Bhawna Chandravanshi

Shielding Engineered Islets with Mesenchymal Stem Cells Enhance Survival Under Hypoxia.

In the present study we focused on the improvisation of islet survival in hypoxia.The Islet like cell aggregates (ICAs) derived from whartons jelly mesenchymal stem cells (WJ MSC) were cultured with and without WJ MSC for 48 h in hypoxia and normoxia and tested for their direct trophic effect on β cell survival. The WJ MSCs themselves secreted insulin upon glucose challenge and expressed the pancreatic markers at both transcription and translational level (C‐peptide, Insulin, Glucagon, and Glut 2). Direct contact of MSCs with ICAs facilitated highest viability under hypoxia as evidenced by fluorescein diacetate/propidium iodide and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The cytokine analysis of the co‐cultured ICAs revealed amplification of anti‐inflammatory cytokine like TGFβ and TNFα accompanied by depletion of pro‐inflammatory cytokines. The increment in VEGF and PDGFa was also seen showing their ability to vascularize upon transplantation. This was further accompanied by reduction in total reactive oxygen species, nitric oxide, and super oxide ions and down regulation of Caspase3, Caspase8, p53, and up regulation of Bcl2 confirming prevention of apoptosis in ICAs. The western blot analysis confirmed the cytoprotective effect of WJ MSC on ICAs as they enhanced the anti‐apoptotic marker BCL2 and reduced the expression of apoptotic markers, Annexin 5 and Caspase 3. There was a significant reduction in the expression of p38 protein in the presence of MSCs making the ICAs responsive to glucose. Taken together our data demonstrate for the first time that the WJ MSC expressed pancreatic markers and their supplementation protected engineered islets against hypoxia and oxidative stress. J. Cell. Biochem. 118: 2672–2683, 2017.

Hypoxia primed placental mesenchymal stem cells for wound healing

Aims: To investigate how Placental Mesenchymal Stem Cells (P‐MSCs) would adapt themselves and survive under hypoxic conditions which are prevalent in most injury sites. Main methods: P‐MSCs were isolated from term placenta and characterised under normoxia and hypoxia (2–2.5% O2). Cells were examined for morphology and surface marker variations by flow cytometry analysis. Glucose stimulated insulin secretion was assayed by Insulin ELISA Kit. Gene expression levels were estimated using Real Time PCR for hypoxia inducible factor1 alpha, Insulin (INS), Glucose transporters (GLUT‐1, GLUT‐2 and GLUT‐3), Adhesion Proteins‐ Integrins, Fibronectin1 (FN1), E‐Cadherin (CDH1), and N‐Cadherin (CDH2) and angiogenesis marker VEGFA. Immunofluorescence assay was done to confirm the presence of C‐Peptide, GLUT 2, E‐Cadherin and ITGB3. Adhesion was confirmed assessed on fibronectin binding. Key findings: We show that insulin secretion is not hampered under hypoxia. We found an upregulation of glucose transporters under hypoxia indicating enhanced glucose uptake needed to cater to metabolic demands of proliferating cells. Up regulation of adhesion molecules was seen under hypoxia indicative of a favoured environment for retention of cells at the injury site. We also found increased level of angiogenesis of P‐MSCs under hypoxia. Significance: Our present study thus demonstrates for the first time that P‐MSCs modulate themselves under hypoxic conditions by secreting insulin, up regulating glucose transporters and adhesion molecules and eventually exhibiting an increased angiogenic potential. We thus infer that priming P‐MSCs under hypoxia, could make them more suitable for wound healing applications. Graphical abstract Figure. No caption available.

Reprogramming mouse embryo fibroblasts to functional islets without genetic manipulation

The constant quest for generation of large number of islets aimed us to explore the differentiation potential of mouse embryo fibroblast cells. Mouse embryo fibroblast cells isolated from 12‐ to 14‐day‐old pregnant mice were characterized for their surface markers and tri‐lineage differentiation potential. They were subjected to serum‐free media containing a cocktail of islet differentiating reagents and analyzed for the expression of pancreatic lineage transcripts. The islet‐like cell aggregates (ICAs) was confirmed for their pancreatic properties via immunofluorecence for C‐peptide, glucagon, and somatostain. They were positive for CD markers—Sca1, CD44, CD73, and CD90 and negative for hematopoietic markers—CD34 and CD45 at both transcription and translational levels. The transcriptional analysis of the ICAs at different day points exhibited up‐regulation of islet markers (Insulin, PDX1, HNF3, Glucagon, and Somatostatin) and down‐regulation of MSC‐markers (Vimentin and Nestin). They positively stained for dithizone, C‐peptide, insulin, glucagon, and somatostatin indicating intact insulin producing machinery. In vitro glucose stimulation assay revealed three‐fold increase in insulin secretion as compared to basal glucose with insulin content being the same in both the conditions. The preliminary in vivo data on ICA transplantation showed reversal of diabetes in streptozotocin induced diabetic mice. Our results demonstrate for the first time that mouse embryo fibroblast cells contain a population of MSC‐like cells which could differentiate into insulin producing cell aggregates. Hence, our study could be extrapolated for isolation of MSC‐like cells from human, medically terminated pregnancies to generate ICAs for treating type 1 diabetic patients.

Small molecules exert anti-apoptotic effect and reduce oxidative stress augmenting insulin secretion in stem cells engineered islets against hypoxia.

Transplantation of pancreatic islets is the most reliable treatment for Type 1 diabetes. However cell death mediated by hypoxia is considered as one of the main difficulties hindering success in islet transplantation. The aim of our experiment was to investigate the role of small molecules in survival of Islet like cell aggregates (ICAs) engineered from umbilical cord matrix under oxygen deprived condition (<5% O2). ICAs were analyzed for cell death via fluoroscein diacetate/propidium iodide (FDA/PI) staining, estimation of Caspase 3 and free radical release in presence and absence of small molecules. The samples were also analyzed for the presence of hypoxia inducible factor 1α (HIF1α) at both transcriptional and translational level. The addition of small molecules showed profound defensive effect on ICAs under hypoxic environment as evidenced by their viability and insulin secretion compared to untreated ICAs. The combinations of Eicosapentaenoic acid (EPA), Docosahexaenoic acid(DHA) and metformin and EPA, DHAandγ amino butyric acid (GABA) acted as anti-apoptotic agents for human ICAs when exposed to 1% O2 for 48h. The combinations of the small molecules reduced the total reactive oxygen species and malonaldehyde (MDA) levels and enhanced the production of glutathione peroxidise (GPx) enzyme under hypoxic conditions. Finally the increase in HIF1α at both protein and gene level confirmed the defensive effect of the additives in hypoxia. These results suggest that the combination of small molecules maintained the viability and functionality of the ICAs in hypoxia by up-regulating HIF1α expression and down regulating the Caspase 3 activity.

Pancreatic Progenitors as Target for Islet Neo genesis to Manage Diabetes

Beta-cell replication and islet neogenesis are a major challenge in diabetes research. Transplantation of islets in diabetic patients has been initiated years ago; however, shortage of donor pancreas and autoimmune rejections has limited their clinical implications. Although attempts are being made to generate islets from pluripotent stem cells, their clinical applications are restricted due to ethical concerns and teratoma formation. To overcome these limitations, transdifferentiation of alpha cells and acinar cells and differentiation of ductal stem cells to beta cells are in the pipeline. The amicable substitute for islet transplantation is the islet neogenesis from pancreatic progenitors. The endogenous islet neogenesis could be accomplished with external clues employing combination of Reg protein/transcription factors/growth factors/mesenchymal stem cells to restore the lost beta cells mass. This chapter focuses on the pancreatic progenitor reservoirs within the pancreas as a target for inducing islet neogenesis in diabetes.

Herbal pre-conditioning induces proliferation and delays senescence in Wharton’s Jelly Mesenchymal Stem Cells

BACKGROUND Mesenchymal Stem Cells (MSCs) are multipotent stem cells which are being explored for various clinical applications. Isolation and in-vitro expansion of MSCs remain important in achieving desired cell number for the therapy. However, in-vitro proliferation of MSCs is often associated with senescence and early onset of apoptosis which limits its therapeutic ability and long term clinical use. Tinospora cordifolia and Withania somnifera are used widely in Ayurveda: the traditional Indian system of medicine and are reported to have rejuvenating and anti-aging potential. In the present study, we investigated the effect of Tinospora cordifolia and Withania somnifera on proliferation and senescence of whartons jelly MSCs (WJMSCs) in-vitro. METHODS WJMSCs were treated in culture medium with Tinospora cordifolia leaf and Withania somnifera root extracts to examine their effect on proliferation and senescence properties of WJMSCs. Proliferation of WJMSCs was assayed by cell count, MTT, BrdU incorporation assay, cell cycle analysis and Ki67 mRNA expression. Senescence was demonstrated using β-galactosidase senescence assay and associated mRNA markers. RESULTS Culture medium supplemented with Tinospora cordifolia leaf and Withania somnifera root extracts exhibited significant increase in proliferation of WJMSCs as evidenced by cell count and MTT assay. Cell cycle analysis using propidium iodide showed increase in G2/M phase and decrease in apoptotic cells. BrdU incorporation and upregulation of proliferation marker ki67 by RT PCR showed increased DNA synthesis/proliferation in Tinospora cordifolia and Withania somnifera extract treated MSCs. Delayed senescence was confirmed by β-galactosidase senescence assay and down regulation of senescence marker p21. CONCLUSION Our results demonstrate for the first time that Tinospora cordifolia and Withania somnifera extracts support proliferation and inhibit senescence in WJMSCs making them suitable candidates as supplements for in-vitro expansion without affecting the cell viability indicating its non-toxic nature.