation of Functional Pancreatic β Cells from Human Pluripotent Stem Cells for Tissue Engineering and Drug Screening Applications

Monday, November 17, 2014: 8:48 AM
207 (Hilton Atlanta)
Jeffrey R. Millman1, Felicia W. Pagliuca1, Mads Gurtler1, Michael Segel1, Alana Van Dervort1, Jennifer Hyoje Ryu1, Quinn Peterson1 and Douglas A. Melton2, (1)HSCRB/HSCI, Harvard University, Cambridge, MA, (2)HSCRB/HSCI/HHMI, Harvard University, Cambridge, MA

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.

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See more of this Session: Stem Cells in Tissue Engineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division