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

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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE60803 | GEO | 2014/08/27

SECONDARY ACCESSION(S): PRJNA259569

REPOSITORIES: GEO

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Redifferentiation of adult human M-NM-2 cells expanded in vitro by inhibition of the WNT pathway

Project description:In vitro expansion of adult human islet M-NM-2 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 M-NM-2-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 M-NM-2-catenin translocation into the nucleus and activation of the WNT pathway. Inhibition of M-NM-2-catenin expression in expanded BCD cells using short hairpin RNA resulted in growth arrest, mesenchymal-epithelial transition, and redifferentiation, as judged by activation of M-NM-2-cell gene expression. Furthermore, inhibition of M-NM-2-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. Gene expression was studied for beta-cells (4 donors). Dedifferentiation was induced by inhibition of M-NM-2-catenin expression using shRNA. The experiment was performed in 4 batches (see the 'Date' characteristic in the Sample records).

2014-08-27 | E-GEOD-60803 | biostudies-arrayexpress

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro

Project description: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, while evidence supports the replicative capacity of adult beta cells in vivo, attempts at expanding human islet cells in tissue culture resulted in loss of beta-cell phenotype. Using a genetic lineage-tracing approach we have 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 a partially open chromatin structure in expanded BCD cells, although they are not transcribed. Here we report that BCD cells can be induced to redifferentiate by a combination of soluble factors. The redifferentiated cells express beta-cell genes, store insulin in typical secretory vesicles, and release it in response to glucose. The redifferentiation process involves mesenchymal-epithelial transition, as judged from changes in gene expression. Moreover, inhibition of the EMT effector SLUG using shRNA results in stimulation of redifferentiation. BCD cells also give rise at a low rate to cells expressing other islet hormones, suggesting transition through an islet progenitor-like stage during redifferentiation. These 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. Gene expression was studied in unexpanded islets (4 donors), expanded and dedifferentiated islet cells (4 donors), and re-differentiated islet cells (3 donors). The experiment was performed in 3 batches (see Date in the description table below).

2011-07-18 | E-GEOD-30732 | biostudies-arrayexpress

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro

2011-07-19 | GSE30732 | GEO

Inhibition of ZEB1 expression induces redifferentiation of adult human β cells expanded in vitro

Project description:In-vitro expansion of functional adult human β-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of β-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded β-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), β-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human β-like cells for cell therapy of diabetes.

2015-07-27 | GSE69658 | GEO

Diverse Targets of β-catenin during the Epithelial-Mesenchymal Transition Define Cancer Stem Cells and Predict Disease Relapse

Project description:Wnt signaling contributes to the reprogramming and maintenance of cancer stem cell (CSC) states that is activated by the epithelial-mesenchymal transition (EMT) program. However, the mechanistic relationship between the EMT and Wnt pathway in CSCs remains unclear. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) indicated that EMT induces a switch from the β-catenin/E-cadherin/Sox15 complex to the β-catenin/Twist1/TCF4 complex, which then binds to CSC-related gene promoters. In tandem co-IP and re-ChIP experiments using epithelial-type cells, Sox15 associated with the β-catenin/E-cadherin complex and then bound to the proximal promoter region of CASP3, consequently resulting in Twist1 cleavage and negatively regulating the β-catenin–elicited promotion of the CSC phenotype. During the EMT, Twist1 in complex with β-catenin enhanced β-catenin/TCF4 transcriptional activity, which includes binding to the proximal promoter region of ABCG2, a marker of CSCs. For clinical application, the five-gene signature nuclear β-cateninHigh/nuclear Twist1High/E-cadherinLow/Sox15Low/CD133High may be a valuable prognostic marker in patients with human lung cancer.

2015-04-04 | GSE67571 | GEO

A Novel Role of Wnt/β Catenin Signaling in Regulating the Nucleolus in Triple Negative Breast CancerA Novel Role of Wnt/β Catenin Signaling in Regulating the Nucleolus in Triple Negative Breast Cancer

Project description:Breast cancer is one of the most common types of cancer in women. One key signaling pathway known to regulate tumor growth, metabolic adaptation, and cellular stress response in breast cancer is Wnt signaling. Breast cancer patients, specifically triple negative breast cancer (TNBC), with upregulated Wnt signaling often have a poor clinical prognosis. However, the effects of Wnt/β-catenin signaling on the nucleolus and the resultant impact on cancer development and progression remain unclear. A notable reduction was observed in the number of nucleoli per nucleus in response to Wnt/β-catenin signaling inhibition in multiple TNBC cell lines. Our comparative proteomic analysis revealed several changes in the composition of the nucleolar proteome of TNBC cells upon inhibition of Wnt signaling. Overall, we demonstrate that Wnt/β-catenin signaling will affects nucleolar functionality and thus influences breast cancer progression. Understanding the role of Wnt signaling in the nucleolus and breast cancer is a critical step towards developing novel therapeutic options for the treatment of breast cancer.

2021-03-05 | PXD021181 | Pride

The NFkB subunit RELA is a master transcriptional regulator of the committed epithelial-mesenchymal transition in airway epithelial cells

Project description:The epithelial-mesenchymal transition (EMT) is a multistep dedifferentiation program important in tissue repair. Here, we examined the role of the transcriptional regulator NFkB in EMT of human primary small airway epithelial cells (hSAECs). Surprisingly, transforming growth factor β (TGFβ) activated NFkB/RELA proto-oncogene, NFkB subunit (RELA) translocation within 1 day of stimulation, yet induction of its downstream gene regulatory network occurred only after 3 days. A time course of TGFβ-induced EMT transition was analyzed by RNA-Seq in the absence or presence of inducible shRNA-mediated silencing of RELA. In WT cells, TGFβ stimulation significantly affected the expression of 2,441 genes. Gene set enrichment analysis identified Wnt, cadherin, and NFkB signaling as the most prominent TGFβ-inducible pathways. By comparison, RELA controlled expression of 3,138 overlapping genes mapping to Wnt, cadherin, and chemokine signaling pathways. Conducting upstream regulator analysis, we found that RELA controls six clusters of upstream transcription factors, many of which overlapped with a transcription factor topology map of EMT developed earlier. RELA triggered expression of three key EMT pathways: (1) the Wnt/β-catenin morphogen pathway, (2) the JUN transcription factor, and (3) the Snail family transcriptional repressor 1 (SNAI1). RELA binding to target genes was confirmed by ChIP. Experiments independently validating Wnt dependence on RELA were performed by silencing RELA via genome editing and indicated that TGFβ-induced WNT5B expression and downstream activation of the Wnt target AXIN2 are RELA-dependent. We conclude that RELA is a master transcriptional regulator of EMT upstream of Wnt morphogen, JUN, SNAI1-ZEB1, and interleukin-6 autocrine loops.

2018-09-26 | GSE84135 | GEO

Characterisation of the Beta-Catenin cytoplasmic/nuclear interactome in leukaemia cells

Project description:Canonical Wnt/B-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no β-catenin nuclear-localization even where cytosolic B-catenin is abundant. Differential propensity for nuclear-localized β-catenin is also observed in cell lines. To investigate the factors mediating the nuclear-localization of B-catenin we carried out a nuclear/cytoplasmic proteomic analysis of the B-catenin interactome in myeloid leukemia cells. From this we identified hundreds of putative novel B-catenin-interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear B-catenin, K562/HEL) versus Wnt-unresponsive cells (low nuclear B-catenin, ML1) suggested the established interactor, LEF1, is a key factor mediating the nuclear-localization of B-catenin in myeloid leukemia. The relative levels of nuclear LEF1 and B-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF1 knockdown inhibited B-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF1 overexpression was able to promote both nuclear-localization and B-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This study is the first to present a B-catenin interactome in hematopoietic cells and reveals LEF1 as a critical regulator of canonical Wnt signaling in myeloid leukemia.

2019-01-11 | PXD009305 | Pride

The beta-catenin interactome in a vascular smooth muscle cell model

Project description:Canonical Wnt/β-catenin signalling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/β-catenin signalling also contributes to various pathologies including cancer, necessitating an understanding of cell context dependent mechanisms regulating this pathway. Since protein-protein interactions underpin β-catenin function and localization, we sought to identify novel β-catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where β-catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC β-catenin interactome.

2018-10-25 | PXD010421 | Pride

Inhibition of canonical Wnt/β-catenin signaling is involved in leflunomide (LEF)-mediated cytotoxic effects on renal carcinoma cells

Project description:Inhibition of canonical Wnt/β-catenin signaling is involved in leflunomide (LEF)-mediated cytotoxic effects on renal carcinoma cells

2016-03-31 | GSE77433 | GEO

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