Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.

Effect of glucose mediated oxidative stress on apoptotic gene expression in gingival mesenchymal stem cells.

BACKGROUND: Diabetes is a common disease that causes gingival and periodontal problems. Stem cells isolated from dental sources are an emerging area of research with a potential to facilitate regenerative medicine. The stem cells retain the property of self-renewal and the ones isolated from dental sources are mainly multipotent mesenchymal stem cells that have the ability to self-renew as well as differentiation towards multiple lineages. OBJECTIVES: We aimed to isolate and characterize gingival mesenchymal stem cells by pluripotency markers and investigated the effect of oxidative stress on growth kinetics and apoptotic gene expression of gingival cells exposed to glucose mediated oxidative stress. METHODS: In this study, we isolated gingival mesenchymal stem cells from gingiva. This was followed by morphologic analysis using inverted phase contrast microscopy and molecular profiling of these cells for the mRNA expression of specific genes. The isolated cells were cultured till passage 3 and then exposed to oxidative stress (high glucose concentration). We measured the apoptotic gene expression and compared their growth kinetics. RESULTS: The results showed that oxidative stress produced by glucose reduced growth kinetics and increased apoptotic gene expression in gingival mesenchymal stem cells. According to the genetic results, glucose activated TNF family to initiate apoptosis. CONCLUSION: In conclusion, the present study demonstrated that high glucose obliterated cellular proliferation testified by evaluating growth kinetics and induced apoptotic gene expression in gingival mesenchymal stem cells. This initiated extrinsic apoptotic pathway mediated by TNF family. Therefore, in diabetes oral health condition is compromised and periodontal disease is common.

Multifaceted roles of Toll-like receptors in acute kidney injury.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) in the first line defense system of our bodies; they are widely expressed on leukocytes and kidney epithelial cells. Infections due to pathogens or danger signals from injured tissues often activate several TLRs and these receptors mediate their signal transduction through the activation of transcription factors that regulate the expression of cytokine interleukin-1ß (IL-1ß), type I interferons (IFNs), and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) dependent cytokines and chemokines. Acute kidney injury (AKI) involves early Toll-like receptors driven immunopathology, while resolution of inflammation is needed for rapid regeneration of injured tubular cells. Despite their well known function in the progression of inflammation; interestingly, activation of TLRs also has been implicated in renal epithelial repair through the induction of certain interleukins and improvement in autophagy mechanism. Studies have found that although the blockade of TLRs during the early injury phase of renal tissues prevented tubular necrosis, suppression of interleukins production and impaired kidney regeneration due to their blockade has been observed during the healing phase of tissue. Taken together, these results suggest that the two danger response programs of renal cells i.e. renal inflammation and regeneration may link at the level of TLRs. This review aims to emphasize on the role of TLRs signaling in different acute kidney injury phases. Understanding of these pathways may turn out to be effective as therapeutic option for kidney diseases.

IL-7 overexpression enhances therapeutic potential of rat bone marrow mesenchymal stem cells for diabetic wounds.

This study was aimed to enhance the healing potential of rat bone marrow mesenchymal stem cells against chronic diabetic wounds through interleukin-7 (IL-7) transfection. IL-7 plays an important role in wound healing and acts as a survival factor in some cell types. This study involves isolation, propagation, and characterization of mesenchymal stem cells (MSCs) and their modification with IL-7 gene via retroviral transfection. Transfected MSCs were assessed for their effect on angiogenic genes by qPCR. Wound healing potential of transfected MSCs was analyzed by scratch assay in vitro and by transplanting these cells in rat diabetic wound models in vivo. Wound area was measured for a period of 15 days and subsequent histological analysis was performed. qPCR results showed increased expression of IL-7 gene (p ≤ 0.05) and also principal angiogenic genes, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), VEGF receptor 1 (FLT-1), and VEGF receptor 2 (FLK-1) (p ≤ 0.05). Neuropilin-1 (NRP-1) did not show any significant change. In vitro analysis of IL-7 MSCs showed intense cell-cell connections and tube formation as compared to the normal MSCs. Rate of wound closure was more (p ≤ 0.001) in case of diabetic group transplanted with IL-7 MSCs. Histological examination revealed enhanced vascular supply in skin tissues of diabetic animals transplanted with IL-7 transfected MSCs as compared to normal MSCs. Immunohistochemical results showed significantly higher expression of IL-7 (p ≤ 0.001) and α-smooth muscle actin(p ≤ 0.001) in the tissue sections of IL-7 transfected group as compared to normal MSCs and the diabetic control group; the latter indicates increase in the number of blood vessels. It is concluded from this study that IL-7 overexpression in MSCs can enhance the healing potential of MSCs and aid in wound closure in diabetic animals through the induction of angiogenic genes.

Transcription profile of genes affected in response to pathological changes in drug-induced rat model of acute kidney injury.

OBJECTIVE: The objective of the present study was to examine the changes in the expression profile of certain genes in rat model of gentamicin-induced acute kidney injury (AKI) and to see whether time period and routes of administration affect their expression levels. METHODS: Rat AKI model was established with gentamicin injection using two different routes of administration and two different time periods. The models were evaluated through histopathological observations. Renal specific genes were selected on the basis of their role during kidney injury. These genes were analyzed through reverse transcriptase (RT) PCR. RESULTS: Marked disorganization of normal structure of proximal and distal tubules was observed in all the gentamicin-treated groups. Many tubules showed loss of brush border and presence of intratubular protein casts. Changes in gene expression levels were observed for kidney injury molecule (KIM-1), osteopontin, bone morphogenic protein-7 (BMP-7), extracellular signal-regulated kinases (ERK), stem cell factor (SCF) and IL-7 receptor with different levels of significance in the renal injury groups studied depending on the time period and route of administration. CONCLUSION: Gene expression seems to be dependent partly on the type of injury, route of administration and time period after induction of injury. An improved mechanistic understanding of gene regulation pathways in AKI may provide basis for potential therapeutic development.

Hypoxic stress and IL-7 gene overexpression enhance the fusion potential of rat bone marrow mesenchymal stem cells with bovine renal epithelial cells.

Transplantation of mesenchymal stem cells (MSCs) has been shown to enhance the improvement in kidney function following injury. However, the poor survival and grafting of the stem cells to the site of injury has restricted their therapeutic efficacy. Accelerated regeneration potential of MSCs has been observed when they were exposed to hypoxic stress or genetic modulation by various cytokines and growth factors. These preconditioning strategies may stimulate endogenous mechanisms resulting in multiple cellular responses. In this study, we used IL-7 gene to transfect MSCs. IL-7 is a hematopoietic growth factor that plays an important role in cell survival, proliferation, and differentiation. MSCs were also subjected to hypoxic stress for 8 and 24 h. These preconditioned MSCs were co-cultured with cisplatin-treated injured Mardin-Darby bovine kidney (MDBK) cells and their fusion potential was analyzed. Flow cytometry of fluorescently labeled preconditioned MSCs and injured MDBK cells revealed evidence of significant (P < 0.001) cell fusion compared to that of the normal MSCs. In addition, we also observed improved migration ability of these preconditioned MSCs in the in vitro wound healing assay, as compared to the normal MSCs. We conclude that hypoxic stress and IL-7 overexpression can enhance the renal regeneration potential of MSCs. This study would help in designing more potent therapeutic strategy in which preconditioned MSCs can be used for renal regeneration.

Dinitrophenol modulates gene expression levels of angiogenic, cell survival and cardiomyogenic factors in bone marrow derived mesenchymal stem cells.

Various preconditioning strategies influence regeneration properties of stem cells. Preconditioned stem cells generally show better cell survival, increased differentiation, enhanced paracrine effects, and improved homing to the injury site by regulating the expression of tissue-protective cytokines and growth factors. In this study, we analyzed gene expression pattern of growth factors through RT-PCR after treatment of mesenchymal stem cells (MSCs) with a metabolic inhibitor, 2,4 dinitrophenol (DNP) and subsequent re-oxygenation for periods of 2, 6, 12 and 24h. These growth factors play important roles in cardiomyogenesis, angiogenesis and cell survival. Mixed pattern of gene expression was observed depending on the period of re-oxygenation. Of the 13 genes analyzed, ankyrin repeat domain 1 (Ankrd1) and GATA6 were downregulated after DNP treatment and subsequent re-oxygenations. Ankrd1 expression was, however, increased after 24h of re-oxygenation. Placental growth factor (Pgf), endoglin (Eng), neuropilin (Nrp1) and jagged 1 (Jag1) were up-regulated after DNP treatment. Gradual increase was observed as re-oxygenation advances and by the end of the re-oxygenation period the expression started to decrease and ultimately regained normal values. Epiregulin (Ereg) was not expressed in normal MSCs but its expression increased gradually from 2 to 24h after re-oxygenation. No change was observed in the expression level of connective tissue growth factor (Ctgf) at any time period after re-oxygenation. Kindlin3, kinase insert domain receptor (Kdr), myogenin (Myog), Tbx20 and endothelial tyrosine kinase (Tek) were not expressed either in normal cells or cells treated with DNP. It can be concluded from the present study that MSCs adjust their gene expression levels under the influence of DNP induced metabolic stress. Their levels of expression vary with varying re-oxygenation periods. Preconditioning of MSCs with DNP can be used for enhancing the potential of these cells for better regeneration.