Insulin-Producing Cells Generated from Dedifferentiated Human Pancreatic Beta Cells Expanded In Vitro

Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening

Blocking of ALK5 activation prevents proliferation and dedifferentiation in cultured islet cells.

<p>A,B, Immunofluorescence analysis of C-peptide expression in human islet cells infected with <i>ALK5</i> or nontarget (NT) shRNA viruses and grown in culture for 3 weeks, compared with uncultured islets and untreated (UTR) cultured cells. Bar = 100 μM. DNA was stained with DAPI. C, Quantitation of immunofluorescence analysis of Ki67 expression in the cells in A. Values in B and C are mean±SE (n = 4–5 donors), based on counting ≥500 cells in each sample. Fold change and P values shown on top of bars are relative to NT shRNA. D, qPCR analysis of RNA extracted from the cells in A. Values are mean±SE (n = 4–5 donors) relative to UTR (RQ = 1) and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤ 0.05, **P≤0.01. Fold change and P values shown on top of bars are relative to NT shRNA.</p

TGFβ Pathway Inhibition Redifferentiates Human Pancreatic Islet β Cells Expanded <i>In Vitro</i>

<div><p><i>In-vitro</i> expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of β-cell-derived (BCD) cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT). Nevertheless, BCD cells maintain open chromatin structure at β-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor β (TGFβ) pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion <i>in vitro</i> involves upregulation of the TGFβ pathway. Blocking TGFβ pathway activation using short hairpin RNA (shRNA) against <i>TGFβ Receptor 1</i> (<i>TGFBR1</i>, <i>ALK5)</i> transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with <i>ALK5</i> shRNA results in their redifferentiation, as judged by expression of β-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. <i>ALK5</i> inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following <i>ex-vivo</i> expansion.</p></div

Differentially expressed genes selected for qPCR validation of microarray results.

<p>Differentially expressed genes selected for qPCR validation of microarray results.</p

ALK5 shRNA inhibits AKT and activates FOXO1.

<p>A, Immunoblotting analysis of phosphorylated AKT, phosphorylated FOXO1, and total FOXO1 proteins in expanded islet cells infected at passage 5 with <i>ALK5</i> or NT shRNA viruses and analyzed 6 days later. Values are mean±SE (n = 4–5 donors) relative to UTR and normalized to HSC70. Fold change and P values are relative to NT shRNA. NS, not significant. B, Immunoblotting analysis of AKT in expanded islet cells infected at passage 5–6 with four <i>AKT</i> shRNA sequences and analyzed 6 days later. Values are mean±SE (n = 4–5 donors) and normalized to HSC70. *P≤ 0.05. Fold change and P values shown on top of bars are relative to NT shRNA. C, qPCR analysis of RNA extracted from expanded islet cells infected at passage 5–6 with <i>AKT</i> or NT shRNA viruses and analyzed 6 days later. Values are mean±SE (n = 4–5 donors) relative to NT shRNA and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤ 0.05. D, qPCR analysis of transcripts encoding cell cycle inhibitors in RNA extracted from expanded islets cells infected at passage 5 with <i>AKT</i> or NT shRNA viruses and analyzed 6 days later. Values are mean±SE (n = 4 donors) relative to NT shRNA and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤ 0.05, **P≤0.01. E, qPCR analysis of RNA extracted from expanded islets cells infected at passage 5 with <i>ALK5</i> shRNA virus and treated with FOXO1 inhibitor, and analyzed 6 days later. Values are mean±SE (n = 3 donors) relative to UTR and normalized to <i>RPLPO</i> and <i>TBP</i>. Fold change and P values are relative to <i>ALK5</i> shRNA. F, Scheme of suggested mechanism for induction of insulin expression and growth arrest by ALK5 downregulation.</p

Donors of islets used in the study.

<p>Donors of islets used in the study.</p

TGFβ pathway is activated in expanded human islet cells.

<p>A, qPCR analysis of RNA extracted from isolated human islets and expanded islet cells at the indicated passages. Values are mean±SE (n = 6 donors) relative to islets (RQ = 1) and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤0.05, **P≤0.01, ***P≤0.001. B, Immunofluorescence analysis of SMAD2/3 in human islets and expanded islet cells at passage 3. SMAD2/3 is localized in the cytoplasm of β-cells, and in the nucleus of GFP⁺ BCD cells. Bar = 25 μm (top), 50 μM (bottom). DNA was stained with DAPI. 100% of GFP⁺ cells showed staining of SMAD2/3 in the nucleus, based on counting ≥500 cells in each of 3 samples from different donors. C, Immunoblotting analysis of phosphorylated SMAD2 in human islets and expanded islet cells at the indicated passages. Values are mean±SE (n = 4–5 donors) relative to islets and normalized to HSC70. *P≤ 0.05.</p

Effect of ALK5 inhibition on redifferentiation of expanded human islet cells.

<p>Cells were infected at passage 5 with <i>ALK5</i> or NT shRNA viruses and analyzed 6 days later. A, qPCR analysis of transcripts encoding β-cell proteins (left), pancreatic hormones (middle), and mesenchymal markers (right).Values are mean±SE (n = 3–8 donors), relative to UTR and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤0.05, **P≤0.01, ***P≤0.001. B, Quantitation of immunofluorescence analysis of C-peptide. Values are mean±SE (n = 3 donors), based on counting ≥500 cells in each sample. C, C-peptide content and glucose-induced secretion. Values are mean±SE (n = 4 donors). D, E, Quantitation of immunofluorescence analysis of ACTA2 and Ki67. Values are mean±SE (n = 3 donors), based on counting ≥500 cells in each sample. F, qPCR analysis of transcripts encoding cell cycle inhibitors. Values are mean±SE (n = 3–5 donors) relative to UTR and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤0.05. Fold change and P values shown on top of bars are relative to NT shRNA.</p

Effect of ALK5 inhibition on redifferentiation of BCD cells.

<p>A, Immunofluorescence analysis of GFP in expanded islet cells at passages 2–3, compared to sorted GFP⁺ cells. Bar = 200 μM. DNA was stained with DAPI. B. Quantitation of cells in A, based on counting ≥500 cells in each of 3 samples from different donors. C, qPCR analysis of transcripts encoding β-cell proteins (left), pancreatic hormones (middle), and mesenchymal markers (right), in RNA extracted from sorted GFP⁺ BCD cells infected at passage 5 with <i>ALK5</i> or NT shRNA viruses, and analyzed 6 days later. Values are mean±SE (n = 3–6 donors) relative to UTR and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤ 0.05, **P≤0.01. Fold change and P values shown on top of bars are relative to NT shRNA. D, Quantitation of immunofluorescence analysis of C-peptide and GFP in cells infected at passage 5 with <i>ALK5</i> or NT shRNA viruses and analyzed 6 days later. Values are mean±SE (n = 4 donors), based on counting ≥500 cells in each sample. Fold change and P value are relative to NT shRNA. E, Co-expression of C-peptide and PDX1 in GFP⁺ BCD cells infected at passage 5 with <i>ALK5</i> or NT shRNA viruses and analyzed 6 days later by immunofluorescence. Bar = 25 μM. DNA was stained with DAPI. F, Hierarchical clustering of 32 upregulated and 20 downregulated (>1.5 fold, pV≤0.05) transcripts in cDNA microarray analyses of RNA extracted from sorted BCD cells infected at passages 4–5 with <i>ALK5</i> (n = 4 donors) or NT (n = 3 donors) shRNA viruses and analyzed 6 days later. G, qPCR validation of cDNA microarray results for selected genes. Values are mean±SE (n = 3–7 donors) relative to NT shRNA and normalized to <i>RPLPO</i> and <i>TBP</i>. *P≤0.05, **P≤0.01, ***P≤0.001. Fold change and P values shown on top of bars are relative to NT shRNA.</p

Redifferentiation of Adult Human β Cells Expanded <i>In Vitro</i> by Inhibition of the WNT Pathway

<div><p>In vitro expansion of adult human islet β cells is an attractive solution for the shortage of tissue for cell replacement therapy of type 1 diabetes. Using a lineage tracing approach we have demonstrated that β-cell-derived (BCD) cells rapidly dedifferentiate in culture and can proliferate for up to 16 population doublings. Dedifferentiation is associated with changes resembling epithelial-mesenchymal transition (EMT). The WNT pathway has been shown to induce EMT and plays key roles in regulating replication and differentiation in many cell types. Here we show that BCD cell dedifferentiation is associated with β-catenin translocation into the nucleus and activation of the WNT pathway. Inhibition of β-catenin expression in expanded BCD cells using short hairpin RNA resulted in growth arrest, mesenchymal-epithelial transition, and redifferentiation, as judged by activation of β-cell gene expression. Furthermore, inhibition of β-catenin expression synergized with redifferentiation induced by a combination of soluble factors, as judged by an increase in the number of C-peptide-positive cells. Simultaneous inhibition of the WNT and NOTCH pathways also resulted in a synergistic effect on redifferentiation. These findings, which were reproducible in cells derived from multiple human donors, suggest that inhibition of the WNT pathway may contribute to a therapeutically applicable way for generation of functional insulin-producing cells following ex-vivo expansion.</p></div