[Show abstract][Hide abstract] ABSTRACT: Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants.
In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3-4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach.
h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes.
[Show abstract][Hide abstract] ABSTRACT: Glomerular parietal epithelial cells (GPECs) are known to revert to embryonic phenotype in response to renal injury. However, the mechanism of de-differentiation in GPECs and the underlying cellular processes are not fully understood. In the present study, we show that cultured GPECs of adult murine kidney undergo epithelial-mesenchymal transition (EMT) to generate cells, which express CD24, CD44 and CD29 surface antigens. Characterization by qRT-PCR and immunostaining of these clonogenic cells demonstrate that they exhibit metastable phenotype with co-expression of both epithelial (cytokeratin-18) and mesenchymal (vimentin) markers. Transcript analysis by qRT-PCR revealed high expression of metanephric mesenchymal (Pax-2, WT-1, Six-1, Eya-1, GDNF) and uteric bud (Hoxb-7, C-Ret) genes in these cells, indicating their bipotent progenitor status. Incubation of GPECs with EMT blocker Prostaglandin E2, resulted in low expression of renal progenitor markers reflecting the correlation between EMT and acquired stemness in these cells. Additional in vitro renal commitment assays confirmed their functional staminality. When injected into E13.5 kidney rudiments, the cells incorporated into the developing kidney primordia and co-culture with E13.5 spinal cord resulted in branching and tubulogenesis in these cells. When implanted under renal capsule of unilaterally nephrectomized mice, these cells differentiated into immature glomeruli and vascular ducts. Our study demonstrates that EMT plays a major role in imparting plasticity to terminally differentiated GPECs by producing metastable cells with traits of kidney progenitors. The present study would improve our understanding on epithelial cell plasticity, furthering our knowledge of its role in renal repair and regeneration.
Full-text · Article · Oct 2009 · Journal of Cellular and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: The success of cell replacement therapy for diabetes depends on the availability and generation of an adequate number of islets, preferably from an autologous origin. Stem cells are now being probed for the generation of physiologically competent, insulin-producing cells. In this investigation, we explored the potential of adipose tissue-derived stem cells (ASCs) to differentiate into pancreatic hormone-expressing islet-like cell aggregates (ICAs). We initiated ASC culture from epididymal fat pads of Swiss albino mice to obtain mesenchymal cells, murine epididymal (mE)-ASCs. Subsequent single-cell cloning resulted in a homogeneous cell population with a CD29(+)CD44(+)Sca-1(+) surface antigen expression profile. We formulated a 10-day differentiation protocol to generate insulin-expressing ICAs from mE-ASCs by progressively changing the differentiation cocktail on day 1, day 3, and day 5. Our stage-specific approach successfully differentiated mesodermic mE-ASCs into definitive endoderm (cells expressing Sox17, Foxa2, GATA-4, and cytokeratin [CK]-19), then into pancreatic endoderm (cells expressing pancreatic and duodenal homeobox [PDX]-1, Ngn3, NeuroD, Pax4, and glucose transporter 2), and finally into cells expressing pancreatic hormones (insulin, glucagon, somatostatin). Fluorescence-activated cell sorting analysis showed that day 5 ICAs contained 64.84% +/- 7.03% PDX-1(+) cells, and in day 10 mature ICAs, 48.17% +/- 3% of cells expressed C-peptide. Day 10 ICAs released C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Electron microscopy of day 10 ICAs revealed the presence of numerous secretory granules within the cell cytoplasm. Calcium alginate-encapsulated day 10 ICAs (1,000-1,200), when transplanted i.p. into streptozotocin-induced diabetic mice, restored normoglycemia within 2 weeks. The data presented here demonstrate the feasibility of using ASCs as a source of autologous stem cells to differentiate into the pancreatic lineage.