Long-term expansion of functional pancreatic islets on a laminin-521 matrix and curative transplantation into diabetic mice. Mus musculus

In this study we show, for the first time, that whole islets can be cultured and significantly expanded long-term in vitro on specific basement membrane (BM) laminin LN-521 which is a normal component of islet BMs. Isolated islets adhere and flatten to form 2-3 cell layer entities with ~10 % of all endocrine cell types proliferating after 2-5 weeks in culture. Time-lapse imaging of freshly isolated islets in suspension labeled with hypoxia and DNA degradation indicators reveals that spherical islets on LN-111 rapidly develop hypoxia and central necrosis, in contrast to islets attached to LN-521, which normal b-cell glucose responsiveness. RNA sequencing analyses reveal that freshly isolated islets express LN-521 binding integrins. Compared to other matrices, islets grown on LN-521 develop a unique expression signature, including expression of specific integrins, involved in cell adhesion and matrix production as well as proliferation. The results reveal specific LN-521-mediated effects into islet cells and suggest that use of in vitro expanded islets may significantly enhance the efficacy of islet transplantation treatment of T1D. Overall design: Isolated mouse pancreatic islets were cultured in wells coated with 3 different matrices, either laminin-521, laminin-421 or laminin-111, for either 0, 3 or 12 days. Islets were seeded to triplicate wells (A, B and C), 20 islets per well. After each time point the islets were collected and stored in RNAlater in -80C until RNA isolation. Total RNA was extracted and sequencing libraries were constructed with 2 ng of total RNA. Barcoded libraries were multiplexed and sequenced as pair-end 75bp reads on HiSeq2000.