It is well established that fibroblasts and mesenchymal stem cells (MSC) share several characteristics with subtle differences. However, no study highlighting the versatility of fibroblasts beyond their multipotentiality has been reported so far. Mouse embryonic fibroblasts (MEFs) are widely used as feeder layers to support the growth of embryonic stem cells (ESC). We hypothesized that MEF may retain ES-like features in concurrence to their developmental hierarchy in addition to their multipotent nature. Hence, we performed a comparative assessment of MEF and ESC to determine their ability to differentiate into cell types other than mesoderm as well as capacity to form teratoma using routine in vitro and in vivo techniques. MEF were derived by trypsin/ EDTA (ethylenediaminetetraacetic acid) digestion from E13.5 embryos after removing heads and viscera following plastic adherence. MEFs robustly proliferated in culture until passage 15 and formed aggregates by hanging drop method. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry revealed the presence of key MSC markers such as CD90, CD73, Sca-1, CD44, CD29, Vimentin and absence of CD45. Additionally, they expressed SSEA-1, Oct-4, Nanog, Sox-2 and ABCG2 as pluripotency markers; Nestin, b-III tubulin, Otx-2 (ectoderm); MEF-2, Mesp2, GATA-2 (mesoderm) and GATA-4, a-amylase, PDX-1 (endoderm) as tri-lineage markers. Furthermore, MEFs formed representative tissues from all three germ layers upon transplantation into Balb/c mice. These unique abilities of MEF to exhibit pluripotency, in addition to fibroblast characteristics and their ready availability with less ethical concerns and low maintenance requirements make them an attractive model for further exploration as a possible tool for regenerative medicine.
Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes.
Over the last century, diabetes has been treated with subcutaneous insulin, a discovery that enabled patients to forego death from hyperglycemia. Despite novel insulin formulations, patients with diabetes continue to suffer morbidity and mortality with unsustainable costs to the health care system. Continuous glucose monitoring, wearable insulin pumps, and closed-loop artificial pancreas systems represent an advance, but still fail to recreate physiologic euglycemia and are not universally available. Islet cell transplantation has evolved into a successful modality for treating a subset of patients with ‘brittle’ diabetes but is limited by organ donor supply and immunosuppression requirements. A novel approach involves generating autologous or immune-protected islet cells for transplant from inducible pluripotent stem cells to eliminate detrimental immune responses and organ supply limitations. In this review, we briefly discuss novel mechanisms for subcutaneous insulin delivery and define their shortfalls. We describe embryological development and physiology of islets to better understand their role in glycemic control and, finally, discuss cell-based therapies for diabetes and barriers to widespread use. In response to these barriers, we present the promise of stem cell therapy, and review the current gaps requiring solutions to enable widespread use of stem cells as a potential cure for diabetes.
Regulatory T cells (Tregs) have become highly relevant in the pathophysiology and treatment of autoimmune diseases, such as type 1 diabetes (T1D). As these cells are known to be defective in T1D, recent efforts have explored ex vivo and in vivo Treg expansion and enhancement as a means for restoring self-tolerance in this disease. Given their capacity to also modulate alloimmune responses, studies using Treg-based therapies have recently been undertaken in transplantation. Islet transplantation provides a unique opportunity to study the critical immunological crossroads between auto and alloimmunity. This procedure has advanced greatly in recent years, and reports of complete abrogation of severe hypoglycemia and long-term insulin independence have become increasingly reported. It is clear that cellular transplantation has the potential to be a true cure in T1D, provided the remaining barriers of cell supply and abrogated need for immune suppression can be overcome. However, the role that Tregs play in islet transplantation remains to be defined. Herein, we synthesize the progress and current state of Treg-based therapies in T1D and islet transplantation. We provide an extensive, but concise, background to understand the physiology and function of these cells and discuss the clinical evidence supporting potency and potential Treg-based therapies in the context of T1D and islet transplantation. Finally, we discuss some areas of opportunity and potential research avenues to guide effective future clinical application. This review provides a basic framework of knowledge for clinicians and researchers involved in the care of patients with T1D and islet transplantation.
The Nr4a subfamily of nuclear receptor comprises three members in mammalian cells: Nur77/Nr4a1, Nurr1/Nr4a2, and Nor1/Nr4a3. Nr4a proteins play key roles in the regulation of glucose homeostasis in peripheral metabolic tissues. However, their biological functions in -cells remain relatively uncharacterized. Here we sought to investigate the potential role of Nor1 in the regulation of -cell mass and, in particular, -cell survival/apoptosis. We used histological analysis to examine the consequences of genetic deletion of either Nur77 and Nor1 on -cell mass, investigated the expression patterns of Nr4as in human islets and INS cells and performed gain-and loss-offunction experiments to further characterize the role of Nor1 in -cell apoptosis. Surprisingly, Nor1 knockout mice displayed increased -cell mass, whereas mice with genetic deletion of Nur77 did not exhibit any significant differences compared with their WT littermates. The increase in -cell mass in Nor1 knockout mice was accompanied by improved glucose toler- ance. A gene expression study performed in both human islets and INS cells revealed that Nor1 expression is significantly increased by pro-inflammatory cytokines and, to a lesser extent, by elevated concentrations of glucose. Nor1 overexpression in both INS and human islet cells caused apoptosis, whereas siRNA-mediated Nor1 knockdown prevented cytokine-induced-cell death. Finally, Nor1 expression was up-regulated in islets of individuals with type 2 diabetes. Altogether, our results uncover that Nor1 negatively regulates -cell mass. Nor1 represents a promising molecular target in diabetes treatment to prevent -cell destruction.Type 2 diabetes is characterized by progressive deterioration of both -cell mass and function, resulting in a relative deficit in insulin secretion (1, 2). Autopsy studies have indeed reported significant reductions in -cell mass in patients with type 2
Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes.
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