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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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Upregulation of the WNT pathway in BCD cells.A, qPCR analysis of RNA extracted from isolated islets (p0) and expanded islet cells at the indicated passage number. 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. C, 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.
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pone-0112914-g001: Upregulation of the WNT pathway in BCD cells.A, qPCR analysis of RNA extracted from isolated islets (p0) and expanded islet cells at the indicated passage number. 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. C, 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.

Mentions: Analysis of WNT pathway gene expression during the first three weeks of islet cell expansion in vitro revealed a significant upregulation of transcripts encoding WNT pathway receptor and target genes (Fig. 1A). Transcripts for the WNT receptor FZD2 were upregulated >30-fold. Activation of the WNT pathway resulted in upregulation of its target genes CCND1 and MYC (20-fold and 3-fold, respectively), which stimulate cell proliferation; PITX2 (130-fold), which activates CCND1 and MYC; and SNAI2 (49-fold), which participates in inhibition of CDH1 expression. Analysis of sorted eGFP+ cells, labeled by an insulin promoter-driven lineage tracing system [4], confirmed the activation of these genes in BCD cells (Fig. 1B). A comparable activation was observed in eGFP-negative cells, which include both non-BCD cells and BCD cells that were not labeled by eGFP [4]. Immunofluorescence analysis showed translocation of β-catenin from its membrane position in islet β-cells into the nucleus in eGFP+-labeled BCD cells (Fig. 1C), where it can activate transcription of target genes. The translocation was associated with a notable epithelial-to-mesenchymal morphological change.


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)

Upregulation of the WNT pathway in BCD cells.A, qPCR analysis of RNA extracted from isolated islets (p0) and expanded islet cells at the indicated passage number. 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. C, 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112914-g001: Upregulation of the WNT pathway in BCD cells.A, qPCR analysis of RNA extracted from isolated islets (p0) and expanded islet cells at the indicated passage number. 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. C, 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.
Mentions: Analysis of WNT pathway gene expression during the first three weeks of islet cell expansion in vitro revealed a significant upregulation of transcripts encoding WNT pathway receptor and target genes (Fig. 1A). Transcripts for the WNT receptor FZD2 were upregulated >30-fold. Activation of the WNT pathway resulted in upregulation of its target genes CCND1 and MYC (20-fold and 3-fold, respectively), which stimulate cell proliferation; PITX2 (130-fold), which activates CCND1 and MYC; and SNAI2 (49-fold), which participates in inhibition of CDH1 expression. Analysis of sorted eGFP+ cells, labeled by an insulin promoter-driven lineage tracing system [4], confirmed the activation of these genes in BCD cells (Fig. 1B). A comparable activation was observed in eGFP-negative cells, which include both non-BCD cells and BCD cells that were not labeled by eGFP [4]. Immunofluorescence analysis showed translocation of β-catenin from its membrane position in islet β-cells into the nucleus in eGFP+-labeled BCD cells (Fig. 1C), where it can activate transcription of target genes. The translocation was associated with a notable epithelial-to-mesenchymal morphological change.

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