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© 2019 National Academy of Pediatric Science and Innovation. All rights reserved. We studied the autopsy material obtained from 7 children who died in the neonatal period in order to evaluate the composition of lymphocytes of the intestinal mucosa against the background of morphological changes in the tissues of the gastrointestinal tract in newborns with sepsis. The main group consisted of 4 children with neonatal sepsis, the control group – of 3 newborns who died from other causes. The research material included the specimen of the small and large intestine. Results. Small intestine: it was found that there were less CD4 + lymphocytes in the small intestinal mucosa in the group of children who died from neonatal sepsis in 75% of cases than in the control group, but this difference was not statistically significant (p=0.1). There were no differences in the number of CD8 + and CD20 + cells in the studied groups. Large intestine: the number of CD4 + lymphocytes of the mucous membrane of the colon was greater in the main group of children than in the control group (p=0.03). An increase in the number of CD4 + cells was registered in 3 of 4 cases of neonatal sepsis. The number of CD8+ and CD20+ lymphocytes in the studied groups was the same (р>0.05). Conclusion. The increase in T-lymphocytes CD4+ in the mucous membrane of the large intestine is probably connected with the antigenic stimulation of opportunistic intestinal bacteria. We found no morphological signs of the suppression of the cells of adaptive immunity associated with the intestinal mucosa.

© 2019 JCBN. Differences in the culturing conditions of mesenchymal stem cells used in regenerative medicine may affect their differentiation ability, genome instability, and therapeutic effects. In particular, bone marrowderived mesenchymal stem cells cultured under hypoxia are known to proliferate while maintaining an undiffer entiated state and the use of deferoxamine, a hypoxia mimetic reagent, has proven to be a suitable strategy to maintain the cells under hypoxic metabolic state. Here, the deferoxamine effects were investigated in mesenchymal stem cells to gain insights into the mechanisms regulating stem cell survival. A 12h deferox amine treatment reduced proliferation, oxygen consumption, mitochondrial activity, and ATP production. Microarray analysis revealed that deferoxamine enhanced the transcription of genes involved in glycolysis and the HIF1α pathway. Among the earliest changes, transcriptional variations were observed in HIF1α, NUPR1, and EGLN, in line with previous reports showing that short deferoxamine treatments induce substantial changes in mesenchymal stem cells glycolysis pathway. NUPR1, which is induced by stress and involved in autophagymediated survival, was upregulated by deferoxamine in a concentrationdependent manner. Consistently, NUPR1 knockdown was found to reduce cell proliferation and increase the proapoptotic effect of stauro sporine, suggesting that deferoxamineinduced NUPR1 promotes mesenchymal stem cell survival and cytoprotective autophagy. Our findings may substantially contribute to improve the effec tiveness of mesenchymal stem cellbased regenerative medicine.

© 2016, Springer Science+Business Media New York.Alcoholic liver disease is a huge medical and social problem that leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Unfortunately, the last stages of the disease do not have efficient treatment, except liver transplantation, and require development of new therapeutical approaches. Transplantation of stem cells might be the most promising approach. In our research, we studied transplantation of autologous hematopoietic stem cells (HSC) into the celiac trunk of patients with alcoholic liver cirrhosis. In this article, we pay particular attention to proliferation and apoptosis—two fundamental processes, which determine the fate of regeneration. Liver biopsy specimens before treatment, 3 and 12 months after transplantation of HSC, were stained immunohistochemically with antibodies against PCNA and Bcl-2. The results showed that treatment was safe and effective, hepatocytes increased proliferation, and inflammatory cells decreased antiapoptotic activity, signifying improvement in liver regeneration. However, effect of treatment after 12 months decreases and requires repeated HSC transplantation.

© 2016, Springer Science+Business Media New York.One the current challenges of modern hepatology is to find new approaches to stimulate liver regeneration and to find new methods for liver disease treatment. Cell therapies, which are based on using regional stem cells for disease treatment, are under active development. However, studies, devoted to their transplantation, are currently scarce. In recent years, hepatic stellate cells are considered to be hepatic stem cells. It is known that activated hepatic stellate cells can transdifferentiate into myofibroblasts and lead to liver fibrosis. The aim of our work was to study the influence of native and activated hepatic stellate cells in vivo by lead nitrate injection after transplantation into partial hepatectomized rats, which is considered to be a classical model to study liver regeneration. Injection of 2-acetylaminofluorene (AAF), which selectively eliminates hepatocyte proliferation, was used to understand the hepatic stellate cells role in liver regeneration process better. Our results suggest that transplanted native and activated hepatic stellate cells can differentiate into hepatocyte-like cells and positively influence liver regeneration without inducing liver fibrosis.

© 2015 John Wiley & Sons, Ltd. Aim: Less invasive therapies using mesenchymal stem cells (MSC) are being developed to treat patients with severe liver cirrhosis. MSC constitute a promising cell source for regenerative therapy and are frequently isolated from bone marrow (BMSC) or adipose tissue (ASC). Therefore, this study assessed the characteristics of these two cell types and their safety for cell infusion. Methods: In vitro, exhaustive genetic analysis was performed using human (h)BMSC and hASC. Subsequently, the expression of mRNA and protein was evaluated. In vivo, mouse (m)BMSC or mASC was infused into serial mice via the peripheral vein, and 24-h survival rate, prothrombin time and cause of death were analyzed. Results: On polymerase chain reaction, western blotting, enzyme-linked immunoassay and fluorescence-activated cell sorting, tissue factor was found to be expressed at higher levels in hASC than in hBMSC. Prothrombin time in mice infused with mASC (>120s) was markedly longer than that of untreated mice (6.5±1.7s) and that of mice infused with BMSC (6.7±0.8s) (P<0.001), indicating that pro-coagulation activity was potently enhanced after ASC infusion. The 24-h survival rates in the mASC- and mBMSC-infused groups were 46.4% (13/28) and 95.5% (21/22), respectively; in the former, the rate decreased with increasing number of infused mASC. This cell number-dependent effect was not observed with mBMSC. A histopathological analysis of mice that died immediately following mASC infusion revealed multiple thrombi in the blood vessels of the lungs. Conclusion: These results indicate that BMSC are a superior and safer cell source for regenerative therapy.

Most of the fundamental studies in the field of developing new methods for treating liver diseases in regenerative medicine are performed with haematopoietic and mesenchymal stem cells. At the same time more and more importance is gaining need for finding new approaches enabling stimulation of regional stem cell compartment and using regional stem cells which are possibly represented by hepatic stellate cells. But studies aimed at their transplantation are rare. Previously, we have established the possibility of these cells to differentiate into hepatocytes after transplantation to rats with partial hepatectomy. In our present research we studied liver regeneration after transplantation of hepatic stellate cells on partial hepatectomy with 2-acetylaminofluorene damage model in rats. 2-acetylaminofluorene blocks hepatocyte proliferation. Results of study confirmed that hepatic stellate cells have the ability to differentiate into hepatocyte and stimulate it's regeneration without the threat of fibrosis after transplantation in rats with partial hepatectomy and administration of 2-acetylaminofluorene.

In 2003, we started autologous bone marrow cell infusion (ABMi) therapy for treating liver cirrhosis. ABMi therapy uses 400 mL of autologous bone marrow obtained under general anesthesia and infused mononuclear cells from the peripheral vein. The clinical study expanded and we treated liver cirrhosis induced by HCV and HBV infection and alcohol consumption. We found that the ABMi therapy was effective for cirrhosis patients and now we are treating patients with combined HIV and HCV infection and with metabolic syndrome-induced liver cirrhosis. Currently, to substantiate our findings that liver cirrhosis can be successfully treated by the ABMi therapy, we are conducting randomized multicenter clinical studies designated "Advanced medical technology B" for HCV-related liver cirrhosis in Japan. On the basis of our clinical study, we developed a proof-of-concept showing that infusion of bone marrow cells (BMCs) improved liver fibrosis and sequentially activated proliferation of hepatic progenitor cells and hepatocytes, further promoting restoration of liver functions. To treat patients with severe forms of liver cirrhosis, we continued translational research to develop less invasive therapies by using mesenchymal stem cells derived from bone marrow. We obtained a small quantity of BMCs under local anesthesia and expanded them into mesenchymal stem cells that will then be used for treating cirrhosis. In this review, we present our strategy to apply the results of our laboratory research to clinical studies. Copyright © 2014, Mary Ann Liebert, Inc.

Evaluation of treatment results of chronic liver diseases should be made on the basis of morphological analysis of liver biopsies. The aim of our study was to investigate the effect of autologous hematopoietic stem cell transplantation on histology activity index and grade of fibrosis in alcoholic liver cirrhosis patients. The study was performed on liver biopsies of 11 patients with alcoholic liver cirrhosis. Biopsies were taken before the injection of autologous peripheral blood stem cells into celiac trunk, 3 and 12 months after the procedure. Liver biopsy specimens were stained with hematoxylin-eosin and Van Gieson's. Results showed improvement of liver structure and decrease in histology activity index in liver biopsies performed 3 and 12 months after transplantation. Our data suggest that autologous transplantation of hematopoietic stem cell in patients with alcoholic liver cirrhosis is effective method that is capable to reduce inflammation activity in the liver, improve its structure and decrease liver fibrogenesis.

Hepatic stellate cells are considered as one of the potential stem cells candidates in the liver. The aim of our work was to study the probability of hepatic stellate cells transplantation to rats after partial hepatectomy, their further homing, the ways of differentiation and hepatocytes repopulation in the recipient liver. For this reason fresh isolated rat's hepatic stellate cells were transplanted into portal vein of intact rats (control group) and rats immediately after partial hepatectomy (experimental group). Before transplantation cells were labeled by adenovirus expressing green fluorescent protein. Our results showed that it was possible to detect 2 types of donor cells in the recipient liver of control and experimental groups:1) hepatocyte-like cells in liver parenchyma;2) small, spindle-shaped, rounded and triangular cells in liver sinusoids and portal areas. Transplantation after partial hepatectomy leads to significant increase of transplanted cells homing and stimulation of their differentiation into hepatocytes. Over the whole experiment there was no hepatic stellate cells transdifferentiation into myofibroblasts, thus there is no risk of liver fibrosis development after this cell type transplantation. In summary hepatic stellate cells after being transplanted are able to differentiate into hepatocytes and do not induce liver fibrosis, that confirms their role in organ regeneration and probable belonging to hepatic progenitor cells.

Nowadays more and more attention is turned to hepatic stellate cells (HSC) and their role in liver regeneration. Nonetheless there are several methodological questions, for example, methods of HSC isolation, their labeling and ways of transplantation. In this work we compared two different methods of HSC isolation: collagenase-pronase liver perfusion with further Histodenz density gradient centrifugation and method of Seglen for isolation of hepatocytes associated with HSC. We also analyzed diverse methods of cells labeling: membrane fluorescent labels PKH26 and with gene of green fluorescent protein (GFP), that could be get into the cells by electroporation, with chemicals like TurboFect or by adenovirus. Then cells were transplanted into rats in two ways: into lien and into system of portal vein. According to our results, we able to conclude that collagenase-pronase liver perfusion with further cells gradient centrifugation in Histodenz is better for HSC isolation than method of Seglen, the most optimal method for cells labeling is with adenovirus, expressing the GFP gene, for HSC transplantation - Transplantation into system of portal vein. © Human stem cells institute, 2013.

Alcoholic liver disease combines various structural and functional impairments of liver caused by excessive alcohol consumption. Alcohol, as a direct hepatotoxic agent, is metabolized in the liver and affects both resident cells and their microenvironment. These changes are reflected in the resulting imbalance of pro- and anti-inflammatory mediators synthesized by the liver macrophages. To date, it is known about the polarization and phenotypic diversity of this cell population, and about macrophages and monocytes involvement in the development of alcoholic hepatitis. These facts allow us to consider macrophages as potential therapeutic targets. However, the available data do not fully disclose the mechanisms of inter- and intradifferon interactions in the human body. The review discusses the results of current studies on the involvement of liver macrophages in the pathomorphogenesis of alcoholic liver disease and the potential for their use in the treatment of this disease.

© 2016, Springer Science+Business Media New York.Alcoholic liver disease is a huge medical and social problem that leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Unfortunately, the last stages of the disease do not have efficient treatment, except liver transplantation, and require development of new therapeutical approaches. Transplantation of stem cells might be the most promising approach. In our research, we studied transplantation of autologous hematopoietic stem cells (HSC) into the celiac trunk of patients with alcoholic liver cirrhosis. In this article, we pay particular attention to proliferation and apoptosis—two fundamental processes, which determine the fate of regeneration. Liver biopsy specimens before treatment, 3 and 12 months after transplantation of HSC, were stained immunohistochemically with antibodies against PCNA and Bcl-2. The results showed that treatment was safe and effective, hepatocytes increased proliferation, and inflammatory cells decreased antiapoptotic activity, signifying improvement in liver regeneration. However, effect of treatment after 12 months decreases and requires repeated HSC transplantation.

© 2016, Springer Science+Business Media New York.One the current challenges of modern hepatology is to find new approaches to stimulate liver regeneration and to find new methods for liver disease treatment. Cell therapies, which are based on using regional stem cells for disease treatment, are under active development. However, studies, devoted to their transplantation, are currently scarce. In recent years, hepatic stellate cells are considered to be hepatic stem cells. It is known that activated hepatic stellate cells can transdifferentiate into myofibroblasts and lead to liver fibrosis. The aim of our work was to study the influence of native and activated hepatic stellate cells in vivo by lead nitrate injection after transplantation into partial hepatectomized rats, which is considered to be a classical model to study liver regeneration. Injection of 2-acetylaminofluorene (AAF), which selectively eliminates hepatocyte proliferation, was used to understand the hepatic stellate cells role in liver regeneration process better. Our results suggest that transplanted native and activated hepatic stellate cells can differentiate into hepatocyte-like cells and positively influence liver regeneration without inducing liver fibrosis.

In 2003, we started autologous bone marrow cell infusion (ABMi) therapy for treating liver cirrhosis. ABMi therapy uses 400 mL of autologous bone marrow obtained under general anesthesia and infused mononuclear cells from the peripheral vein. The clinical study expanded and we treated liver cirrhosis induced by HCV and HBV infection and alcohol consumption. We found that the ABMi therapy was effective for cirrhosis patients and now we are treating patients with combined HIV and HCV infection and with metabolic syndrome-induced liver cirrhosis. Currently, to substantiate our findings that liver cirrhosis can be successfully treated by the ABMi therapy, we are conducting randomized multicenter clinical studies designated "Advanced medical technology B" for HCV-related liver cirrhosis in Japan. On the basis of our clinical study, we developed a proof-of-concept showing that infusion of bone marrow cells (BMCs) improved liver fibrosis and sequentially activated proliferation of hepatic progenitor cells and hepatocytes, further promoting restoration of liver functions. To treat patients with severe forms of liver cirrhosis, we continued translational research to develop less invasive therapies by using mesenchymal stem cells derived from bone marrow. We obtained a small quantity of BMCs under local anesthesia and expanded them into mesenchymal stem cells that will then be used for treating cirrhosis. In this review, we present our strategy to apply the results of our laboratory research to clinical studies. Copyright © 2014, Mary Ann Liebert, Inc.

Most of the fundamental studies in the field of developing new methods for treating liver diseases in regenerative medicine are performed with haematopoietic and mesenchymal stem cells. At the same time more and more importance is gaining need for finding new approaches enabling stimulation of regional stem cell compartment and using regional stem cells which are possibly represented by hepatic stellate cells. But studies aimed at their transplantation are rare. Previously, we have established the possibility of these cells to differentiate into hepatocytes after transplantation to rats with partial hepatectomy. In our present research we studied liver regeneration after transplantation of hepatic stellate cells on partial hepatectomy with 2-acetylaminofluorene damage model in rats. 2-acetylaminofluorene blocks hepatocyte proliferation. Results of study confirmed that hepatic stellate cells have the ability to differentiate into hepatocyte and stimulate it's regeneration without the threat of fibrosis after transplantation in rats with partial hepatectomy and administration of 2-acetylaminofluorene.

Hepatic stellate cells are considered as one of the potential stem cells candidates in the liver. The aim of our work was to study the probability of hepatic stellate cells transplantation to rats after partial hepatectomy, their further homing, the ways of differentiation and hepatocytes repopulation in the recipient liver. For this reason fresh isolated rat's hepatic stellate cells were transplanted into portal vein of intact rats (control group) and rats immediately after partial hepatectomy (experimental group). Before transplantation cells were labeled by adenovirus expressing green fluorescent protein. Our results showed that it was possible to detect 2 types of donor cells in the recipient liver of control and experimental groups:1) hepatocyte-like cells in liver parenchyma;2) small, spindle-shaped, rounded and triangular cells in liver sinusoids and portal areas. Transplantation after partial hepatectomy leads to significant increase of transplanted cells homing and stimulation of their differentiation into hepatocytes. Over the whole experiment there was no hepatic stellate cells transdifferentiation into myofibroblasts, thus there is no risk of liver fibrosis development after this cell type transplantation. In summary hepatic stellate cells after being transplanted are able to differentiate into hepatocytes and do not induce liver fibrosis, that confirms their role in organ regeneration and probable belonging to hepatic progenitor cells.

© 2015 John Wiley & Sons, Ltd. Aim: Less invasive therapies using mesenchymal stem cells (MSC) are being developed to treat patients with severe liver cirrhosis. MSC constitute a promising cell source for regenerative therapy and are frequently isolated from bone marrow (BMSC) or adipose tissue (ASC). Therefore, this study assessed the characteristics of these two cell types and their safety for cell infusion. Methods: In vitro, exhaustive genetic analysis was performed using human (h)BMSC and hASC. Subsequently, the expression of mRNA and protein was evaluated. In vivo, mouse (m)BMSC or mASC was infused into serial mice via the peripheral vein, and 24-h survival rate, prothrombin time and cause of death were analyzed. Results: On polymerase chain reaction, western blotting, enzyme-linked immunoassay and fluorescence-activated cell sorting, tissue factor was found to be expressed at higher levels in hASC than in hBMSC. Prothrombin time in mice infused with mASC (>120s) was markedly longer than that of untreated mice (6.5±1.7s) and that of mice infused with BMSC (6.7±0.8s) (P<0.001), indicating that pro-coagulation activity was potently enhanced after ASC infusion. The 24-h survival rates in the mASC- and mBMSC-infused groups were 46.4% (13/28) and 95.5% (21/22), respectively; in the former, the rate decreased with increasing number of infused mASC. This cell number-dependent effect was not observed with mBMSC. A histopathological analysis of mice that died immediately following mASC infusion revealed multiple thrombi in the blood vessels of the lungs. Conclusion: These results indicate that BMSC are a superior and safer cell source for regenerative therapy.

Nowadays more and more attention is turned to hepatic stellate cells (HSC) and their role in liver regeneration. Nonetheless there are several methodological questions, for example, methods of HSC isolation, their labeling and ways of transplantation. In this work we compared two different methods of HSC isolation: collagenase-pronase liver perfusion with further Histodenz density gradient centrifugation and method of Seglen for isolation of hepatocytes associated with HSC. We also analyzed diverse methods of cells labeling: membrane fluorescent labels PKH26 and with gene of green fluorescent protein (GFP), that could be get into the cells by electroporation, with chemicals like TurboFect or by adenovirus. Then cells were transplanted into rats in two ways: into lien and into system of portal vein. According to our results, we able to conclude that collagenase-pronase liver perfusion with further cells gradient centrifugation in Histodenz is better for HSC isolation than method of Seglen, the most optimal method for cells labeling is with adenovirus, expressing the GFP gene, for HSC transplantation - Transplantation into system of portal vein. © Human stem cells institute, 2013.

Evaluation of treatment results of chronic liver diseases should be made on the basis of morphological analysis of liver biopsies. The aim of our study was to investigate the effect of autologous hematopoietic stem cell transplantation on histology activity index and grade of fibrosis in alcoholic liver cirrhosis patients. The study was performed on liver biopsies of 11 patients with alcoholic liver cirrhosis. Biopsies were taken before the injection of autologous peripheral blood stem cells into celiac trunk, 3 and 12 months after the procedure. Liver biopsy specimens were stained with hematoxylin-eosin and Van Gieson's. Results showed improvement of liver structure and decrease in histology activity index in liver biopsies performed 3 and 12 months after transplantation. Our data suggest that autologous transplantation of hematopoietic stem cell in patients with alcoholic liver cirrhosis is effective method that is capable to reduce inflammation activity in the liver, improve its structure and decrease liver fibrogenesis.