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Effect of miR-375 overexpression on BCD cell redifferentiation.

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posted on 13.04.2015, 06:11 by Gili Nathan, Sharon Kredo-Russo, Tamar Geiger, Ayelet Lenz, Haggai Kaspi, Eran Hornstein, Shimon Efrat

A. miR-375 in-situ hybridization in human islets. DNA was stained with DAPI. Bar = 75 μm. B. Overexpression of miR-375. Expanded islet cells, or sorted GFP+ BCD cells, at passages 5–12 were infected with miR-375 or empty viral vectors and analyzed 5 days later by qPCR. Data are mean±SE (for expanded islet cells, n = 8 donors; for BCD cells, n = 3 donors), relative to empty viral vector, and normalized to miR-24 and U6-snRNA. C, D. Changes in expression of mesenchymal genes (C) and islet cell genes (D) in expanded islet cells infected at passages 4–12 with miR-375 or empty viral vectors, and analyzed by qRT-PCR. Data are mean±SE (n = 4–8 donors). E. Changes in expression of β-cell genes in sorted GFP+ BCD cells infected at passages 4–7 with miR-375 or empty viral vectors, and analyzed 5 days later by qPCR. Data are mean±SE (n = 3 donors), relative to empty viral vector. F-H. Immunofluorescence analysis of C-peptide in expanded islet cells infected at passages 5–6 with miR-375 or empty viral vectors. F,G. Quantitation of C-peptide+ cells among total expanded islet cells (F), or GFP+ BCD cells (G). Values are mean±SD (n = 3 donors), based on counting >500 cells in each condition. H. Bar = 30 μm. I. Changes in proliferation of expanded islet cells infected at passages 3–5 with miR-375 or empty viral vectors, and analyzed 5 days later by immunofluorescence for Ki67. Values are mean±SD (n = 4 donors), based on counting >500 cells in each condition.

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