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

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

Synergistic effects of β-catenin downregulation and RC treatment.

<p><b>A</b>, Effect of a 4-day RC treatment on levels of WNT pathway gene transcripts in expanded islet cells at passages 5–6. Data are mean±SE (n = 4–5 donors). *p<0.05, compared with UTR cells. <b>B</b>, Effect of a 4-day RC treatment on subcellular localization of β-catenin in expanded islet cells at passage 6. Beta-catenin is localized throughout the cell in >98% of expanded untreated cells, while >98% of cells treated with β-catenin shRNA show β-catenin membrane localization. Bar = 50 µm. <b>C</b>, Effect of a 4-day RC treatment on levels of WNT pathway gene transcripts in sorted eGFP⁺ BCD cells at passages 5–6. Data are mean±SE (n = 3–4 donors). *p<0.05, compared with UTR cells. <b>D</b>,<b>E</b>, Synergistic effect of a 8-day RC treatment and β-catenin shRNA on levels of WNT pathway target gene (D) and β-cell transcripts (E) in expanded islet cells at passages 5–6. Data are mean±SE (n = 3–5 donors). *p<0.05, relative to nontarget shRNA. <b>F</b>, C-peptide immunofluorescence in expanded islet cells infected at passages 5–6 with β-catenin or NT shRNA viruses, and treated for 4 days with RC. Bar = 75 µm. Values are mean±SD (n = 3 donors), based on quantitation of >1000 cells in each group. *p<0.005, relative NT shRNA.</p

Genes selected for validation of microarray results by qPCR.

<p>Genes selected for validation of microarray results by qPCR.</p

Global changes in gene expression in BCD cells infected with β-catenin shRNA.

<p><b>A</b>, Heat map of cDNA microarray analysis of RNA extracted from sorted eGFP⁺ BCD cells at passages 4–5, 7 days following infection with β-catenin or NT shRNA viruses. n = 4 donors for β-catenin shRNA; n = 3 donors for NT shRNA. <b>B</b>, Gene ontology analysis of cDNA microarray results. <b>C</b>, Validation of candidate genes from the microarray analysis by qPCR analysis of RNA extracted from eGFP⁺ BCD cells at passages 5–6, 7 days following infection with β-catenin or NT shRNA viruses. Data are mean±SE (n = 3–4 donors). *p<0.05, **p<0.005, relative to NT shRNA. Validation of IAPP transcripts is seen in D.</p

Effect of β-catenin downregulation on BCD cell redifferentiation.

<p><b>A</b>, qPCR analysis of RNA extracted from expanded islet cells at passages 4–5, 7 days following infection with increasing amounts of β-catenin or NT shRNA viruses. Data are mean±SE (n = 3 donors). *p<0.05, **p<0.005, relative to NT shRNA. <b>B</b>, qPCR analysis of RNA extracted from expanded islet cells at passages 5–7, 7 days following infection with β-catenin or NT shRNA viruses. Data are mean±SE (n = 3–8 donors). *p<0.05, **p<0.005, relative NT shRNA. <b>C</b>, C-peptide immunofluorescence in expanded islet cells infected at passages 5–7 with β-catenin or NT shRNA viruses, and analyzed 7 days later. Bar = 50 µm Values are mean±SD (n = 4 donors), based on quantitation of >1000 cells in each group. *p<0.005, relative NT shRNA. <b>D</b>, qPCR analysis of RNA extracted from sorted eGFP⁺ BCD cells at passages 6–8, 7 days following infection with β-catenin or NT shRNA viruses. Data are mean±SE (n = 3–4 donors). *p<0.05, relative to NT shRNA.</p

Upregulation of the WNT pathway in BCD cells.

<p><b>A</b>, qPCR analysis of RNA extracted from isolated islets (p0) and expanded islet cells at the indicated passage number. <b>B</b>, qPCR analysis of RNA extracted from sorted eGFP⁺ BCD cells at passages 5–6. Data in A and C are mean±SE (n = 3–5 donors). *p<0.05, **p<0.005, compared with P0. <b>C</b>, Immunofluorescence analysis of isolated islets, and expanded islet cells at passage 6. Beta-catenin is localized in the membrane region in >98% of islet cells, while >98% of cells at passage 6 show β-catenin nuclear localization. Active β-catenin was not detected in islet β cells. Bar = 50 µm.</p

Donors of islets used in the study.

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

Synegistic effects of β-catenin and HES1 downregulation.

<p><b>A</b>, qPCR analysis of RNA extracted from expanded islet cells at passage 6, 7 days following infection with β-catenin shRNA, HES1 shRNA, or both. Data are mean±SE (n = 3 donors). *p<0.05, **p<0.005. <b>B</b>, Effect of β-catenin downregulation on levels of NOTCH pathway gene transcripts. qPCR analysis of RNA extracted from expanded islet cells at passages 5–6, 7 days following infection with β-catenin or NT shRNA viruses. Data are mean±SE (n = 3–8 donors). *p<0.05, **p<0.005, relative to NT shRNA.</p

Primer sequences for qPCR analyses.

<p>Primer sequences for qPCR analyses.</p