Prakash Baligar

Bone marrow stem cell therapy partially ameliorates pathological consequences in livers of mice expressing mutant human α1‐antitrypsin

Alpha‐1‐antitrypsin (AAT) deficiency (AATD) is a genetic disease, caused by mutation of the AAT gene. Accumulation of mutated AAT protein aggregates in hepatocytes leads to endoplasmic reticulum stress, resulting in impairment of liver functions and, in some cases, hepatocellular carcinoma, whereas decline of AAT levels in sera is responsible for pulmonary emphysema. In advanced liver disease, the only option for treatment is liver transplantation, whereas AAT replacement therapy is therapeutic for emphysema. Given that hepatocytes are the primary affected cells in AATD, we investigated whether transplantation of bone marrow (BM)‐derived stem cells in transgenic mice expressing human AATZ (the Z variant of AAT) confers any competitive advantages compared to host cells that could lead to pathological improvement. Mouse BM progenitors and human mesenchymal stem cells (MSCs) appeared to contribute in replacement of 40% and 13% host hepatocytes, respectively. Transplantation of cells resulted in decline of globule‐containing hepatocytes, improvement in proliferation of globule‐devoid hepatocytes from the host‐derived hepatocytes, and apparently, donor‐derived cells. Further analyses revealed that transplantation partially improves liver pathology as reflected by inflammatory response, fibrosis, and apoptotic death of hepatocytes. Cell therapy was also found to improve liver glycogen storage and sera glucose level in mice expressing human AATZ mice. These overall improvements in liver pathology were not restricted to transplantation of mouse BM cells. Preliminary results also showed that following transplantation of human BM‐derived MSCs, globule‐containing hepatocytes declined and donor‐derived cells expressed human AAT protein. Conclusion: These results suggest that BM stem cell transplantation may be a promising therapy for AATD‐related liver disease. (Hepatology 2017;65:1319‐1335).

JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule GSK-J4. Our results propose a potential role of JMJD3 in lineage conversion of BM cells into hepatic lineage.

Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells

IntroductionCell replacement therapy may be considered as an alternate approach to provide therapeutic dose of plasma factor VIII (FVIII) in patients with hemophilia A (HA). However, immune rejection limits the use of allogeneic cells in this mode of therapy. Here, we have examined the role of donor major histocompatibility complex (MHC)-stimulated host CD4+CD25+ regulatory T (Treg) cells in suppressing immune responses against allogeneic uncommitted (Lin−) bone marrow cells (BMCs) for correction of bleeding disorder in HA mice.MethodsAllogeneic donor Lin− BMCs were co-transplanted with allo-antigen sensitized Treg cells in HA mice having acetaminophen-induced acute liver injury. Plasma FVIII activity was determined by in vitro functional assay, and correction of bleeding phenotype was assessed on the basis of capillary blood clotting time and tail-clip challenge. The immunosuppression potential of the sensitized Treg cells on CD4+ T cells was studied both in vitro and in vivo. Suppression of inflammatory reactions in the liver against the homed donor cells by sensitized Treg cells was analysed by histopathological scoring. Allo-specificity of sensitized Treg cells and long-term retention of immunosuppression were examined against a third-party donor and by secondary challenge of allogeneic donor cells, respectively. The engraftment and phenotype change of donor BMCs in the liver and their role in synthesis of FVIII and liver regeneration were also determined.ResultsCo-transplantation of allogeneic Lin− BMCs with sensitized Treg cells led to systemic immune modulation and suppression of inflammatory reactions in the liver, allowing better engraftment of allogeneic cells in the liver. Allo-antigen priming led to allo-specific immune suppression even after 1 year of transplantation. Donor-derived endothelial cells expressed FVIII in HA mice, leading to the correction of bleeding phenotype. Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury.ConclusionsA highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin− BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells. This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.

Bone marrow stem-cell therapy for genetic and chronic liver diseases

There are no permanent remedies for patients suffering from genetic liver diseases (GLDs) and liver cirrhosis (LC). In such cases, liver transplantation has resulted in improved quality of life, but it is not affordable by most patients. Therefore, a cost-effective, safe, and permanent cure for these diseases is desirable. Cell therapy seems an encouraging option for treatment of these liver diseases in the future. Animal experiments and clinical studies have demonstrated that, depending on the nature of the liver disease and the patient, autologous and/or allogeneic bone marrow (BM)-derived stem-cell therapy could be a promising treatment option. Although no clinical trials using BM-derived stem cells for treatment of GLD have yet been conducted, many phase I clinical trials have been conducted and a few such trials for the treatment of LC by use of autologous and/or allogeneic cells are in progress. Overall, the results of these trials are indicative of clinical benefits with no adverse effect on the patients. This review focuses on the current status of BM stem-cell therapy for treatment of GLD and LC in experimental animals and human subjects. It also proposes safer approaches to immune-regulation in allogeneic transplantation of cells.

Human kidney stone matrix: Latent potential to restrain COM induced cytotoxicity and inflammatory response

Kidney stone disease is a multi-factorial disorder resulting from the interplay of various risk factors including lifestyle, environment and genetics along with metabolic activities inside the body. However, it is difficult to determine how these factors converge to promote stone disease. Extensive investigations of kidney stone composition at the molecular level have been carried out however; its impact on the complex mechanism of stone formation is still obscure. Hence, an in vitro study was designed to investigate the attenuation of calcium oxalate toxicity by human kidney stone matrix proteins on NRK-52E cells using flowcytometry, Western blotting, RT-PCR and immunofluorescence assays. Morphological alterations in cell-crystal interaction were assessed using scanning electron microscopy. Microscopic studies showed profound impairment of COM crystal structure as a consequence of protein-crystal interactions. RT-PCR analysis and immunocytochemistry of NRK-52E cells revealed the up-regulation of inflammatory and stress biomarkers OPN and HSP-70, respectively, in response to COM toxicity; which diminished significantly in the presence of kidney stone matrix proteins. The results of present study propose that the mechanism undertaken by matrix proteins to attenuate COM induced cytotoxicity could be attributed to the modulation of crystal structure, which subsequently restraint the inflammatory response and apoptotic cell death. The inference drawn through this study could provide better understanding of the intricate process of kidney stone formation.

Role of antigen presenting cell invariant chain in the development of hepatic steatosis in mouse model

The role of Invariant chain (CD74 or Ii) in antigen presentation via Antigen Presenting Cells (APC), macrophage recruitment as well as survival, T cell activation and B cell differentiation has been well recognized. However, the aspect of CD74 which is involved in the development of hepatic steatosis and the pathways through which it acts remain to be studied. In this study, we investigated the role of CD74 in the inflammatory pathway and its contribution to development of hepatic steatosis. For this, wild type C57BL/6J and CD74 deficient mice (Ii(-/-) mice) were fed with high fat high fructose (HFHF) diet for 12 weeks. Chronic consumption of this feed did not develop hepatic steatosis, glucose intolerance or change in the level of immune cells in Ii(-/-) mice. Moreover, there was relatively delayed expression of genes involved in development of non alcoholic fatty liver disease (NAFLD) in HFHF fed Ii(-/-) mice as compared to that of C57BL/6J phenotype. Taken together, the data suggest that HFHF diet fed Ii(-/-) mice fail to develop hepatic steatosis, suggesting that Ii mediated pathways play a vital role in the initiation and propagation of liver inflammation.

Therapeutic Potential of Bone Marrow-Derived Stem Cells in Treatment of Genetic Diseases of the Liver

Genetic liver diseases are the greatest challenge of modern health care that have no proven treatment. End-stage patients are advised for transplantation of liver. Unlike kidney, donor liver is difficult to obtain. Therefore, an alternate route of generating hepatocytes has been sought. BM-derived stem cells have been shown to differentiate into hepatocytes in culture and in vivo. It has been proposed that small increase in missing protein may cause significant improvement in the pathology of genetic liver diseases. Experimental findings from our and other groups heighten the scope for exploitation of this novel phenomenon of adult stem cells plasticity for the treatment of such disease. It has been presumed that BM-stem cell therapy not only helps for the synthesis of missing protein, it may protect liver from hepatocellular carcinoma or other adverse pathological changes of the liver.