Epithelial to mesenchymal transition in human endocrine islet cells

Abstract

Backgroundβ-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells.ObjectiveTo investigate whether EMT takes place in the endocrine non-β cells of human islets.MethodologyHuman islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer.ResultsCo-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers.ConclusionsIn adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype.

Direct suppression of human islet dedifferentiation, progenitor genes, but not epithelial to mesenchymal transition by liraglutide

“…Interestingly, transcripts of progenitor markers: NEUROG3 , and ALDH1A3 in ex vivo dedifferentiated islets were upregulated in day 4 ( Figure 2 E) similar to the findings in diabetic islets of murine models. During islet dysfunctional process, epithelial-mesenchymal transition genes such as Nestin and Vimentin were observed during in vivo β−cell dedifferentiation [ 4 ], and expanded cultured human isolated islets [ 7 , 9 ]. Similarly, we found the rises of mesenchymal gene expressions: ACTA2, CDH-2, SNAIL2, and VIM , in the day 4 high glucose ex vivo cultured islets ( Figure 2 F).…”

“…NanoDrop SPectrophotometer was used to quantify the cDNAs. Gene-specific primers were ordered according to [ 9 , 27 , 28 ,] and then used to perform qPCR using SYBR Green PCR Master mix (Thermo Fisher) and an QuantStudio 3 (Thermo Fisher). Normalization of qPCR values was with values from qPCR of GAPDH values in the same samples.…”

“…β-cell dedifferentiation also occurs when cultured islets ex vivo but it was thought to involve genes important in β−cell function while the endocrine islets undergo epithelial-mesenchymal transition (EMT) [ 7 ] or loss of β−cell maintenance [ 8 ]. In our lineage tracing study during beta-cell dedifferentiation and conversion of islet endocrine cells [ 4 ], we observed evidence of epithelial mesenchymal transition such as immunoreactive Nestin + , Vimentin + and glucagon + cells [ 4 ] while other works have shown EMT gene expressions elevated in cultured human islets [ 7 , 9 ]. Whether or not ex vivo human islet dedifferentiation share molecular mechanisms of diabetic β−cell dedifferentiation has not been clearly elucidated.…”

Dysregulation of the Pdx1/Ovol2/Zeb2 axis in dedifferentiated β-cells triggers the induction of genes associated with epithelial–mesenchymal transition in diabetes

“…This suggests changes in islet cell:cell contacts do not provide any migratory advantages to β-cells as seen in cancer and metastasis. However, given that the dissociation of mouse and human islets is known to induce EMT in vitro [ 76 ], β-cell dedifferentiation may weaken islet cell:cell contacts and contribute in part to the mesenchymal reprogramming of β-cells. Interestingly, islets from diabetic subjects present increased vasculature density caused by the dilation of pre-existing islet blood vessels, which is thought to enable the adaptation of pancreatic islets to insulin requirements [ 77 , 78 ].…”

MicroRNA Expression Analysis of In Vitro Dedifferentiated Human Pancreatic Islet Cells Reveals the Activation of the Pluripotency-Related MicroRNA Cluster miR-302s

“…It has been previously demonstrated that in vitro culture of native adult human pancreatic islets derived from non-diabetic donors resulted into the generation/expansion of an undifferentiated cell population [ 1 ]. Several studies demonstrated that the resulting cell population derives from an epithelial to mesenchymal transition (EMT) program which induces specialized islet cells to lose endocrine pancreatic markers (dedifferentiation) while acquiring a mesenchymal/multipotent phenotype [ 2 , 3 , 4 ]. Moreover, additional evidence from in vitro human β-cell lineage tracing experiments demonstrated that dedifferentiated cells derive also from β-cells, thus representing a potentially ideal in vitro model of β-cell dedifferentiation [ 5 , 6 , 7 ].…”