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Redifferentiation of adult human β cells expanded in vitro by inhibition of the WNT pathway.

Lenz A, Toren-Haritan G, Efrat S - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
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.

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Synergistic effects of β-catenin downregulation and RC treatment.A, 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, 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. C, 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. D,E, 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. F, 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.
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pone-0112914-g007: Synergistic effects of β-catenin downregulation and RC treatment.A, 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, 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. C, 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. D,E, 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. F, 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.

Mentions: We have previously shown that BCD cells can be redifferentiated by treatment with a combination of soluble factors in serum-free medium, termed Redifferentiation Cocktail (RC) [6]. These factors include activin A, exendin-4, nicotinamide, and high glucose concentrations, which have been shown to promote β-cell differentiation, in serum-free medium supplemented with B27 and ITS. RC treatment resulted in a significant reduction in transcripts encoding WNT pathway receptor and target genes (Fig. 7A), and in translocation of β-catenin from the cytoplasm and nucleus in eGFP+-labeled BCD cells to the membrane in redifferentiated C-peptide+ cells (Fig. 7B). Analysis of sorted eGFP+ BCD cells showed changes in transcripts encoding WNT pathway gene expression similar to those observed in total expanded islet cells (Fig. 7C). Expanded islet cells subjected to both RC treatment and β-catenin shRNA showed a synergistic 2-fold decrease in transcripts encoding FZD2 and WNT pathway target genes, compared with cells treated with RC and NT shRNA (Fig. 7D). A 3.6–7-fold increase in transcripts encoding CDH1, insulin gene transcription factors, insulin, and IAPP, was also observed in cells treated with RC and β-catenin shRNA, compared with those treated with RC and NT shRNA (Fig. 7E). Finally, the number of C-peptide+ cells more than doubled following the dual treatment, compared with cells treated with RC alone (Fig. 7F), suggesting that the synergistic effect was manifested in induction of redifferentiation in a larger number of BCD cells, rather than stimulation of higher insulin expression in cells already induced to differentiate. Overall, these findings suggest that a further decrease in WNT pathway activity, compared with that induced by RC alone, results in enhanced BCD cell redifferentiation. A second β-catenin shRNA sequence was used to confirm that the observed changes were due to β-catenin-specific inhibition. RC treatment combined with β-catenin shRNA TRCN-3843, which reduced β-catenin protein levels in HeLa cells by 76%, resulted in an increase in INS, IAPP and PDX1 transcripts comparable to that observed using β-catenin shRNA TRCN-3845 (Fig. 7E), indicating that the effect on cell redifferentiation was caused by specific inhibition of β-catenin expression.


Redifferentiation of adult human β cells expanded in vitro by inhibition of the WNT pathway.

Lenz A, Toren-Haritan G, Efrat S - PLoS ONE (2014)

Synergistic effects of β-catenin downregulation and RC treatment.A, 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, 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. C, 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. D,E, 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. F, 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.
© Copyright Policy
Related In: Results  -  Collection

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pone-0112914-g007: Synergistic effects of β-catenin downregulation and RC treatment.A, 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, 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. C, 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. D,E, 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. F, 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.
Mentions: We have previously shown that BCD cells can be redifferentiated by treatment with a combination of soluble factors in serum-free medium, termed Redifferentiation Cocktail (RC) [6]. These factors include activin A, exendin-4, nicotinamide, and high glucose concentrations, which have been shown to promote β-cell differentiation, in serum-free medium supplemented with B27 and ITS. RC treatment resulted in a significant reduction in transcripts encoding WNT pathway receptor and target genes (Fig. 7A), and in translocation of β-catenin from the cytoplasm and nucleus in eGFP+-labeled BCD cells to the membrane in redifferentiated C-peptide+ cells (Fig. 7B). Analysis of sorted eGFP+ BCD cells showed changes in transcripts encoding WNT pathway gene expression similar to those observed in total expanded islet cells (Fig. 7C). Expanded islet cells subjected to both RC treatment and β-catenin shRNA showed a synergistic 2-fold decrease in transcripts encoding FZD2 and WNT pathway target genes, compared with cells treated with RC and NT shRNA (Fig. 7D). A 3.6–7-fold increase in transcripts encoding CDH1, insulin gene transcription factors, insulin, and IAPP, was also observed in cells treated with RC and β-catenin shRNA, compared with those treated with RC and NT shRNA (Fig. 7E). Finally, the number of C-peptide+ cells more than doubled following the dual treatment, compared with cells treated with RC alone (Fig. 7F), suggesting that the synergistic effect was manifested in induction of redifferentiation in a larger number of BCD cells, rather than stimulation of higher insulin expression in cells already induced to differentiate. Overall, these findings suggest that a further decrease in WNT pathway activity, compared with that induced by RC alone, results in enhanced BCD cell redifferentiation. A second β-catenin shRNA sequence was used to confirm that the observed changes were due to β-catenin-specific inhibition. RC treatment combined with β-catenin shRNA TRCN-3843, which reduced β-catenin protein levels in HeLa cells by 76%, resulted in an increase in INS, IAPP and PDX1 transcripts comparable to that observed using β-catenin shRNA TRCN-3845 (Fig. 7E), indicating that the effect on cell redifferentiation was caused by specific inhibition of β-catenin expression.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus