Anandwardhan A. Hardikar

pH-sensitive freeze-dried chitosan-polyvinyl pyrrolidone hydrogels as controlled release system for antibiotic delivery.

The aim of this study was to develop a pH-sensitive chitosan/polyvinyl pyrrolidone (PVP) based controlled drug release system for antibiotic delivery. The hydrogels were synthesised by crosslinking chitosan and PVP blend with glutaraldehyde to form a semi-interpenetrating polymer network (semi-IPN). The semi-IPN formation was confirmed by Fourier transform infrared spectroscopic (FTIR) analysis. Semi-IPNs, viz, air-dried and freeze-dried, were compared for their surface morphology, wettability, swelling properties and pH-dependent swelling. Air- and freeze-dried membranes were also incorporated with amoxicillin and antibiotic release was studied. Porous freeze-dried hydrogels (pore diameter, 39.20+/-2.66 microm) exhibited superior pH-dependent swelling properties over non-porous air-dried hydrogels. A high octane contact angle (144.20+/-0.580) of hydrogel was indicative of its hydrophilic nature. Increased swelling of hydrogels, under acidic conditions, was due to the protonation of a primary amino group on chitosan, as confirmed by FTIR analysis. Freeze-dried membranes released around 73% of the amoxicillin (33% by air-dried) in 3 h at pH 1.0 and, thus, had superior drug-release properties to air-dried hydrogels. Freeze-dried membranes could serve as potent candidates for antibiotic delivery in an acidic environment.

Expression of islet-specific microRNAs during human pancreatic development

During pancreatic islet development, sequential changes in gene expression are known to be necessary for efficient differentiation and function of the endocrine pancreas. Several studies till now have demonstrated that microRNAs (miRNAs), which regulate translation of gene transcripts, influence gene expression cascades involved in pancreas development. Some of these miRNAs; miR-7 and miR-375 have been known to be expressed at high levels in pancreas and are also known to be involved in Zebrafish pancreas development as well as insulin secretion in mice. We demonstrate here that 4 different islet-specific microRNAs (miR-7, miR-9, miR-375 and miR-376) are expressed at high levels during human pancreatic islet development. Of these, miR-375, is seen to be differentially expressed in human islet beta- as well as non-beta-cells. Though no significant difference in abundance of miR-375 was noted in either cell type, analysis of islet-specific miRNA and mRNA in single cells show that non-beta cells contain higher levels of miR-375. Our data demonstrate that miRNAs that are known to be regulated during Zebrafish pancreatic development may play similar role in human pancreatic islet development.

Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates

Development of the endocrine pancreas includes a series of early events wherein precursor cells cluster, that is migrate to form cell aggregates, which subsequently differentiate into islets of Langerhans. We show that PANC-1 cells, a human pancreatic cell line, differentiates into hormone-producing islet-like cell aggregates after exposure to a defined serum-free medium. These cells were used to provide the following evidence that fibroblast growth factor (FGF)2 is a paracrine chemoattractant during PANC-1 cell clustering: (i) FGF2 is secreted and remains bound to the extracellular matrix from where it may diffuse to form chemoattractive gradients; (ii) a subset of cells expresses FGF receptors (FGFRs) -1, -2, -3, and -4; (iii) inhibition of FGFR tyrosine kinase inhibits cell clustering; and (iv) FGF2 neutralizing antibody inhibits clustering. In addition, adult human islet-derived precursor cells, which cluster and differentiate in a manner similar to PANC-1 cells, also secrete FGF2 and express FGFRs. We conclude that FGF2, acting as a paracrine chemoattractant, stimulates clustering of precursor cells, an early step leading to islet-like cell aggregate formation. Similar processes may occur during development of the islet of Langerhans in humans.

The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells

Epithelial-to-mesenchymal transition is a phenomenon necessary for embryonic development and also seen during certain pathological conditions. We show here for the first time that reduction in miR-30 family microRNAs, is responsible for mesenchymal transition of primary cultures of human pancreatic epithelial cells. We found that miR-30 family microRNAs target mesenchymal gene transcripts and maintain them in a translationally inactive state. Forced depletion using miR-30 family specific anti-miRs leads to mesenchymal transition while ectopic overexpression maintains the epithelial phenotype. We also show that miR-30 family microRNAs increase in abundance during differentiation of pancreatic islet-derived mesenchymal cells into hormone-producing islet-like cell aggregates. Our studies in human adult diseased pancreas also demonstrate that miR-30 family microRNAs are expressed at lower abundance in fibrotic lesions during pancreatitis. Together, our data confirm that miR-30 family microRNAs form a part of the regulatory signaling events involved in cellular response of pancreatic epithelial cells during mesenchymal transition.

Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo

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.

Growth modulation of fibroblasts by chitosan-polyvinyl pyrrolidone hydrogel: Implications for wound management?

Wounds in adults and fetuses differ in their healing ability with respect to scar formation. In adults, wounds lacking the epidermis exhibit excess collagen production and scar formation. Fibroblasts synthesize and deposit a collagen rich extracellular matrix. The early migration and proliferation of fibroblasts in the wound area is implicated in wound scarring. We have synthesized a hydrogel from chitosan-polyvinyl pyrrolidone (PVP) and examined its effect on fibroblast growth modulationin vitro. The hydrogel was found to be hydrophilic as seen from its octane contact angle (141.2 ± 0.37ℴ). The hydrogel was non-toxic and biocompatible with fibroblasts and epithelial cells as confirmed by the 3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. It showed dual properties by supporting growth of epithelial cells (SiHa) and selectively inhibiting fibroblast (NIH3T3) growth. Growth inhibition of fibroblasts resulted from their inability to attach on to the hydrogel. These findings are supported by image analysis, which revealed a significant difference (P < 0.05) between the number of fibroblasts attached to the hydrogel in tissue culture as compared to tissue culture treated polystyrene (TCPS) controls. However, no significant difference was observed (P > 0.05) in the number of epithelial (SiHa) cells attached on to the hydrogel as compared to the TCPS control. Althoughin vivo experiments are awaited, these findings point to the possible use of chitosan PVP hydrogels in wound-management.

Differential placental methylation and expression of VEGF, FLT-1 and KDR genes in human term and preterm preeclampsia

BackgroundPreeclampsia, a pregnancy complication of placental origin is associated with altered expression of angiogenic factors and their receptors. Recently, there is considerable interest in understanding the role of adverse intrauterine conditions in placental dysfunction and adverse pregnancy outcomes. Since we have observed changes in placental global DNA methylation levels in preeclampsia, this study was undertaken to examine gene promoter CpG methylation and expression of several angiogenic genes.We recruited 139 women comprising, 46 normotensive women with term delivery (≥37 weeks), 45 women with preeclampsia delivering preterm (<37 weeks) and 48 women with preeclampsia delivering at term. Expression levels and promoter CpG methylation of VEGF, FLT-1 and KDR genes in placentae from respective groups were determined by Taqman-based quantitative real time PCR and by the Sequenom® EpiTYPER™ technology respectively.ResultsWe observed several differentially methylated CpG sites in the promoter regions of VEGF, FLT-1 and KDR between the normotensive and preeclampsia groups. We specifically observed hypomethylated CpGs in the promoter region and an increased expression of VEGF gene between term and preterm preeclampsia. However, mean promoter CpG methylation could not account for the higher expression of FLT-1 and KDR in preterm preeclampsia as compared to normotensive group.ConclusionsOur data indicates altered DNA methylation patterns in the VEGF, FLT-1 and KDR genes in preeclampsia as compared to the normotensive group, which could be involved in the pathophysiology of preeclampsia. Hypomethylation of VEGF promoter and consequent upregulation of VEGF mRNA levels could be a compensatory mechanism to restore normal angiogenesis and blood flow in preterm preeclampsia. This study suggests a role of altered DNA methylation in placental angiogenesis and in determining adverse pregnancy outcomes.

Multigenerational Undernutrition Increases Susceptibility to Obesity and Diabetes that Is Not Reversed after Dietary Recuperation

People in developing countries have faced multigenerational undernutrition and are currently undergoing major lifestyle changes, contributing to an epidemic of metabolic diseases, though the underlying mechanisms remain unclear. Using a Wistar rat model of undernutrition over 50 generations, we show that Undernourished rats exhibit low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age-/gender-matched control rats. Undernourished rats also have higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an 8-fold increased susceptibility to Streptozotocin-induced diabetes compared to control rats. Importantly, these metabolic abnormalities are not reversed after two generations of unrestricted access to commercial chow (nutrient recuperation). Altered epigenetic signatures in insulin-2 gene promoter region of Undernourished rats are not reversed by nutrient recuperation, and may contribute to the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations.

Human fetal pancreatic insulin-producing cells proliferate in vitro

There have been considerable efforts towards understanding the potential of human pancreatic endocrine cells to proliferate and transition into mesenchymal cell populations. Since rodent studies have demonstrated that mouse insulin-producing cells do not proliferate in vitro, a similar possibility has been considered for human islet endocrine cells. Considering the inherent differences in mouse and human pancreatic islets, we decided to assess the potential of human fetal pancreatic insulin-producing cells to proliferate in vitro. We studied the proliferative potential of human fetal pancreatic islet-derived populations from second or third trimester fetal pancreas and characterized the cells that grow out during their expansion. We have used seven different approaches including in situ hybridization and immunostaining, quantitative estimation of multiple gene transcripts in populations as well as in single cells, clonal analysis of islet cells, assessment of heritable marks of active insulin promoter, and thymidine analog-based lineage tracing. Our studies demonstrate that human fetal pancreatic insulin-producing cells proliferate in vitro to generate mesenchymal cell populations. Interestingly, epigenetic modifications that mark open chromatin conformation of insulin promoter regions are retained even after a million fold expansion/proliferation in vitro. These findings demonstrate that hormone-producing cells in pancreatic islets proliferate in vitro and retain epigenetic marks that characterize an active insulin promoter. Such in vitro-derived mesenchymal cells may be of potential use in cell-replacement therapy for diabetes.

HUMAN UMBILICAL CORD BLOOD SERUM PROMOTES GROWTH, PROLIFERATION, AS WELL AS DIFFERENTIATION OF HUMAN BONE MARROW–DERIVED PROGENITOR CELLS

SummaryFelal calf serum (FCS) is conventionally used for animal cell cultures due to its inherent growth-promoting activities. However animal welfare issues and stringent requirements for human transplantation studies demand a suitable alternative for FCS. With this view, we studied the effect of FCS, human AB serum (ABS), and human umbilical cord blood serum (UCBS) on murine islets of Langerhans and human bone marrow-derived mesenchymal-like cells (hBMCs). We found that there was no difference in morphology and functionality of mouse islets cultured in any of these three different serum supplements as indicated by insulin immunostaining. A comparative analysis of hBMCs maintained in each of these three different serum supplements demonstrated that UCBS supplemented media better supported proliferation of hBMCs. Moreover, a modification of adipogenic differentiation protocol using UCBS indicates that it can be used as a supplement to support differentiation of hBMCs into adipocytes. Our results demonstrate that UCBS not only is suitable for maintenance of murine pancreatic islets, but also supports attachment, propagation, and differentiation of hBMCs in vitro. We conclude that UCBS can serve as a better serum supplement for growth, maintenance, and differentiation of hBMCs, making it a more suitable supplement in cell systems that have therapeutic potential in human transplantation programs.