Limbal tissues can be cultured on various types of scaffolds to create a sheet of limbal-corneal epithelium for research as well as clinical transplantation. An optically clear, biocompatible, biomimetic scaffold would be an ideal replacement graft for transplanting limbal stem cells. In this study, we evaluated the physical and culture characteristics of the recombinant human cross-linked collagen scaffold (RHC-III scaffold) and compared it with denuded human amniotic membrane (HAM). Optical/mechanical properties and microbial susceptibility were measured for the scaffolds. With the approval of the institutional review board, 2 mm fresh human limbal tissues were cultured on 2.5 x 2.5 cm(2) scaffolds in a medium containing autologous serum in a feeder cell-free submerged system. The cultured cell systems were characterized by morphology and immunohistochemistry for putative stem cells and differentiated cell markers. The refractive index (RI) and tensile strength of the RHC-III scaffold were comparable to human cornea, with delayed in vitro degradation compared to HAM. RHC-III scaffolds were 10-fold less susceptible to microbial growth. Cultures were initiated on day 1, expanded to form a monolayer by day 3 and covered the entire growth surface in 10 days. Stratified epithelium on the scaffolds was visualized by transmission electron microscopy. The cultured cells showed p63 and ABCG2 positivity in the basal layer and were immunoreactive for cytokeratin K3 and K12 in the suprabasal layers. RHC-III scaffold supports and retains the growth and stemness of limbal stem cells, in addition to resembling human cornea; thus, it could be a good replacement scaffold for growing cells for clinical transplantation.

BackgroundDevelopment of model systems have helped to a large extent, in bridging gap to understand the mechanism(s) of disease including diabetes. Interestingly, WNIN/GR-Ob rats (Mutants), established at National Centre for Laboratory Animals (NCLAS) of National Institute of Nutrition (NIN), form a suitable model system to study obesity with Type 2 diabetes (T2D) demonstrating several secondary complications (cataract, cardiovascular complications, infertility, nephropathy etc). The present study has been carried out to explore the potent application(s) of multipotent stem cells such as bone marrow mesenchymal stem cells (BM-MSCs), to portray features of pre-diabetic/T2D vis-à-vis featuring obesity, with impaired glucose tolerance (IGT), hyperinsulinemia (HI) and insulin resistance (IR) seen with Mutant rats akin to human situation.Methodology/Principal FindingsPrimary cultures of BM-MSCs (third passage) from Mutants, its lean littermate (Lean) and parental control (Control) were characterized for: proliferation markers, disease memory to mark obesity/T2D/HI/IR which included phased gene expression studies for adipogenic/pancreatic lineages, inflammatory markers and differentiation ability to form mature adipocytes/Insulin-like cellular aggregates (ILCAs). The data showed that BM-MSCs from Mutant demonstrated a state of disease memory, depicted by an upregulated expression of inflammatory markers (IL-6, TNFα); increased stem cell recruitment (Oct-4, Sox-2) and proliferation rates (CD90+/CD29+, PDA, ‘S’ phase of cell cycle by FACS and BrdU incorporation); accelerated preadipocyte induction (Dact-1, PPARγ2) with a quantitative increase in mature adipocyte formation (Leptin); ILCAs, which were non-responsive to high glucose did confer the Obese/T2D memory in Mutants. Further, these observations were in compliance with the anthropometric data.ConclusionsGiven the ease of accessibility and availability of MSCs, the present study form the basis to report for the first time, application of BM-MSCs as a feasible in vitro model system to portray the disease memory of pre-clinical/T2D with IR - a major metabolic disorder of global concern.

Adipose tissue development is a highly regulated phenomenon orchestrated by several check points (recruitment of mesenchymal stem cells and their lineage commitment) to form mature adipocytes. Once committed to obesity, expansion of adipose tissue occurs either by hypertrophy or hyperplasia or by both resulting in an altered physiological status. This precipitates as inflammatory responses, leading to endoplasmic reticulum and oxidative stress altering the gene expression of adipose tissue in a depot-specific manner. However, such studies reporting a phased gene expression profile in conditions of rodent obesity are not reported so far. WNIN/Ob mutant obese rat, developed at our institute is an excellent model to study the pathophysiological changes underlying obesity. Here, we report the gene expression profile of this mutant rat (obese and lean), compared with the parental control, with reference to markers of embryonic stem cells, adipogenesis, inflammation, and senescence in both subcutaneous (SCAT) and retroperitoneal (RPAT) adipose depots representing abdominal fat. We demonstrate an upregulation of genes such as Sox-2, Pref-1, PPARγ2, LPL, IRS-1, GLUT-4, IL-6, TNFα, and telomerase in SCAT and RPAT depots of the obese rat compared to its lean counterpart indicating no difference in fat depots at different locations. This is suggestive of a similar phenotypic expression of mutant gene. Data form the phased gene expression changes of adipogenesis (embryonic/adipogenic/inflammatory) in the present obese rat model system advocate for inflammatory mediated response(s) associated with obesity-a condition often seen in humans.

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