Prakash Baligar

Hepatocyte transplantation from stem cells holds a promising approach for treating liver failure, but inefficient differentiation into functional hepatocytes limits therapeutic applications. Recent research highlights the role of non-parenchymal cells, such as hepatic stellate cells (HSCs) and human umbilical vein endothelial cells (HUVECs), in supporting hepatocyte function through paracrine signaling. This study investigates the synergistic effects of HUVEC and HSC-conditioned media on the differentiation of mouse embryonic stem cells (mESCs) into hepatocyte-like cells (HLCs) by using a 3D gelatin scaffold. mESCs were cultured on a gelatin scaffold with conditioned media from HUVECs and HSCs (1:1:1), and differentiation was assessed through immunohistochemistry and gene expression analysis. Results showed that mouse embryonic stem cell-derived hepatocyte-like cells (mESC-HLCs) cultured with conditioned media exhibited increased expression of mature hepatic markers, including albumin, cytokeratin 18, and hepatocyte nuclear factor 4α, alongside markers of hepatoblasts and cholangiocytes. Functional assays demonstrated enhanced albumin secretion, creatinine production, and urea synthesis compared to cells treated with single conditioned media or standard differentiation media. These findings suggest that combining scaffold-based culture with niche-mimicking conditioned media effectively enhances hepatocyte differentiation, providing a promising approach for regenerative medicine and drug discovery applications.

Breast cancer (BCa) is marked by uncontrolled cell division, causing the reactivation of the multi-unit ribonucleoprotein telomerase enzyme by expression of the catalytic telomerase reverse transcriptase (hTERT) subunit. Targeting hTERT can therefore offer an advantage in BCa management. Medicinal plants such as Hydrastis canadensis, used in homeopathy has therefore been explored for BCa treatment. Hydrastis canadensis mother tincture (MT) showed the presence of phytoactive components exhibiting anti-cancerous potential. The components were identified by GC-MS analysis and 45 phytochemicals were then screened using in-silico tools. The protein targets of the active components were predicted by submitting the Simplified Molecular Input Line Entry System (SMILES) of the compounds to SwissTargetPrediction, giving active components targeting the hTERT protein. Molecular docking studies were performed on AutoDock 4.2 and Molecular Dynamics (MD) simulation studies on GROningen MAchine for Chemical Simulations (GROMACS) 2023.2. The MM/PBSA calculations were performed with the gmxMMPBSA package. The data was then validated through wet lab experiments, Linoleic acid and Gadoleic acid, as modulators of human telomerase reverse transcriptase (hTERT) were identified, influencing cancer cell survival. The results highlighted the mechanistic role of Hydrastis candensis to target the key oncogenic protein hTERT operative in breast cancer with potential therapeutic applications in cancer treatment.

Stem cell-based tissue engineering is an emerging tool for developing functional tissues of choice. To understand pluripotency and hepatic differentiation of mouse embryonic stem cells (mESCs) on a three-dimensional (3D) scaffold, we established an efficient approach for generating hepatocyte-like cells (HLCs) from hepatoblast cells. We developed porous and biodegradable scaffold, which was stimulated with exogenous growth factors and investigated stemness and differentiation capacity of mESCs into HLCs on the scaffold in-vitro. In animal studies, we had cultured mESCs-derived hepatoblast-like cells on the scaffold and then, transplanted them into the partially hepatectomized C57BL/6 male mice model to evaluate the effect of gelatin scaffold on hepatic regeneration. The 3D culture system allowed maintenance of stemness properties in mESCs. The step-wise induction of mESCs with differentiation factors leads to the formation of HLCs and expressed liver-specific genes, including albumin, hepatocyte nucleic factor 4 alpha, and cytokeratin 18. In addition, cells also expressed Ki67, indicating cells are proliferating. The secretome showed expression of albumin, urea, creatinine, alanine transaminase, and aspartate aminotransferase. However, the volume of the excised liver which aids regeneration has not been studied. Our results indicate that hepatoblast cells on the scaffold implanted in PH mouse indicates that these cells efficiently differentiate into HLCs and cholangiocytes, forming hepatic lobules with central and portal veins, and bile duct-like structures with neovascularization. The gelatin scaffold provides an efficient microenvironment for liver differentiation and regeneration both in-vitro and in-vivo. These hepatoblasts cells would be a valuable source for 3D liver tissue engineering/transplantation in liver diseases.

Stem cell based toxicity prediction plays a very important role in the development of the drug. Unexpected adverse effects of the drugs during clinical trials are a major reason for the termination or withdrawal of drugs. Methods for predicting toxicity employ in vitro as well as in vivo models; however, the major drawback seen in the data derived from these animal models is the lack of extrapolation, owing to interspecies variations. Due to these limitations, researchers have been striving to develop more robust drug screening platforms based on stem cells. The application of stem cells based toxicity testing has opened up robust methods to study the impact of new chemical entities on not only specific cell types, but also organs. Pluripotent stem cells, as well as cells derived from them, can be evaluated for modulation of cell function in response to drugs. Moreover, the combination of state-of-the -art techniques such as tissue engineering and microfluidics to fabricate organ- on-a-chip, has led to assays which are amenable to high throughput screening to understand the adverse and toxic effects of chemicals and drugs. This review summarizes the important aspects of the establishment of the embryonic stem cell test (EST), use of stem cells, pluripotent, induced pluripotent stem cells and organoids for toxicity prediction and drug development.

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.

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. These results suggest that BM stem cell transplantation may be a promising therapy for AATD-related liver disease. (Hepatology 2017;65:1319-1335).

Therapeutic potential of bone marrow (BM)-derived cells in acute and chronic liver injury model has been investigated. Apart from pathological improvement of the liver, we also studied liver regeneration and genetic alteration in donor cells towards neoplasia. Acetaminophen-induced acute and CCl4-induced chronic liver injured male mice were transplanted with BM-derived uncommitted and CD45+ cells, respectively from female mice. Transplantation of CD45 cells resulted in enhanced recovery of fibrotic liver during initial pro-fibrogenic condition as compared with sham control. Significant recovery from apoptotic death of hepatocytes was also observed. After 3 months of transplantation, donor-derived hepatocytes in acute and chronic liver injury models were found to be about 5% and 17%, respectively.