Vitamin D and vitamin A receptor expression and the proliferative effects of ligand activation of these receptors on the development of pancreatic progenitor cells derived from human fetal pancreas.
ABSTRACT The growth and development of pancreatic islet cells are regulated by various morphogens. Vitamin A modulates in vitro differentiation of islet cells and vitamin D affects beta-cell insulin secretion, while both vitamin ligands act through heterodimerization with the retinoid X receptor (RXR). However, their effects in modulating pancreatic development have not been determined. In this study, cultured human pancreatic progenitor cells (PPCs) isolated from human fetal pancreas were stimulated to differentiate into islet-like cell clusters (ICCs). RT-PCR, Western blotting and immunocytochemistry were used to examine the expression and localization of vitamin D receptor (VDR), retinoic acid receptor (RAR), and RXR in PPCs. The effects of added all-trans retinoic acid (atRA, a form of vitamin A), calcitriol (activated vitamin D) and of these ligands together on PPC cell viability, proliferation and apoptosis were assessed by MTT, BrdU and ELISA assays, respectively. Post-treatment neurogenin-3 (NGN3) expression, necessary for islet-cell lineage development, was examined by real-time RT-PCR. Results showed that RAR, RXR and VDR were expressed in PPCs. RAR and RXR were localized in nuclei, and the VDR in nuclei, cytoplasm and plasma membrane. atRA and calcitriol each increased PPC viability and proliferation; atRA additionally decreased PPC apoptosis. Co-addition of atRA and calcitriol had no additive effects on cell viability but did increase ngn3 responses. In conclusion, RAR, RXR and VDR are expressed in human fetal PPCs and PPC proliferation can be promoted by calcitriol, atRA or both together, data valuable for elucidating mechanisms underlying islet development and for developing clinical islet transplantation.
- SourceAvailable from: Chi Chiu Wang[Show abstract] [Hide abstract]
ABSTRACT: Recent advance in directed differentiation of pancreatic stem cells offers potential to the development of replacement therapy for diabetic patients. However, the existing differentiation protocols are complex, time-consuming, and costly; thus there is a need for alternative protocols. Given the common developmental origins of liver and pancreas, we sought to develop a novel protocol, devoid of growth factors, by using liver stromal cells (LSCs) derived from human fetal liver. We examined the effects of the LSCs on the differentiation of pancreatic progenitor cells (PPCs) into islet-like cell clusters (ICCs). PPCs and LSCs isolated from 1st to 2nd trimester human fetal tissues underwent co-cultures; differentiation and functionality of ICCs were determined by examining expression of critical markers and secretion of insulin. Co-culture with 2nd but not 1st trimester LSCs enhanced ICC differentiation and functionality without the use of exogenous differentiation 'cocktails'. Differential expression profiles of growth factors from 1st versus 2nd trimester fetal liver were compared. Many morphogenic factors were expressed by LSCs, while insulin-like growth factor 1 (IGF1) was identified as one of the key molecules responsible for the ICC differentiation. This is the first report showing that an LSC-induced microenvironment can enhance ICC differentiation and functionality. Further modifications of the stroma microenvironment may offer an alternative, efficient and cost-effective approach to providing islets for transplantation.Stem cell reviews 01/2014; · 5.08 Impact Factor
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ABSTRACT: Aldehyde dehydrogenase 1 (ALDH1), a human stem-cell marker, is an enzyme responsible for converting retinaldehydes to retinoic acids (RAs) to modulate cell differentiation. However, data on expression levels and functional roles of ALDH1 during human fetal pancreatic development are limited. The focus of this study was to characterise ALDH1 expression patterns and to determine its functional role in islet cell differentiation. The presence of ALDH1 in the human fetal pancreas (8-22 weeks) was characterised by microarray, quantitative RT-PCR, western blotting and immunohistological approaches. Isolated human fetal islet-epithelial cell clusters were treated with ALDH1 inhibitors, retinoic acid receptor (RAR) agonists and ALDH1A1 small interfering (si)RNA. In the developing human pancreatic cells, high ALDH1 activity frequently co-localised with key stem-cell markers as well as endocrine transcription factors. A high level of ALDH1 was expressed in newly differentiated insulin(+) cells and this decreased as development progressed. Pharmacological inhibition of ALDH1 activity in human fetal islet-epithelial cell clusters resulted in reduced endocrine cell differentiation and increased cell apoptosis, and was reversed with co-treatment of RAR/RXR agonists. Furthermore, siRNA knockdown of ALDH1A1 significantly decreased RAR expression and induced cell apoptosis via suppression of the phosphoinositide 3-kinase (PI3K) pathway and activation of caspase signals. Our findings indicate that ALDH1(+) cells represent a pool of endocrine precursors in the developing human pancreas and that ALDH1 activity is required during endocrine cell differentiation. Inhibition of ALDH1-mediated retinoid signalling impairs human fetal islet cell differentiation and survival.Diabetologia 12/2013; · 6.49 Impact Factor
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ABSTRACT: Many studies in different biological systems have revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) modulates signaling pathways triggered at the plasma membrane by agents such as Wnt, transforming growth factor (TGF)-β, epidermal growth factor (EGF), and others. In addition, 1α,25(OH)2D3 may affect gene expression by paracrine mechanisms that involve the regulation of cytokine or growth factor secretion by neighboring cells. Moreover, post-transcriptional and post-translational effects of 1α,25(OH)2D3 add to or overlap with its classical modulation of gene transcription rate. Together, these findings show that vitamin D receptor (VDR) cannot be considered only as a nuclear-acting, ligand-modulated transcription factor that binds to and controls the transcription of target genes. Instead, available data support the view that much of the complex biological activity of 1α,25(OH)2D3 resides in its capacity to interact with membrane-based signaling pathways and to modulate the expression and secretion of paracrine factors. Therefore, we propose that future research in the vitamin D field should focus on the interplay between 1α,25(OH)2D3 and agents that act at the plasma membrane, and on the analysis of intercellular communication. Global analyses such as RNA-Seq, transcriptomic arrays, and genome-wide ChIP are expected to dissect the interactions at the gene and molecular levels.Frontiers in Physiology 01/2014; 5:60.