Papers by Anandwardhan Hardikar
Non-Coding RNA, Dec 13, 2018
In this review, we provide an overview of the current knowledge on the role of different classes ... more In this review, we provide an overview of the current knowledge on the role of different classes of non-coding RNAs for islet and β-cell development, maturation and function. MicroRNAs (miRNAs), a prominent class of small RNAs, have been investigated for more than two decades and patterns of the roles of different miRNAs in pancreatic fetal development, islet and β-cell maturation and function are now emerging. Specific miRNAs are dynamically regulated throughout the period of pancreas development, during islet and β-cell differentiation as well as in the perinatal period, where a burst of β-cell replication takes place. The role of long non-coding RNAs (lncRNA) in islet and β-cells is less investigated than for miRNAs, but knowledge is increasing rapidly. The advent of ultra-deep RNA sequencing has enabled the identification of highly islet-or β-cell-selective lncRNA transcripts expressed at low levels. Their roles in islet cells are currently only characterized for a few of these lncRNAs, and these are often associated with β-cell super-enhancers and regulate neighboring gene activity. Moreover, ncRNAs present in imprinted regions are involved in pancreas development and β-cell function. Altogether, these observations support significant and important actions of ncRNAs in β-cell development and function.
Baghdad Science Journal
Background: Adipose derived-mesenchymal stem cells have been used as an alternative to bone marro... more Background: Adipose derived-mesenchymal stem cells have been used as an alternative to bone marrow cells in this study. Objective: We investigated the in vitro isolation, identification, and differentiation of stem cells into neuron cells, in order to produce neuron cells via cell culture, which would be useful in nerve injury treatment. Method: Mouse adipose mesenchymal stem cells were dissected from the abdominal subcutaneous region. Neural differentiation was induced using β-mercaptoethanol. This study included two different neural stage markers, i.e. nestin and neurofilament light-chain, to detect immature and mature neurons, respectively. Results: The immunocytochemistry results showed that the use of β-mercaptoethanol resulted in the successful production of neuron cells. This was attributable to the increase and significant overexpression of the nestin protein during the early exposure period, which resulted in the expression of the highest levels of nestin. In comparison, th...
Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1
Background Islet cell death is a common feature of type 1 diabetes (T1D), including after islet c... more Background Islet cell death is a common feature of type 1 diabetes (T1D), including after islet cell transplantation, as well as of type 2 diabetes (T2D). As of today, we lack tools to quantitatively detect islet cell loss prior to the clinical onset of diabetes, or to predict the progression of established diabetes. Our preliminary data demonstrates that a signature of 20 different non-coding (nc) RNAs (including microRNAs) reflects beta cell death (RAPID: RNA-based Analysis for Prediction of Islet Death signature). Objectives 1)To identify a high-throughput platform for detection of ncRNAs/microRNAs 2)To validate the RAPID signature of 20 ncRNAs using this high-throughput platform 3)To test the suitability of these ncRNA biomarkers in predicting the progression to T1D Method Next Generation Sequencing (NGS) studies on developing human pancreas have led to the identification of specific ncRNAs that are found to be expressed specifically in the human pancreatic islets. We use combined FISH and immunostaining to confirm the localization of each miRNA in human islets. TaqMan-based real-time PCR on plasma from at-risk diabetic individuals and age/gender matched controls in a longitudinal study, is used to measure the abundance of specific ncRNAs in plasma-EDTA samples of individuals at risk of T1D. I will discuss the comparison of 2 different ultra-high-throughput TaqMan real-time PCR platforms that we use to analyze these samples and present the data demonstrating the suitability of these RNA-based biomarkers in predicting the progression to T1D. Results Present study has allowed us to validate a microRNA-based signature of islet cell death in diabetes. Using a cohort of kids at risk of T1D, we demonstrate the suitability and advantages of these ncRNA biomarkers over traditional / conventional antibody- and glucose-based detection. The development of an assay for early detection of islet injury and death has direct applications in clinical medicine. Hopefully, this study results will inform medical researchers as to how to predict the development of Type 1 diabetes, monitor response to interventions such as islet transplantation, vaccines and drugs that aim to retard beta cell loss or promote beta cell regeneration.
Journal of Diabetes Research
Background. Women with previous gestational diabetes mellitus (GDM) have evidence of postpartum β... more Background. Women with previous gestational diabetes mellitus (GDM) have evidence of postpartum β-cell dysfunction, which increases their risk of developing type 2 diabetes (T2DM) later in life. Elevated levels of circulating cell-free preproinsulin (INS) DNA correlate with dying β-cells in both mice and humans. The aim of this study was to determine if cell-free circulating INS DNA levels are higher in women with previous GDM who develop T2DM. Methods. We used droplet digital (dd) PCR to measure the levels of cell-free circulating methylated and unmethylated INS DNA in plasma from 97 women with normal glucose tolerance (NGT), 12 weeks following an index GDM pregnancy. Women were assessed for up to 10 years for the development of T2DM. Results. In the follow-up period, 22% of women developed T2DM. Compared with NGT women, total cell-free INS DNA levels were significantly higher in women who developed T2DM (P=0.02). There was no difference in cell-free circulating unmethylated and me...
Cell Transplantation
Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. ... more Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. T1D can be treated using cadaveric islet transplantation, but this therapy is severely limited by a lack of pancreas donors. To develop an alternative cell source for transplantation therapy, we carried out the epigenetic characterization in nine different adult mouse tissues and identified visceral adipose-derived progenitors as a candidate cell population. Chromatin conformation, assessed using chromatin immunoprecipitation (ChIP) sequencing and validated by ChIP-polymerase chain reaction (PCR) at key endocrine pancreatic gene promoters, revealed similarities between visceral fat and endocrine pancreas. Multiple techniques involving quantitative PCR, in-situ PCR, confocal microscopy, and flow cytometry confirmed the presence of measurable (2–1000-fold over detectable limits) pancreatic gene transcripts and mesenchymal progenitor cell markers (CD73, CD90 and CD105; >98%) in visceral ...
Islets, Sep 3, 2017
Pancreatic β-cells are connected to neighboring endocrine cells through the adherin proteins and ... more Pancreatic β-cells are connected to neighboring endocrine cells through the adherin proteins and gap junctions. Connexin 36 (Cx36) is one of the most well-studied and abundantly expressed gap-junction proteins within rodent islets, which is important in coordinated insulin secretion. The expression of connexins is regulated at various levels and by several mechanisms; one of which is via microRNAs. In past 2 decades, microRNAs (miRNAs) have emerged as key molecules in developmental, physiologic and pathological processes. However, very few studies have demonstrated miRNA-mediated regulation of connexins. Even though there are no reports yet on miRNAs and Cx36; we envisage that considering the important role of connexins and microRNAs in insulin secretion, there would be common pathways interlinking these biomolecules. Here, we discuss the current literature on connexins and miRNAs specifically with reference to islet function.
J Endocrinol, 2009
The treatment of diabetes by islet transplantation is presently hampered by the shortage of organ... more The treatment of diabetes by islet transplantation is presently hampered by the shortage of organ donors. The generation of insulin-producing cells is therefore a major objective in the long-term goal of curing diabetes. Alternative sources of pancreatic β-cells include existing pancreatic cells, embryonic stem cells, and cells from other tissues such as liver. This commentary considers evidence for two new sources of β-cells: intrahepatic biliary epithelial cells and gall bladder epithelium. These observations raise the possibility that a patient's own cells may be used as a source of insulin-producing cells for cell replacement in diabetes.
Cell Transplantation
ABSTRACT
Principles of Developmental Genetics, 2015
Abstract The pancreas is composed of two main cell types: exocrine cells and endocrine cells, bot... more Abstract The pancreas is composed of two main cell types: exocrine cells and endocrine cells, both of which are derived from the endodermal germ layer. It is currently unclear if there exists a multipotent progenitor cell within the embryonic pancreas that is capable of giving rise to both exocrine and endocrine cell lineages. Progenitor cells for both the exocrine and endocrine cells reside within the embryonic pancreatic ductal epithelium. Many transcription factors have been identified that are critical for pancreas formation and the differentiation of the endocrine cell populations. However, less is known about the genes that control exocrine cell fate and differentiation. The β-cell mass has the capacity to expand and contract throughout the life of the organism in response to changing metabolic demands. In adults, the majority of new insulin-producing β-cells come from the proliferation of pre-existing β-cells, although, under some conditions (e.g., pancreatic injury, obesity), β-cell neogenesis may occur from cells that are located within the ductal epithelium. There are currently no good protocols for generating mature, glucose-responsive insulin-producing β-cells in vitro from embryonic stem (ES) or iPS cells. Pancreatic progenitor cells generated in vitro from ES or iPS cells can differentiate into mature, glucose-responsice β-cells when transplanted in vivo . Thus, as yet unidentified in vivo factors promote terminal differentiation of β-cells from stem cell sources.
Cell transplantation
The success of immunoisolation devices for islet transplantation depends on the nature of semiper... more The success of immunoisolation devices for islet transplantation depends on the nature of semipermeable membranes, which permit the crossover of micronutrients, glucose, and insulin and prevent the entry of immunocytes and other transplant rejection mechanisms. In the present study we examined the properties of chitosan-polyvinyl pyrrolidone (PVP) hydrogels for possible application as an immunoisolation device. Hydrogels with two different proportions of chitosan-PVP (M1 1:1 and M2 2:1, v/v) were synthesized by cross-linking with glutaraldehyde. Hydrogels were characterized for their hydrophilic nature, protein adsorption, diffusion properties, cytotoxicity, and islet compatibility. Hydrogel membranes were found to be hydrophilic as determined by high octane contact angle value (M1: 142.9 +/- 0.46; M2: 143.6 +/- 0.49). Protein adsorption on the hydrogels was found to be low (0.0143 +/- 0.0027 mg for M1 and 0.0136 +/- 0.0049 mg for M2) compared to tissue culture polystyrene (TCPS) (0...
Stem Cells in Clinic and Research, 2011
PLoS ONE, 2010
Insulin-producing pancreatic islet b cells (b-cells) are destroyed, severely depleted or function... more Insulin-producing pancreatic islet b cells (b-cells) are destroyed, severely depleted or functionally impaired in diabetes. Therefore, replacing functional b-cell mass would advance clinical diabetes management. We have previously demonstrated the importance of Cdk4 in regulating b-cell mass. Cdk4-deficient mice display b-cell hypoplasia and develop diabetes, whereas b-cell hyperplasia is observed in mice expressing an active Cdk4R24C kinase. While b-cell replication appears to be the primary mechanism responsible for b-cell mass increase, considerable evidence also supports a contribution from the pancreatic ductal epithelium in generation of new b-cells. Further, while it is believed that majority of b-cells are in a state of 'dormancy', it is unclear if and to what extent the quiescent cells can be coaxed to participate in the b-cell regenerative response. Here, we address these queries using a model of partial pancreatectomy (PX) in Cdk4 mutant mice. To investigate the kinetics of the regeneration process precisely, we performed DNA analog-based lineage-tracing studies followed by mathematical modeling. Within a week after PX, we observed considerable proliferation of islet b-cells and ductal epithelial cells. Interestingly, the mathematical model showed that recruitment of quiescent cells into the active cell cycle promotes bcell mass reconstitution in the Cdk4R24C pancreas. Moreover, within 24-48 hours post-PX, ductal epithelial cells expressing the transcription factor Pdx-1 dramatically increased. We also detected insulin-positive cells in the ductal epithelium along with a significant increase of islet-like cell clusters in the Cdk4R24C pancreas. We conclude that Cdk4 not only promotes bcell replication, but also facilitates the activation of b-cell progenitors in the ductal epithelium. In addition, we show that Cdk4 controls b-cell mass by recruiting quiescent cells to enter the cell cycle. Comparing the contribution of cell proliferation and islet-like clusters to the total increase in insulin-positive cells suggests a hitherto uncharacterized large non-proliferative contribution.
Laboratory Investigation, 2010
Recent evidence has shown that stem cell factor (SCF) and its receptor, c-Kit, have an important ... more Recent evidence has shown that stem cell factor (SCF) and its receptor, c-Kit, have an important role in pancreatic islet development by promoting islet cell differentiation and proliferation. In this study, we examined the role of c-Kit and SCF in the differentiation and proliferation of insulin-and glucagon-producing cells using a human pancreatic duct cell line (PANC-1). Our study showed that increased expression of endocrine cell markers (such as insulin and glucagon) and transcription factors (such as PDX-1 and PAX-6) coincided with a decrease in CK19 þ and c-Kit þ cells (Po0.001) during PANC-1 cell differentiation, determined by immunofluorescence and qRT-PCR. Cells cultured with exogenous SCF showed an increase in insulin þ (26%) and glucagon þ (35%) cell differentiation (Po0.01), an increase in cell proliferation (Po0.05) and a decrease in cell apoptosis (Po0.01). siRNA knockdown of c-Kit resulted in a decrease in endocrine cell differentiation with a reduction in PDX-1 and insulin mRNA, as well as the number of cells immunostaining for PDX-1 and insulin. Taken together, these results show that c-Kit/SCF interactions are involved in mediating islet-like cluster formation and islet-like cell differentiation in a human pancreatic duct cell line.
Journal of Endocrinology, 2009
There have been considerable efforts towards understanding the potential of human pancreatic endo... more 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 acti...
Journal of Biosciences, 1999
ABSTRACT
Heart, Lung and Circulation, 2010
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Papers by Anandwardhan Hardikar