Redifferentiation of expanded human islet β cells by inhibition of ARX
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE73433 | GEO | 2016/06/20
SECONDARY ACCESSION(S): PRJNA296955
REPOSITORIES: GEO
Similar Datasets
Project description:Ex-vivo expansion of adult human islet β cells has been evaluated for generation of abundant insulin-producing cells for transplantation; however, lineage-tracing has demonstrated that this process results in β-cell dedifferentiation. Redifferentiation of β-cell-derived (BCD) cells can be achieved using a combination of soluble factors termed Redifferentiation Cocktail (RC); however, this treatment leads to redifferentiation of only a fraction of BCD cells. This study aimed at improving redifferentiation efficiency by affecting the balance of islet progenitor-cell transcription factors activated by RC treatment. Specifically, RC treatment induces the transcription factors PAX4 and ARX, which play key roles in directing pancreas endocrine progenitor cells into the β/δ or α/PP developmental pathways, respectively. Misactivation of ARX in RC-treated BCD cells may inhibit their redifferentiation into β cells. Blocking ARX expression by shRNA elevated insulin mRNA levels 12.8-fold, and more than doubled the number of insulin-positive BCD cells. ARX inhibition in expanded α-cell-derived cells treated with RC did not cause their transdifferentiation into insulin-producing cells. The combination of RC and ARX shRNA treatment may facilitate the generation of abundant insulin-producing cells for transplantation into patients with type 1 diabetes.
| S-EPMC4746595 | biostudies-literature
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
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
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
Project description: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.
2014-08-27 | GSE60803 | GEO
Project description:After their destruction in adult mice, insulin-producing pancreatic beta-cells slowly regenerate from other islet cells, like glucagon-producing alpha-cells. However the molecular basis of this conversion is unknown. Moreover it remains unclear if this intra-islet cell conversion is relevant to human diseases with extensive beta-cell loss, like in type 1 diabetes (T1D). Here, we show that subsets of glucagon-expressing cells in subjects with T1D produce Insulin and other molecular features of beta-cells, accompanied by loss of the alpha-cell regulators DNA methyltransferase 1 (Dnmt1) and Aristaless-related homeobox (Arx). We generated mice permitting lineage tracing and inactivation of Dnmt1 and Arx in adult alpha-cells. Within 3 months of Dnmt1 and Arx loss, 50% of alpha-cells converted into cells producing insulin protein but not glucagon, changes not observed in alpha-cells after only Arx or Dnmt1 loss. Single cell isolation and high-throughput RNA sequencing revealed efficient and extensive alpha-cell conversion into progeny indistinguishable by global gene expression from native beta-cells. Our work reveals pathways regulated by Arx and Dnmt1 sufficient for achieving targeted generation of beta-cells from adult pancreatic alpha-cells.
2017-02-16 | GSE79457 | GEO
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.
2011-07-19 | GSE30732 | GEO
Project description:Redifferentiation of expanded human islet β cells by inhibition of ARX
| PRJNA296955 | ENA
Project description:Aristaless-related homeobox (ARX) is an X-linked gene encoding a bi-functional morphogenetic transcription factor with a key role in neuronal migration and brain development. Mutations in ARX have been identified in patients with X-linked Lissencephaly with Abnormal Genitalia (XLAG) and Early-infantile epileptic encephalopathy (EIEE). The aim of this project was to perform a proteomic analysis in whole neonatal brains isolated from XLAG and EIEE mouse models, ArxKO/Y and Arx(GCG)7/Y, to compare the mutant proteome profiles versus the wild-type ones by LC-MS/MS. By comparing the two proteomics profiles, common and different protein regulations emerged. Collectively, these findings could provide novel molecular insights into the proteomic mechanisms underlying XLAG and EIEE pathogenesis and may accelerate the design of pathway-guided therapeutic interventions for ARX-endophenotypes.
2024-06-14 | PXD021358 | Pride
Project description:Arx is a paired-box homeodomain transcription factor and the vertebrate ortholog to the Drosophila aristaless (al) gene. Mutations in Arx are associated with a variety of human diseases, including X-linked infantile spasm syndrome (OMIM: 308350), X-linked myoclonic epilepsy with mental retardation and spasticity (OMIM: 300432), X-linked lissencephaly with ambiguous genitalia (OMIM: 300215), X-linked mental retardation 54 (OMIM: 300419), and agenesis of the corpus callosum with abnormal genitalia (OMIM: 300004). Arx-deficient mice exhibit a complex, pleiotrophic phenotype, including decreased proliferation of neuroepithelial cells of the cortex, dysgenesis of the thalamus and olfactory bulbs, and abnormal nonradial migration of GABAergic interneurons. It has been suggested that deficits in interneuron specification, migration, or function lead to loss of inhibitory neurotransmission, which then fails to control excitatory activity and leads to epilepsy or spasticities. Given that Arx mutations are associated with developmental disorders in which epilepsy and spasticity predominate and that Arx-deficient mice exhibit deficits in interneuron migration, understanding the function of Arx in interneuron migration will prove crucial to understanding the pathology underlying interneuronopathies. Yet, downstream transcriptional targets of Arx, to date, remain unidentified. The aim of this project is to identify bona fide transcriptional targets for the Arx, a transcription factor required for normal migration of interneurons from the ganglionic eminences to the cortex, and to investigate the functions of these genes in the Arx-dependent pathway regulating nonradial neuronal migration. We hypothesize that the genes regulated by the Arx transcription factor will play a critical role in the nonradial migration of interneurons and that the results of this study will provide novel insights into the molecular mechanisms of nonradial neuronal migration, in particular, and possibly the molecular and biochemical pathogenesis underlying epilepsy, mental retardation, infantile spasm syndromes, and other so-called interneuronopathies;; We have recently generated a transgenic mouse with a floxed Arx allele (Arxflox). We have generated conditional knockouts in which Arx is removed specifically from the brain by mating Arxflox mice with transgenic mice expressing Cre behind the neural tube-specific transcriptional regulatory elements of the POU domain, class 3, transcription factor 4 promoter. Preliminary analyses of these mice suggest that conditional knockout mice recapitulate the nonradial migration defects associated with conventional knockout mice. We will compare the gene expression profiles of ganglion eminences (GEs; the anatomical source for nonradially migrating interneurons) from male Arxflox mice that express Pou3f-Cre to those from male mice without Arxflox allele. Animals will be prepared and sacrificed following our institutional protocol. Tissue will be rapidly dissected from E14.5 (the temporal peak of interneuron migration) GEs (MGE and LGE from both left and right hemispheres). Preliminary experiments suggest that the amounts of RNA that can be isolated from a pair of GEs is in the range of 2000-3500 ng, which should be sufficient for microarray analysis following linear amplification of RNA. The GEs from each animal will be combined, snap frozen in liquid nitrogen, and stored at -80 C until RNA is extracted. Total RNA will be extracted using Trizol followed by RNA purification with the RNeasy cleanup kit. We will be providing total RNA samples from four wildtype and four transgenic animals (true biological replicates) from three separate litters to mitigate any expression differences resulting from mouse to mouse or litter to litter variation.
2008-10-25 | E-GEOD-12609 | biostudies-arrayexpress