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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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Global changes in gene expression in BCD cells infected with β-catenin shRNA.A, 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, Gene ontology analysis of cDNA microarray results. C, 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.
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pone-0112914-g005: Global changes in gene expression in BCD cells infected with β-catenin shRNA.A, 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, Gene ontology analysis of cDNA microarray results. C, 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.

Mentions: Analysis of β-cell gene expression revealed a 8-fold increase in insulin transcript levels (Fig. 4A,B). The levels of insulin transcripts were inversely proportional to the levels of β-catenin transcripts, which were a function of the β-catenin shRNA virus MOI (Fig. 4A). This increase was manifested in a >3.5-fold elevation in the number of C-peptide+ cells, compared with cells treated with NT shRNA (Fig. 4C). This change reflected induction of redifferentiation in cells which did not express detectable insulin levels in the presence of high β-catenin levels, rather than upregulation of insulin expression in the minority of cells (∼0.5%) which did not undergo dedifferentiation. The increase in insulin transcripts was accompanied by induction of transcripts encoding the insulin gene transcription factors PDX1 and MAFA, as well as IAPP transcripts (Fig. 4B). In addition, GCG and SST transcripts were also elevated in expanded islet cells infected with the β-catenin shRNA virus (Fig. 4B). Our previous results have documented the potential of expanded islet cells to give rise to GCG- and SST-positive cells, which are distinct from insulin-positive cells generated in these cultures, and likely originate from non-BCD cells [7]. Analysis of RNA extracted from sorted eGFP+ BCD cells confirmed the activation of β-cell transcripts in BCD cells (Fig. 4D), and the lack of activation of GCG, SST, or PPY transcripts (Fig. S2). To profile global changes in gene expression induced in BCD cells by reducing β-catenin expression, RNA extracted from sorted eGFP+ cells infected with β-catenin or NT shRNA viruses was subjected to cDNA microarray analysis. A total of 297 genes were upregulated >1.5-fold, while 257 genes were downregulated >1.5-fold (Fig. 5A). Gene ontology analyses identified expression of genes involved in cell proliferation, cell adhesion, and cell-extracellular matrix interactions to have changed most significatly (Fig. 5B). The microarray results were validated by qPCR analyses of selected genes (Table 3, Fig. 5C). Taken together, these findings suggest that downregulation of β-catenin levels alone induces profound phenotypic changes in BCD cells, including growth arrest, MET, and 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)

Global changes in gene expression in BCD cells infected with β-catenin shRNA.A, 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, Gene ontology analysis of cDNA microarray results. C, 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.
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pone-0112914-g005: Global changes in gene expression in BCD cells infected with β-catenin shRNA.A, 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, Gene ontology analysis of cDNA microarray results. C, 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.
Mentions: Analysis of β-cell gene expression revealed a 8-fold increase in insulin transcript levels (Fig. 4A,B). The levels of insulin transcripts were inversely proportional to the levels of β-catenin transcripts, which were a function of the β-catenin shRNA virus MOI (Fig. 4A). This increase was manifested in a >3.5-fold elevation in the number of C-peptide+ cells, compared with cells treated with NT shRNA (Fig. 4C). This change reflected induction of redifferentiation in cells which did not express detectable insulin levels in the presence of high β-catenin levels, rather than upregulation of insulin expression in the minority of cells (∼0.5%) which did not undergo dedifferentiation. The increase in insulin transcripts was accompanied by induction of transcripts encoding the insulin gene transcription factors PDX1 and MAFA, as well as IAPP transcripts (Fig. 4B). In addition, GCG and SST transcripts were also elevated in expanded islet cells infected with the β-catenin shRNA virus (Fig. 4B). Our previous results have documented the potential of expanded islet cells to give rise to GCG- and SST-positive cells, which are distinct from insulin-positive cells generated in these cultures, and likely originate from non-BCD cells [7]. Analysis of RNA extracted from sorted eGFP+ BCD cells confirmed the activation of β-cell transcripts in BCD cells (Fig. 4D), and the lack of activation of GCG, SST, or PPY transcripts (Fig. S2). To profile global changes in gene expression induced in BCD cells by reducing β-catenin expression, RNA extracted from sorted eGFP+ cells infected with β-catenin or NT shRNA viruses was subjected to cDNA microarray analysis. A total of 297 genes were upregulated >1.5-fold, while 257 genes were downregulated >1.5-fold (Fig. 5A). Gene ontology analyses identified expression of genes involved in cell proliferation, cell adhesion, and cell-extracellular matrix interactions to have changed most significatly (Fig. 5B). The microarray results were validated by qPCR analyses of selected genes (Table 3, Fig. 5C). Taken together, these findings suggest that downregulation of β-catenin levels alone induces profound phenotypic changes in BCD cells, including growth arrest, MET, and 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