Functional maturation and in vitro differentiation of neonatal porcine islet grafts

Background There is a strong rationale to pursue the use of neonatal porcine islets (NPIs) as an unlimited source of islets for clinical xenotransplantation. Since NPIs are composed of immature insulin producing beta (ß) cells and ductal precursor cells, they provide an ideal model to examine culture conditions to enhance ß cell proliferation and/or ß cell neo-formation from ductal cells. In an attempt to optimize the potential of NPIs as a source of ß cell grafts, we utilized an in vitro differentiation protocol and measured its effect on the functional maturation and differentiation of NPIs. Methods Pancreata from 1 to 3-day old neonatal pigs were digested and cultured in standard Ham's-F10 media for 5 days. Each independent preparation was then further cultured in DMEM-F12 differentiation media containing growth factors added in a stepwise fashion, or cultured in control Ham's-F10 media. After 20 days in culture, islets were assessed for insulin secretory capacity, cellular composition, gene expression and metabolic activity after transplantation in immunedeficient diabetic mice. Results Compared to control islets, differentiated islets exhibited a significantly higher proportion of endocrine cells, proliferating cell nuclear antigen double positive ß cells, and an enhanced glucose stimulated insulin secretory activity. Mice transplanted with differentiated islets had significantly lower blood glucose values at weeks 18 and 20 compared to nondifferentiated controls and were shown to be more glucose tolerant. Conclusions Culturing NPIs in a 20-day step-wise differentiation media increases the proportion of endocrine cells and augments both in vitro and in vivo function of the islets. Received 13 February 2018. Revision received 13 June 2018. Accepted 26 June 2018. * Contributed equally Corresponding Author Dr. Gregory S. Korbutt, Alberta Diabetes Institute, 5-002 Li Ka Shing Center, University of Alberta, Edmonton, Alberta, Canada, T6G 2E1. T: (780) 492-4657 F: (780) 492-5501. Email: korbutt@ualberta.ca Authorship TH and LKS performed experiments, presented and analyzed the data and contributed to writing the manuscript. BS conducted all transplants and glucose tolerance tests. GSK designed the study, financial support, data interpretation, manuscript review and final editing of manuscript. Disclosure The authors declare no conflicts of interest associated with this manuscript. Funding This study is supported by the Canadian Institutes of Health Research (Grant #MOP 119500). Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

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