Histochemical analysis of self-organizing 3D spheroid-like cultures of adipose-derived mesenchymal stem cells

Histochemical staining demonstrated fusion of cells with formation of syncytium-like structures. On the periphery cells were polygonal, round, had epithelial morphology. Groups of cells organized in line inside spheroid remained elongated AD-MSC shape. Most of the cells did not proliferate. There were no migrating SMA+ cells. Polygonal cells on the periphery and a few cells inside the spheroid were desmin+. Interestingly, peripheral and elongated shape cells expressed epithelial marker CK-19+. Probably, within spheroid there was spontaneous mesenchymal-epithelial transdifferentiation. When spheroids were placed in a new culture dish we observed explantational outgrowth of spindle-shaped AD-MSC suggesting that cells within spheroids were alive, able to outgrow and revert through epithelial-mesenchymal transdifferentiation back to MSC phenotype. Investigation was funded by RFBR grant 18-04-01133 and supported by Program of Competitive Growth of KFU

Influence of long-term cultivation and cryopreservation on phenotype of rat hepatic stellate cells

Many organs and tissues have been reported to harbor regional stem cells. One of the candidates for this role in the liver is hepatic stellate cells (HSC). Cultivation of cells in vitro may lead to changes in their morphology and phenotype. The risk of changes increases with long-term cultivation (passage 5 and above), as well as with cryopreservation of cells. Freezing allows storage of isolated cell; however, the method can influence cells phenotype and properties. The aim of research was to study phenotype of rat HSC during long-term cultivation and cryopreservation. Three cultures of HSC (I - after cryopreservation, II - long-term culture, passage 6, and III - control, passage 4) were stained immunocytochemically with antibodies to desmin, -SMA, CK19, Ki-67; quantitative real-time PCR analysis was also performed to evaluate desmin and -SMA gene expression. It was observed that morphology of cells was similar in all three groups. In groups with long-term cultivation and cryopreserA134-A13

Gene Therapy Using Plasmid DNA Encoding VEGF164 and FGF2 Genes: A Novel Treatment of Naturally Occurring Tendinitis and Desmitis in Horses

This clinical study describes the intralesional application of the plasmid DNA encoding two therapeutic species-specific growth factors: vascular endothelial growth factor (VEGF164) and fibroblast growth factor 2 (FGF2) in seven horses to restore naturally occurring injuries of the superficial digital flexor tendon (SDFT) (tendinitis) and in three horses with suspensory ligament branch desmitis. Following application all horses were able to commence a more rapid exercise program in comparison to standardized exercise programs. Clinical observation and ultrasonic imaging was used to evaluate the regeneration rate of the tendon and ligament injury recovery and to confirm the safety of this gene therapy in horses, throughout a 12 month period. Follow-up data of the horses revealed a positive outcome including significant ultrasonographic and clinical improvements in 8 out of 10 horses with SDFT and suspensory ligament branch lesions, with return to their pre-injury level of performance by 2–6 months after the completion of treatment. The ninth horse initially presenting with severe suspensory ligament branch desmopathy, showed no significant ultrasonographic improvements in the first 2 months after treatment, however, it improved clinically and became less lame. The final horse, presenting with severe tendinitis of the SDFT returned to their pre-injury level of performance, but experienced re-injury 6 months after treatment. This data is highly promising, however, further research in experimental models, with the histopathological, immunohistochemical and gene expression evaluation of the equine tendon/ligament after gene therapy application is required in order to fully understand the mechanisms of action. This treatment and the significant clinical impacts observed represents an important advancement in the field of medicine

Histopathological characterization of skeletal muscle during postnatal ontogenesis of dysferlin-deficient mice

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Role of microvesicles derived from adipose mesenchymal stem cells in liver regeneration after partial hepatectomy in rats

Introduction. There are numerous studies of stimulation of liver regeneration by transplantation of mesenchymal stem cells. Their effect is explained by direct intercellular interactions and paracrine communications. One of the alternative ways to influence on liver regeneration is injection of microvesicles. Microvesicles are extracellular vesicles that contain growth factors and cytokines and play a major role in intercellular paracrine communications. It is said, that in compare to cells microvesicles transplantation is not accompanied by risk of metaplasia and mutations, because they do not contain any genetic information. To study the effect of transplantation of mesenchymal stem cells microvesicles derived from adipose fat tissue on liver regeneration after partial hepatectomy in rats. Methods. Mesenchymal stem cells, isolated from visceral rat fatty tissue (adMSC), were treated by Cytochalasin B to derive microvesicles. These microvesicles were transplanted into portal vein of rats after partial hepatectomy. Control group of rats after partial hepatectomy received injection of PBS. On 2, 5, 7 and 14 days after operation rats were sacrificed. Functional parameters of liver were analyzed by biochemical tests, morphological changes were studied by immunohistochemical staining of liver slices with antibodies to desmin (HSC marker), Ki-67 (proliferation marker), α-SMA (myofibroblasts marker), CK-19 (cholangiocytes marker). Results. According to immunohistochemistry results injection of microvesicles: 1) inhibits activation of HSC - there were 20% less desmin+ cells; 2) there were no transformation of HSC into α-SMA+ myofibroblasts and no risk of liver fibrosis; 3) inhibition of cellular proliferation. So, Ki-67+ hepatocytes number (area of portal tract and central vein) decreased in compare to control group. Number of Ki-67+ nonparenchymal cells in portal tract area was 2 times less, than in control. 4) there were no differences in CK-19 expression in experimental and control groups. As far as the cellular proliferation and thus liver regeneration was inhibited, we`ve seen higher numbers of ALT levels in experimental group. Decreased regeneration could be also visualized by lower triglycerides and cholesterol levels, there were less, than normal values. Blood urea nitrogen normalized in control group on 14th day, but in experimental - on 7th day already. Conclusion. Injection of adMSC microvesicles inhibits general cellular response to partial hepatectomy. Inhibition of hepatocytes activation and proliferation slows down liver regeneration, that is proved by biochemical tests. Inhibition of HSC activation means also lesser risk of their transformation into myofibroblasts and fibrosis development. Thus microvesicles transplantation is not for stimulation of liver regeneration. Probably these inhibitory effects could be applied for treatment of liver fibrosis, that needs to be studied. Work supported by Program of Competitive Growth of KFU.466-46

Effect of curcumin and gliotoxin on rat liver myofibroblasts culture

Since 1990s, when it was demonstrated by Hammel and others, that liver fibrosis is reversible, researchers and physicians actively search for new antifibrotic therapies. In recent years knowledge of liver fibrosis pathophysiology has greatly advanced and new cellular and molecular mechanisms were described. The cells that determine extracellular matrix components distribution are myofibroblasts, but their origin is diverse. They can be activated hepatic stellate cells (HSC), portal fibroblasts (PF) or circulating mesenchymal stem cells of bone marrow. Activation of HSC and PF and their fibrogenic potential is upregulated by transcription factor NF-B. Among large number of substrates to inhibit NF-B and induce apoptosis we chose curcumin and gliotoxin. Primarily in current work we optimized the explantation culture method for isolation of hepatic myofibroblasts and received two different cultures - myofibroblasts of HSC and PF origin. Exposition of 50 ?M curcumin and 0,1 ?M gliotoxi

Influence of adenoviral transduction with Adv5-optHGF-RFP on phenotype of hepatic stellate cells

Adenoviral transduction of hepatic stellate cells is applicable for short time stimulation of genes expression, safety for phenotype and cells proliferation. At the same time adenoviral transduction with growth factors genes leads to expression of hepatoblasts markers