Nitric oxide-mediated inhibition of phenylephrine-induced contraction in response to hypothermia is partially modulated by endothelial Rho-kinase.

This study examined the possible upstream cellular signaling pathway associated with nitric oxide (NO)-mediated inhibition of phenylephrine-induced contraction in isolated rat aortae in response to mild hypothermia, with a particular focus on endothelial Rho-kinase. We examined the effects of mild hypothermia (33°C), wortmannin, Nω-nitro-L-arginine methyl ester (L-NAME), Y-27632, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and methylene blue, alone and combined, on phenylephrine-induced contraction in isolated rat aortae. Finally, we examined the effects of mild hypothermia, wortmannin, Y-27632 and L-NAME, alone and combined, on endothelial nitric oxide synthase (eNOS) and endothelial Rho-kinase membrane translocation induced by phenylephrine. Mild hypothermia attenuated phenylephrine-induced contraction only in endothelium-intact aortae. L-NAME, wortmannin, ODQ and methylene blue increased phenylephrine-induced contraction of endothelium-intact aortae pretreated at 33°C. Wortmannin did not significantly alter the L-NAME-induced enhancement of phenylephrine-induced maximal contraction of endothelium-intact aortae pretreated at 33°C. Wortmannin abolished the ability of Y-27632 to magnify the hypothermic inhibition of maximal phenylephrine-induced contraction. Wortmannin and L-NAME inhibited the enhancing effect of mild hypothermia on phenylephrine-induced eNOS phosphorylation. Y-27632 and L-NAME attenuated the enhancing effect of hypothermia on phenylephrine-induced endothelial Rho-kinase membrane translocation. The results suggest that hypothermia-induced, NO-dependent inhibition of phenylephrine-induced contraction is mediated by phosphoinositide 3-kinase and inhibited by endothelial Rho-kinase activation.

Linolenic Acid Attenuates the Vasodilation Induced by Acetylcholine in Isolated Rat Aortae.

This study aims to examine the effect of linolenic acid on the vasodilation or vasoconstriction induced by acetylcholine and bupivacaine in isolated rat aortae and its underlying mechanism. The effect of linolenic acid on the vasodilation induced by acetylcholine, the calcium ionophore A23187, sodium nitroprusside, and 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (bromo-cyclic guanosine monophosphate [bromo-cGMP]) in endothelium-intact and endothelium-denuded aortae was examined. Linolenic acid inhibited vasodilation induced by acetylcholine, calcium ionophore A23187, and sodium nitroprusside. However, this fatty acid increased bromo-cGMP-induced vasodilation in endothelium-denuded aortae. Linolenic acid increased bupivacaine-induced contraction in endothelium-intact aortae, whereas it decreased bupivacaine-induced contraction in endothelium-intact aortae with Nω-nitro-l-arginine methyl ester and endothelium-denuded aortae. Linolenic acid inhibited acetylcholine- and bupivacaine-induced phosphorylation of endothelial nitric oxide synthase. Sodium nitroprusside increased cGMP in endothelium-denuded aortic strips, whereas bupivacaine decreased cGMP in endothelium-intact aortic strips. Linolenic acid decreased cGMP levels produced by bupivacaine and sodium nitroprusside. Together, these results suggest that linolenic acid inhibits acetylcholine-induced relaxation by inhibiting a step just prior to nitric oxide-induced cGMP formation. In addition, linolenic acid-mediated inhibition of vasodilation induced by a toxic concentration (3 × 10-4 M) of bupivacaine seems to be partially associated with inhibition of the nitric oxide-cGMP pathway.

CD105+ Porcine Endometrial Stromal Mesenchymal Stem Cells Possess Differentiation Potential Toward Cardiomyocyte-Like Cells and Insulin-Producing ß Cell-Like Cells In Vitro.

Porcine mesenchymal stem cells (MSCs) are similar to human MSCs, hence considered a valuable model for assessing potential for cell therapy. Porcine adipose-derived MSCs (AD-MSCs) and endometrial stromal MSCs (EMSCs) displayed fibroblast-like morphology and were positive for MSC markers CD73, CD90, and CD105 and negative for hematopoietic markers CD34 and CD45. The EMSCs had similar or slightly higher growth rate compared to AD-MSCs, and similar percentage of cells of both EMSCs and AD-MSCs were at G0/G1 and G2/M phases; however, EMSCs had significantly ( P < .05) higher percentage of cells at S phase of cell cycle than AD-MSCs. Transdifferentiation ability to cardiomyocyte-like cells was confirmed in differentiated cells by the expression of lineage-specific marker genes such as DES, ACTA2, cTnT, and ACTC1 by real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, cardiomyocyte-specific protein markers cTnT and ACTC1 were expressed in completely differentiated cells. Endodermal differentiation capacity of EMSCs to pancreatic ß cell-like cells was evident with the changes in morphology and the expression of ß-cell-specific marker genes such as PDX1, GLUT2, SST, NKX6.1, PAX4, and NGN3 as analyzed by RT-qPCR. The differentiated cells secreted insulin and C-peptide upon glucose challenge and also they expressed insulin, PDX1, PAX4, NGN3, and GLUT2 at protein level as assessed by immunostaining confirming the successful differentiation to ß cell-like cells. Porcine EMSCs possess all the characteristics of MSCs and are suitable model for studying molecular mechanisms of cellular differentiation.

Lipid Emulsion Inhibits the Late Apoptosis/Cardiotoxicity Induced by Doxorubicin in Rat Cardiomyoblasts.

This study aimed to examine the effect of lipid emulsion on the cardiotoxicity induced by doxorubicin in H9c2 rat cardiomyoblasts and elucidates the associated cellular mechanism. The effects of lipid emulsion on cell viability, Bax, cleaved caspase-8, cleaved capase-3, Bcl-XL, apoptosis, reactive oxygen species (ROS), malondialdehyde, superoxide dismutase (SOD), catalase and mitochondrial membrane potential induced by doxorubicin were examined. Treatment with doxorubicin decreased cell viability, whereas pretreatment with lipid emulsion reduced the effect of doxorubicin by increasing cell viability. Lipid emulsion also suppressed the increased expression of cleaved caspase-3, cleaved caspase-8, and Bax induced by doxorubicin. Moreover, pretreatment with lipid emulsion decreased the increased Bax/Bcl-XL ratio induced by doxorubicin. Doxorubicin-induced late apoptosis was reduced by treatment with lipid emulsion. In addition, pretreatment with lipid emulsion prior to doxorubicin enhanced glycogen synthase kinase-3ß phosphorylation. The increased malondialdehyde and ROS levels by doxorubicin were reduced by lipid emulsion pretreatment. Furthermore, lipid emulsion attenuated the reduced SOD and catalase activity and the decreased mitochondrial membrane potential induced by doxorubicin. Taken together, these results suggest that lipid emulsion attenuates doxorubicin-induced late apoptosis, which appears to be associated with the inhibition of oxidative stress induced by doxorubicin.

Comparative analysis of human Wharton's jelly mesenchymal stem cells derived from different parts of the same umbilical cord.

Easy isolation, lack of ethical issues, high proliferation, multi-lineage differentiation potential and immunomodulatory properties of umbilical cord (UC)-derived mesenchymal stem cells (MSCs) make them a valuable tool in stem cell research. Recently, Wharton's jelly (WJ) was proven as the best MSC source among various compartments of UC. However, it is still unclear whether or not Wharton's jelly-derived MSCs (WJMSCs) from different parts of the whole cord exhibit the same characteristics. There may be varied MSCs present in different parts of WJ throughout the length of the UC. For this purpose, using an explant attachment method, WJMSCs were isolated from three different parts of the UC, mainly present towards the placenta (mother part), the center of the whole cord (central part) and the part attached to the fetus (baby part). WJMSCs from all three parts were maintained in normal growth conditions (10% ADMEM) and analyzed for mesenchymal markers, pluripotent genes, proliferation rate and tri-lineage differentiation potential. All WJMSCs were highly proliferative, positively expressed CD90, CD105, CD73 and vimentin, while not expressing CD34, CD45, CD14, CD19 or HLA-DR, differentiated into adipocytes, osteocytes and chondrocytes and expressed pluripotency markers OCT-4, SOX-2 and NANOG at gene and protein levels. Furthermore, MSCs derived from all the parts were shown to have potency towards hepatocyte-like cell differentiation. Human bone marrow-derived MSCs were used as a positive control. Finally, we conclude that WJMSCs derived from all the parts are valuable sources and can be efficiently used in various fields of regenerative medicine.

Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos.

To study gene expression and to determine distinctive characteristics of embryos produced by different methods, normalisation of the gene(s) of interest against reference gene(s) has commonly been employed. Therefore, the present study aimed to assess which reference genes tend to express more stably in single porcine blastocysts produced in vivo (IVO) or by parthenogenetic activation (PA), in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) using different analysis programs, namely geNorm, Normfinder and Bestkeeper. Commonly used reference genes including 18S rRNA (18S), H2A histone family, member Z (H2A), hypoxanthine phosphoribosyltransferase1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein 4 (RPL4), peptidylprolyl isomerase A (PPIA), beta actin (ACTB), succinate dehydrogenase complex, subunit A (SDHA) and hydroxymethylbilane synthase (HMBS2) were analysed; most of them resulted in significantly (P<0.05) different cycle threshold (CT) values in porcine embryos except for SDHA and H2A. In evaluation of stable reference genes across in vivo and in vitro porcine blastocysts, three kinds of programs showed slightly different results; however, there were similar patterns about the rankings of more or less stability overall. In conclusion, SDHA and H2A were determined as the most appropriate reference genes for reliable normalisation in order to find the comparative gene expression in porcine blastocysts produced by different methods, whereas 18S was regarded as a less-stable reference gene. The present study has evaluated the stability of commonly used reference genes for accurate normalisation in porcine embryos to obtain reliable results.

DMSO- and Serum-Free Cryopreservation of Wharton's Jelly Tissue Isolated From Human Umbilical Cord.

The facile nature of mesenchymal stem cell (MSC) acquisition in relatively large numbers has made Wharton's jelly (WJ) tissue an alternative source of MSCs for regenerative medicine. However, freezing of such tissue using dimethyl sulfoxide (DMSO) for future use impedes its clinical utility. In this study, we compared the effect of two different cryoprotectants (DMSO and cocktail solution) on post-thaw cell behavior upon freezing of WJ tissue following two different freezing protocols (Conventional [-1°C/min] and programmed). The programmed method showed higher cell survival rate compared to conventional method of freezing. Further, cocktail solution showed better cryoprotection than DMSO. Post-thaw growth characteristics and stem cell behavior of Wharton's jelly mesenchymal stem cells (WJMSCs) from WJ tissue cryopreserved with a cocktail solution in conjunction with programmed method (Prog-Cock) were comparable with WJMSCs from fresh WJ tissue. They preserved their expression of surface markers, pluripotent factors, and successfully differentiated in vitro into osteocytes, adipocytes, chondrocytes, and hepatocytes. They also produced lesser annexin-V-positive cells compared to cells from WJ tissue stored using cocktail solution in conjunction with the conventional method (Conv-Cock). Real-time PCR and Western blot analysis of post-thaw WJMSCs from Conv-Cock group showed significantly increased expression of pro-apoptotic factors (BAX, p53, and p21) and reduced expression of anti-apoptotic factor (BCL2) compared to WJMSCs from the fresh and Prog-Cock group. Therefore, we conclude that freezing of fresh WJ tissue using cocktail solution in conjunction with programmed freezing method allows for an efficient WJ tissue banking for future MSC-based regenerative therapies. J. Cell. Biochem. 117: 2397-2412, 2016. © 2016 Wiley Periodicals, Inc.

In vitro comparative analysis of human dental stem cells from a single donor and its neuronal differentiation potential evaluated by electrophysiology.

AIMS: The aim of this study was to find out a mesenchymal stem cells (MSCs) source from human dental tissues of the same donor (follicle, papilla and pulp), which exhibits higher neurogenic differentiation potential in vitro. MAIN METHODS: MSCs were isolated from dental tissues (follicle, papilla and pulp) by digestion method. All MSCs were analyzed for pluripotent makers by western blot, cell surface markers by flow cytometry, adipo- and osteocytes markers by RT-qPCR. The neuronal differentiated MSCs were characterized for neuronal specific markers by RT-qPCR and immunofluorescence. Functional neuronal properties were analyzed by electrophysiology and synaptic markers expression. KEY FINDINGS: All MSCs expressed pluripotent markers (Oct4, Sox2 and Nanog) and were found positive for mesenymal markers (CD44, CD90, CD105) while negative for hematopoietic markers (CD34 and CD45). Furthermore, MSCs were successfully differentiated into adipocytes, osteocytes and trans-differentiated into neuronal cells. Among them, dental pulp derived MSCs exhibits higher neurogenic differentiation potential, in term of expression of neuronal specific markers at both gene and protein level, and having higher Na(+) and K(+) current with the expression of synaptic markers. SIGNIFICANCE: The three types of dental MSCs from a single donor broadly possessed similar cellular properties and can differentiate into neuronal cells; however, pulp derived MSCs showed higher neurogenic potential than the follicle and papilla, suggesting their use in future stem cells therapy for the treatment of neurodegenerative disorders.

Research Advancements in Porcine Derived Mesenchymal Stem Cells.

In the present era of stem cell biology, various animals such as Mouse, Bovine, Rabbit and Porcine have been tested for the efficiency of their mesenchymal stem cells (MSCs before their actual use for stem cell based application in humans. Among them pigs have many similarities to humans in the form of organ size, physiology and their functioning, therefore they have been considered as a valuable model system for in vitro studies and preclinical assessments. Easy assessability, few ethical issues, successful MSC isolation from different origins like bone marrow, skin, umbilical cord blood, Wharton's jelly, endometrium, amniotic fluid and peripheral blood make porcine a good model for stem cell therapy. Porcine derived MSCs (pMSCs have shown greater in vitro differentiation and transdifferention potential towards mesenchymal lineages and specialized lineages such as cardiomyocytes, neurons, hepatocytes and pancreatic beta cells. Immunomodulatory and low immunogenic profiles as shown by autologous and heterologous MSCs proves them safe and appropriate models for xenotransplantation purposes. Furthermore, tissue engineered stem cell constructs can be of immense importance in relation to various osteochondral defects which are difficult to treat otherwise. Using pMSCs successful treatment of various disorders like Parkinson's disease, cardiac ischemia, hepatic failure, has been reported by many studies. Here, in this review we highlight current research findings in the area of porcine mesenchymal stem cells dealing with their isolation methods, differentiation ability, transplantation applications and their therapeutic potential towards various diseases.

Characterization and evaluation of neuronal trans-differentiation with electrophysiological properties of mesenchymal stem cells isolated from porcine endometrium.

Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K+ currents (Ito), at holding potential of -80 mV, Na+ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.

Human mesenchymal stem cells - current trends and future prospective.

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.