University of Wisconsin-Madison
Maturation of human pluripotent stem cell-derived pancreatic progenitors into monohormonal islet-like cells after transplantation to mice
Sara Sackett1, Dan Tremmel1, Xiang Li1, Drew Roenneburg1, Cori O'Brien1, Jon S Odorico1.
1Surgery, University of Wisconsin-Madison, Medical School and Public Health, Madison, WI, United States
Stem cell based therapies, such as the differentiation of beta or islet-like cells from human pluripotent stem cells (hPSCs) hold great potential for the treatment of Type I diabetes. After years of research it is now well established that hPSCs can be directed to differentiate into enriched populations of pancreatic progenitors and beta-like cells in vitro and are capable of curing diabetes in mice. However, a variety of published protocols, all different from one another, appear to achieve the same endpoint.
The objective of our study is to analyze the impact from different in vitro differentiation protocols on maturation and function of cells after transplantation. Using multistage 18-32 day protocols which begin with bFGF, Activin A and BMP4 treatment to generate definitive endoderm, we show H1 cells begin to express key stage specific pancreatic developmental gene signatures (Sox17, Foxa2, Pdx1, Ngn3, Nkx6.1, Gcg, Ins) as cells differentiate through various protocols as characterized by immunocytochemical (ICC) and QPCR analyses. We evaluated multiple different media formulations in vitro and then transplanted 2-5x10^6 cells derived by different protocols under the kidney capsule of immunocompromised mice (SCID/Beige or NSG) with and without decellularized human pancreatic extracellular matrix (P-ECM) to determine if these pancreatic progenitors are capable of differentiating into mature, functional beta-like cells in vivo. The mice were monitored for changes in blood glucose, c-peptide secretion and received glucose tolerance tests over a 32-week time course.
We find that some protocols generate monohormonal insulin-expressing beta cells in vitro while others produce polyhormonal cells that express both insulin and glucagon at the end of the in vitro differentiation period. Of the latter group, monohormonal cells may evolve during in vivo differentiation as shown by ICC staining, in some cases, whereas with other protocols they remain predominantly polyhormonal. Moreover, some protocols produce few hormone positive cells while generating grafts containing non-endodermal tissues or teratomas with a relatively high frequency. ELISA analyses show detectable levels of c-peptide (>120 pM by 20 weeks) in some transplanted mice, while others do not, with some intra-protocol variability. In some cases glucose tolerance tests show improved glucose clearance beginning as early as 12 weeks and increased c-peptide following glucose stimulation.
Although the precise mechanism of the in vivo maturation remains undefined, it is clear that some in vitro derived cell populations have significantly greater potential for differentiating into insulin-secreting beta cells than others. The incorporation of P-ECM into transplantation assays may further promote functional maturation of progenitor cell populations.
This work was supported by the Juvenile Diabetes Research Foundation. Thanks to Dr. Luis Fernandez for the provision of islets. Thanks to the University of Wisconsin Organ and Tissue Donation for the provision of pancreata.
11:00 - 12:30
|Alternative Sources of Beta Cells: Xenoislet, Stem Cells, Tissue Engineering||Maturation of human pluripotent stem cell-derived pancreatic progenitors into monohormonal islet-like cells after transplantation to mice||Room 110|