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Functional Maturation and In Vitro Differentiation of Neonatal Porcine Islet Grafts

Hassouna, Tarek, MD1,2; Seeberger, Karen L.1,2; Salama, Bassem, MD1,2; Korbutt, Gregory S., PhD1,2

doi: 10.1097/TP.0000000000002354
Original Basic Science—General
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Background There is a strong rationale to pursue the use of neonatal porcine islets (NPIs) as an unlimited source of islets for clinical xenotransplantation. Because 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 neoformation from ductal cells. In an attempt to optimize the potential of NPIs as a source of ß cell grafts, we used 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 Dulbecco's modified Eagle medium nutrient mixture-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 immunodeficient mice with diabetes.

Results Compared with 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 with nondifferentiated controls and were shown to be more glucose tolerant.

Conclusions Culturing NPIs in a 20-day stepwise differentiation media increases the proportion of endocrine cells and augments both in vitro and in vivo function of the islets.

The optimization of an in vitro differentiation protocol to induce the functional maturation and differentiation of neonatal porcine islets into ß cells, for their ultimate use in clinical xenotransplantation.

1 Alberta Diabetes Institute, Alberta, Canada.

2 Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.

Received 13 February 2018. Revision received 13 June 2018.

Accepted 26 June 2018.

This study is supported by the Canadian Institutes of Health Research (grant MOP 119500).

The authors declare no conflicts of interest.

T.H., K.L.S., and G.S.K. have contributed equally.

T.H. and L.K.S. performed experiments, presented and analyzed the data and contributed to writing the article. B.S. conducted all transplants and glucose tolerance tests. G.S.K. designed the study, financial support, data interpretation, article review, and final editing of the article.

Correspondence: Gregory S. Korbutt, PhD, Alberta Diabetes Institute, 5-002 Li Ka Shing Center, University of Alberta, Edmonton, Alberta, Canada T6G 2E1. (korbutt@ualberta.ca).

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