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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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Synegistic effects of β-catenin and HES1 downregulation.A, 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, 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.
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pone-0112914-g008: Synegistic effects of β-catenin and HES1 downregulation.A, 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, 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.

Mentions: Our previous work has shown that inhibition of the NOTCH pathway mediator HES1 induces BCD cell redifferentiation [9]. We therefore investigated a possible synergistic effect of inhibiting both β-catenin and HES1 on redifferentiation. As seen in Fig. 8A, a combination of the two treatments resulted in 1.4–7.6-fold higher levels of transcripts encoding insulin, IAPP, and the insulin gene transcription factors PDX1 and NEUROD1. β-catenin has been shown to affect expression of genes encoding NOTCH pathway components [28]. Treatment with β-catenin shRNA resulted in a significant upregulation of JAG2 and NOTCH4 (Fig. 8B), both of which were found to be downregulated in expanded islet cells, compared with human islets [8]. In addition, β-catenin shRNA induced downregulation of NOTCH2, which was upregulated in expanded islet cells, compared with human islets, and of JAG1 (Fig. 8B). Thus, part of the synergistic effect of the two shRNAs on redifferentiation could be due to blocking of β-catenin effects on expression of NOTCH pathway components. Taken together, these findings suggest that simultaneous inhibition of WNT and NOTCH pathways can contribute to BCD cell redifferentiation.


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)

Synegistic effects of β-catenin and HES1 downregulation.A, 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, 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231080&req=5

pone-0112914-g008: Synegistic effects of β-catenin and HES1 downregulation.A, 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, 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.
Mentions: Our previous work has shown that inhibition of the NOTCH pathway mediator HES1 induces BCD cell redifferentiation [9]. We therefore investigated a possible synergistic effect of inhibiting both β-catenin and HES1 on redifferentiation. As seen in Fig. 8A, a combination of the two treatments resulted in 1.4–7.6-fold higher levels of transcripts encoding insulin, IAPP, and the insulin gene transcription factors PDX1 and NEUROD1. β-catenin has been shown to affect expression of genes encoding NOTCH pathway components [28]. Treatment with β-catenin shRNA resulted in a significant upregulation of JAG2 and NOTCH4 (Fig. 8B), both of which were found to be downregulated in expanded islet cells, compared with human islets [8]. In addition, β-catenin shRNA induced downregulation of NOTCH2, which was upregulated in expanded islet cells, compared with human islets, and of JAG1 (Fig. 8B). Thus, part of the synergistic effect of the two shRNAs on redifferentiation could be due to blocking of β-catenin effects on expression of NOTCH pathway components. Taken together, these findings suggest that simultaneous inhibition of WNT and NOTCH pathways can contribute to BCD cell redifferentiation.

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