Browsing by Author "Bhonde, RR"

Now showing 1 - 6 of 6
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    Generation of Pancreatic Hormone-Expressing Islet-Like Cell Aggregates from Murine Adipose Tissue-Derived Stem Cells
    (STEM CELLS, 2009) Chandra, V; Swetha, G; Phadnis, S; Nair, PD; Bhonde, RR
    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(+)Sca1(+) 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 stagespecific 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. STEM CELLS 2009; 27: 1941-1953
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    Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo
    (CYTOTHERAPY, 2011) Phadnis, SM; Joglekar, MV; Dalvi, MP; Muthyala, S; Nair, PD; Ghaskadbi, SM; Bhonde, RR; Hardikar, AA
    Background aims. The scarcity of human islets for transplantation remains a major limitation of cell replacement therapy for diabetes. Bone marrow-derived progenitor cells are of interest because they can be isolated, expanded and offered for such therapy under autologous/allogeneic settings. Methods. We characterized and compared human bone marrow-derived mesenchymal cells (hBMC) obtained from (second trimester), young (1--24 years) and adult (34--81 years) donors. We propose a novel protocol that involves assessment of paracrine factors from regenerating pancreas in differentiation and maturation of hBMC into endocrine pancreatic lineage in vivo. Results. We observed that donor age was inversely related to growth potential of hBMC. Following in vitro expansion and exposure to specific growth factors involved in pancreatic development, hBMC migrated and formed islet-like cell aggregates (ICA). ICA show increased abundance of pancreatic transcription factors (Ngn3, Brn4, Nkx6.1, Pax6 and Isl1). Although efficient differentiation was not achieved in vitro, we observed significant maturation and secretion of human c-peptide (insulin) upon transplantation into pancreactomized and Streptozotocin (STZ)-induced diabetic mice. Transplanted ICA responded to glucose and maintained normoglycemia in diabetic mice. Conclusions. Our data demonstrate that hBMC have tremendous in vitro expansion potential and can be differentiated into multiple lineages, including the endocrine pancreatic lineage. Paracrine factors secreted from regenerating pancreas help in efficient differentiation and maturation of hBMC, possibly via recruiting chromatin modulators, to generate glucose-responsive insulin-secreting cells.
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    Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice
    (PLOS ONE, 2011) Chandra, V; Swetha, G; Muthyala, S; Jaiswal, AK; Bellare, JR; Nair, PD; Bhonde, RR
    Background: 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. Methodology/Principal Findings: 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 (Hnf3 beta, 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. Conclusions: 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.
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    Nonporous polyurethane membranes as islet immunoisolation matrices - Biocompatibility studies
    (JOURNAL OF BIOMATERIALS APPLICATIONS, 2002)
    Novel elastomeric nonporous polyurethane membranes were synthesised with differing hard segment contents for evaluation as possible islet encapsulation matrices. Physico-chemical properties of these membranes were reported earlier by authors and have been found suitable for immunoisolation. In the present study, membranes were evaluated for their in vitro biocompatibility. Membranes T1, T4, T5 and T6 did not show toxicity in direct cell contact study towards L929 fibroblasts. However, T2 and T3 were found cytotoxic and were excluded from further testing. NIH3T3 cells when exposed to leach out products of T4, T5 and T6 showed no cytotoxicity, while T1 decreased cellular viability as confirmed by MTT assay. T4 and T5 alone were seen to be compatible with mouse islets while T6 was incompatible to the mouse islets. Digital image analysis (DIA) studies showed intact morphology of islets cultured on the T4 and T5 with viability (88.4 and 91% respectively) comparable to islets on tissue culture polystyrene (TOPS) control. Islets on T4 and T5 also retained their functionality, as judged by insulin secretion in response to in vitro glucose challenge (16.0 mM). These studies point out the crucial role of surface free energy and hydrophilicity in deciding compatibility of polyurethane membranes with islets of Langerhans. Studies indicate that polyurethane membranes T4 and T5 could be potential candidates for islet immunoisolation.
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    Reversal of experimental diabetes in mice by transplantation of neo-islets generated from human amnion-derived mesenchymal stromal cells using immuno-isolatory macrocapsules
    (CYTOTHERAPY, 2010) Kadam, SS; Sudhakar, M; Nair, PD; Bhonde, RR
    Background aims. The ethical and biologic limitations with current sources of stem cells have resulted in a quest to look for alternative sources of multipotent stem cells of human origin. Amniotic membrane is of interest as a source of cells for regenerative medicine because of its ease of availability, plasticity and inexhaustible source that does not violate the sanctity of independent life. Although researchers have shown the stem cell-like potential of human amniotic epithelial cells, the mesenchymal part of amnion has remained less explored. Methods. We established a long-term culture of mesenchymal-like stem cells derived from full-term human amniotic membrane and their differentiation into functional pancreatic lineage. Results. The amnion-derived mesenchymal-like stem cells expressed various mesenchymal markers and demonstrated multilineage differentiation capacity. We also observed that these cells could form islet-like clusters (ILC) on exposure to serum-free defined media containing specific growth factor and differentiating agents. Differentiated ILC showed expression of human insulin, glucagon and somatostatin by immunocytochemistry, while quantitative reverse transcription/real-time-polymerase chain reaction (qRT-PCR) data demonstrated the expression of insulin, glucagon, somatostatin, Ngn3 and Isl1. Moreover, encapsulation of the ILC in polyurethane-polyvinyl pyrrolidone macrocapsules and their subsequent transplantation in experimental diabetic mice resulted in restoration of normoglycemia, indicating their ability to respond to high glucose without immunorejection. Conclusions. Our results demonstrate that amnion-derived mesenchymal stromal cells can undergo islet neogenesis, indicating amnion as an alternative source of islets for cell replacement therapy in diabetes.
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    Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice
    (CYTOTHERAPY, 2013) Kanafi, MM; Rajeshwari, YB; Gupta, S; Dadheech, N; Nair, PD; Gupta, PK; Bhonde, RR
    Background aims. The success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol. Methods. We compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets. Results. SHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3-4 weeks, which persisted for >60 days. Conclusions. Our results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.