Azra Mehmood

Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation and anti-apoptosis capabilities

Decline in the function of stem cells with age, such as other cells of the body, results in an imbalance between loss and renewal. Increasing age of the donor thus diminishes the effectiveness of MSCs (mesenchymal stem cells) transplantation in age‐related diseases. The clinical use of stem cell therapies needs autologous stem cell transplantation; it is essential therefore to study the repair ability and survivability of cells before transplantation. Bone marrow derived MSCs possess multi‐lineage differentiation potential, but aging adversely affects their therapeutic efficacy. MSCs from young (2–3 months) and aged (23–24 months) GFP (green fluorescent protein)‐expressing C57BL/6 mice were isolated and their regenerative potential was assessed in vitro. Real‐time RT—PCR (reverse transcriptase—PCR) showed significantly higher expression of Sirt1 in MSCs isolated from young than older animals. Down‐regulation of VEGF (vascular endothelial growth factor), SDF‐1 (stromal‐cell‐derived factor 1), AKT (also known as protein kinase B) and up‐regulation of p53, p21, Bax and p16 occurred in aged cells. Tube formation, wound healing and proliferative abilities of the young MSCs were better than the aged MSCs. The results suggest that age‐related increased expression of apoptotic and senescent genes, with concomitant decrease in Sirt1 gene expression, inhibits to some extent stem cell functioning.

Preconditioning diabetic mesenchymal stem cells with myogenic medium increases their ability to repair diabetic heart

IntroductionMesenchymal stem cells (MSCs) have the potential for treatment of diabetic cardiomyopathy; however, the repair capability of MSCs declines with age and disease. MSCs from diabetic animals exhibit impaired survival, proliferation, and differentiation and therefore require a strategy to improve their function. The aim of the study was to develop a preconditioning strategy to augment the ability of MSCs from diabetes patients to repair the diabetic heart.MethodsDiabetes was induced in C57BL/6 mice (6 to 8 weeks) with streptozotocin injections (55 mg/kg) for 5 consecutive days. MSCs isolated from diabetic animals were preconditioned with medium from cardiomyocytes exposed to oxidative stress and high glucose (HG/H-CCM).ResultsGene expression of VEGF, ANG-1, GATA-4, NKx2.5 MEF2c, PCNA, and eNOS was upregulated after preconditioning with HG/H-CCM, as evidenced by reverse transcriptase/polymerase chain reaction (RT-PCR). Concurrently, increased AKT phosphorylation, proliferation, angiogenic ability, and reduced levels of apoptosis were observed in HG/H-CCM-preconditioned diabetic MSCs compared with nontreated controls. HG/H-CCM-preconditioned diabetic-mouse-derived MSCs (dmMSCs) were transplanted in diabetic animals and demonstrated increased homing concomitant with augmented heart function. Gene expression of angiogenic and cardiac markers was significantly upregulated in conjunction with paracrine factors (IGF-1, HGF, SDF-1, FGF-2) and, in addition, reduced fibrosis, apoptosis, and increased angiogenesis was observed in diabetic hearts 4 weeks after transplantation of preconditioned dmMSCs compared with hearts with nontreated diabetic MSCs.ConclusionsPreconditioning with HG/H-CCM enhances survival, proliferation, and the angiogenic ability of dmMSCs, augmenting their ability to improve function in a diabetic heart.

Pre-conditioned mesenchymal stem cells ameliorate renal ischemic injury in rats by augmented survival and engraftment.

BackgroundIschemia is the major cause of acute kidney injury (AKI), associated with high mortality and morbidity. Mesenchymal stem cells (MSCs) have multilineage differentiation potential and can be a potent therapeutic option for the cure of AKI.MethodsMSCs were cultured in four groups SNAP (S-nitroso N-acetyl penicillamine), SNAP + Methylene Blue (MB), MB and a control for in vitro analysis. Cultured MSCs were pre-conditioned with either SNAP (100 μM) or MB (1 μM) or both for 6 hours. Renal ischemia was induced in four groups (as in in vitro study) of rats by clamping the left renal padicle for 45 minutes and then different pre-conditioned stem cells were transplanted.ResultsWe report that pre-conditioning of MSCs with SNAP enhances their proliferation, survival and engraftment in ischemic kidney. Rat MSCs pre-conditioned with SNAP decreased cell apoptosis and increased proliferation and cytoprotective genes’ expression in vitro. Our in vivo data showed enhanced survival and engraftment, proliferation, reduction in fibrosis, significant improvement in renal function and higher expression of pro-survival and pro-angiogenic factors in ischemic renal tissue in SNAP pre-conditioned group of animals. Cytoprotective effects of SNAP pre-conditioning were abrogated by MB, an inhibitor of nitric oxide synthase (NOS) and guanylate cyclase.ConclusionThe results of these studies demonstrate that SNAP pre-conditioning might be useful to enhance therapeutic potential of MSCs in attenuating renal ischemia reperfusion injury.

Stromal cell derived factor-1alpha protects stem cell derived insulin-producing cells from glucotoxicity under high glucose conditions in-vitro and ameliorates drug induced diabetes in rats

BackgroundDiabetes mellitus is affecting more than 300 million people worldwide. Current treatment strategies cannot prevent secondary complications. Stem cells due to their regenerative power have long been the attractive target for the cell-based therapies. Mesenchymal stem cells (MSCs) possess the ability to differentiate into several cell types and to escape immune recognition in vitro. MSCs can be differentiated into insulin-producing cells (IPCs) and could be an exciting therapy for diabetes but problems like poor engraftment and survivability need to be confronted. It was hypothesized that stromal cell derived factor- 1alpha (SDF-1alpha) will enhance therapeutic potential of stem cell derived IPCs by increasing their survival and proliferation rate.MethodsNovel culture conditions were developed to differentiate bone marrow derived mesenchymal stem cells (BMSCs) into IPCs by using endocrine differentiation inducers and growth factors via a three stage protocol. In order to enhance their therapeutic potential, we preconditioned IPCs with SDF-1alpha.ResultsOur results showed that SDF-1alpha increases survival and proliferation of IPCs and protects them from glucotoxicity under high glucose conditions in vitro. SDF-1alpha also enhances the glucose responsive insulin secretion in IPCs in vitro. SDF-1alpha preconditioning reverses hyperglycemia and increase serum insulin in drug induced diabetic rats.ConclusionsThe differentiation of BMSCs into IPCs and enhancement of their therapeutic potential by SDF-1alpha preconditioning may contribute to cell based therapies for diabetes.

Identification of ionotrophic purinergic receptors in Huh-7 cells and their response towards structural proteins of HCV genotype 3a

Hepatitis C virus (HCV) is a major health problem in developing countries including Pakistan. Chronic HCV infection results in progressive liver disease including fibrosis, cirrhosis, insulin resistance and eventually hepatocellular carcinoma (HCC). Ionotrophic purinergic (P2X) receptors are identified to involve in a spectrum of physiological and pathophysiological processes. However, the role of P2X receptors in HCV liver associated diseases still remains to be investigated. The current study was designed to identify the presence of P2X receptors in human liver cells. Furthermore, it investigates the response of P2X receptors towards HCV structural proteins (E1E2). To determine that how many isoforms of P2X receptors are expressed in human liver cells, human hepatoma cell line (Huh-7) was used. Transcripts (mRNA) of five different isoforms of P2X receptors were identified in Huh-7 cells. To examine the gene expression of identified isoforms of P2X receptors in presence of HCV structural proteins E1E2, Huh-7/E1E2 cell line (stably expressing HCV structural proteins E1E2) was used. The results showed significant increase (6.2 fold) in gene expression of P2X4 receptors in Huh-7/E1E2 cells as compared to control Huh-7 cells. The findings of present study confirmed the presence of transcripts of five different isoforms of P2X receptors in human liver cells and suggest that P2X4 receptors could be represented an important component of the purinergic signaling complex in HCV induced liver pathogenesis.

Diazoxide preconditioning of endothelial progenitor cells improves their ability to repair the infarcted myocardium

Reduced survival and homing of the transplanted cells in the oxidative stressed ischemic environment limits the potential outcome of cell therapies for myocardial ischemia. Diazoxide (DZ), a highly selective mitochondrial ATP sensitive K+ channel opener, is known to improve the survival and therapeutic ability of mesenchymal stem cells and skeletal myoblasts for the repair of heart failure. The current study explored the effect of DZ preconditioning in improving the ability of endothelial progenitor cells (EPCs) to counteract, in vitro oxidative stress, and to repair the infarcted myocardium. The EPCs were preconditioned by 30 min incubation with 200 μM DZ followed by exposure to 200 μM hydrogen peroxide (H2O2) for 2 h. Non‐preconditioned EPCs with and without exposure to H2O2 were used as control. DZ preconditioning of EPCs resulted in significantly reduced cell injury as shown by reduced lactate dehydrogenase release and expression of annexin V‐PE in comparison to untreated EPCs. Furthermore, DZ preconditioned EPCs exhibited upregulated expression of prosurvival genes (VEGF, SDF‐1α, PCNA, and Bcl2), improved chemokines release (VEGF, IGF, and SDF‐1α), viability, Akt phosphorylation and tube formation. In vivo experiments involved transplantation of DZ preconditioned and untreated EPCs in the left ventricle after permanent ligation of left anterior descending coronary artery in rats. The results demonstrated that DZ EPCs transplanted group showed significant reduction in infarct size along with robust cell proliferation, angiogenesis and improvement in cardiac function. The current study demonstrates that DZ preconditioning enhances EPCs survival under oxidative stress in vitro and their ability to treat myocardial infarction.

Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro

Aims Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. Main methods Fibroblasts were treated with 100 &mgr;M vitamin E for 24 h at 37 °C followed by heat shock for 10 min at 51 °C in fresh serum free medium. Key findings Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin‐V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down‐regulated expression of pro‐apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor‐1alpha (SDF‐1&agr;) and also showed significantly up‐regulated levels of PCNA, VEGF, BCL‐XL, FGF7, FGF23, FLN&bgr; and Col7&agr; genes presumably through activation of phosphatidylinositol 3‐kinase (PI3‐K)/Akt pathway. Significance The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds.

Transplantation of stromal-derived factor 1α and basic fibroblast growth factor primed insulin-producing cells reverses hyperglycaemia in diabetic rats

Abstract The defective insulin production is associated with severely reduced islet cell mass leading to diabetes. Growth factors preconditioned stem cells have arisen as an effective therapy to treat many diseases including diabetes. The current study was designed to assess the effect of pretreatment of ASCs derived IPCs with combination of stromal cell derived factor 1 alpha (SDF1α) and basic fibroblast growth factor (bFGF) in improving glucose tolerance in streptozotocin induced diabetic rats. The results showed maximally significant reduction in hyperglycaemia and fibrosis, while up-regulation of survival and pancreas-specific genes, insulin levels and homing of transplanted cells in SDF-1α + bFGF IPCs transplanted rats as compared with other groups. Moreover, increased expression of insulin, glucagon and Glut-2 in pancreas of the SDF-1α + bFGF IPCs transplanted group indicated more regeneration of pancreas. Hence, the use of IPCs preconditioned with SDF-1α + bFGF would be more effective for treating diabetes.

Serum from CCl4-induced acute rat injury model induces differentiation of ADSCs towards hepatic cells and reduces liver fibrosis

Abstract Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl4 injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl4 which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl4-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson’s trichome staining was observed.