A novel strategy to increase the proliferative potential of adult human Î2-cells while maintaining their differentiated phenotype

Abstract

Our previous studies demonstrated that Wnt/GSK-3/b-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human b-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases b-catenin nuclear translocation and b-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human b-cell proliferation while maintaining a b-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ,6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human b-cell proliferation ,20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptomewide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced b-catenin-mediated and b-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/bcatenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human b-cell proliferation while maintaining the b-cell phenotype. Citation: Aly H, Rohatgi N, Marshall CA, Grossenheider TC, Miyoshi H, et al. (2013) A Novel Strategy to Increase the Proliferative Potential of Adult Human b-Cells While Maintaining Their Differentiated Phenotype. PLoS ONE 8(6): e66131. doi:10.1371/journal.pone.0066131 Editor: Chunming Liu, University of Kentucky, United States of America Received February 14, 2013; Accepted May 1, 2013; Published June 12, 2013 Copyright: 2013 Aly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding provided by NIH Grants DK006181 (MLM), NIH DK00618146S1 (MLM), DK07296 (MLM), NIH Washington University Diabetes Research Center Morphology and Metabolic Analysis Core DK20579 (MLM), American Diabetes Association Grant 7-04-MN-32 (MLM), Barnes-Jewish Hospital Foundation Grant 7401-00 (MLM). Human islets were obtained from the Integrated Islet Distribution Program (IIDP) sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the Juvenile Diabetes Research Foundation International (JDRFI) and the JDRFI Sponsored Islets for Research Program at Washington University (JDRF-31-2008-382). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mmcdaniel@wustl.edu

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@inproceedings{Aly2013ANS, title={A novel strategy to increase the proliferative potential of adult human Î2-cells while maintaining their differentiated phenotype}, author={Haytham Aly and Nidhi Rohatgi and Connie A . Marshall and Tiffani C . Grossenheider and Hiroyuki Miyoshi and Thaddeus S Stappenbeck and Scot J Matkovich and Michael L. McDaniel}, year={2013} }