Liver stem cells (LSCs) are precursors of hepatoblasts, which are presumed to be the transit-amplifying cells that first give rise to committed progenitors, and then to hepatocytes and cholangiocytes. Liver stem/progenitor cells appear and undergo a massive expansion in chronic liver damage and become cancer stem cells (CSC) to further initiate tumor formation. These cells are also known to be responsible for tumor relapse, metastasis, and chemoresistance in liver cancer. Therefore, understanding how they form, how they are sustained and how to defeat them is among the most intensive areas of cancer research today. Additionally, liver stem/ progenitor cells are candidates for ex-vivo liver cell replacement as an alternative method to orthologous liver transplantation in the treatment of end-stage liver pathologies. Recent advances allow us to obtain patient’s hepatocytes derived from inducible pluripotent stem cells (iPSCs) reprogrammed from his own fibroblast and use them for tissue replacement and gene therapy. The first step of hepatic development from iPSCs is the induction of definitive endoderm containing liver stem/progenitor cells by using chemicals. Further treatment with some growth factors can then direct cells towards the hepatic lineage. Even if the limitations of technology still remain (i.e., the potential for teratoma formation and low efficiency), iPS-derived hepatocytes are a very promising population for cell therapies in hepatology.RESEARCH INTERESTS
Our research concentrates on two main themes: 1.Understanding which and how molecular signaling events regulate the expansion and activation of EpCAM+/ CD133+ subset of liver stem/progenitor cells in the development of Hepatocellular Carcinoma (HCC), 2. Developing a method to produce highly pure, functional and fully-differentiated hepatocytes from non-transgenic iPSCs to be potentially used for cell-based therapies.
1. Hepatic Stem Cells in HCC: Here, we have two main lines of research First; we investigate the importance of pluripotency genes on the stem cell phenotype in HCC. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology; because the effects of such reprogramming may remain dormant until engaged later in response to (epi) genetic and/or (micro) environmental events. To test this hypothesis, we utilized an in vitro model of Oct4, Sox-2, KLF4 and c-myc (OSKM) overexpressing cancer stem cell (CSC)-like cellular state in HuH7 cell line. After overexpressing OSKM genes in non-hepatic stem cell population (EpCAM-/CD133- cells) we analyze cell behaviors such as proliferation, migration, EMT and in vivo tumor formation.
Secondly, we aim to understand the role of Polycomb repressive complex 2 (PRC2) on the expansion and activation of the EpCAM+/CD133+ subset of liver stem/progenitor cells in HCC.PRC2 has a crucial role in epigenetic gene silencing and regulation of developmental pathways. EZH2 encodes histone methyltransferase enzyme as the catalytic component of PRC2, creating H2K27Me3 histone mark which results in transcriptional silencing. EZH2 is overexpressed in HCC and mostly associated with the progression and aggressiveness of HCC. Our goal is to identify transcriptomal changes regulating H3K27Me3 mark on the liver cancer stem cells.
2. Stem Cell Based Therapies for Liver Diseases: Differentiated hepatocytes produced from a patient’s inducible pluripotent stem cells (iPSCs) have many potential therapeutic applications, including their use in tissue replacement and gene therapy as well as disease modeling. In our lab, disease-specific iPSC colonies have been reprogrammed chemically from skin fibroblasts isolated from urea cycle disease (Citrullinemia) patients, then produce iPSC-derived hepatocytes by optimized differentiation protocols. Further, a functional copy of the mutant gene will be transferred into such hepatocytes demonstrating proof of principle of an ex vivo gene therapy approach. Lastly, the corrected hepatocytes will be functionally validated in an in-vivo cell transplantation model.RESEARCH HIGHLIGHTS
Liver stem/progenitor cells recently defined as sources of cancer stem cells responsible tumor initiation, relapse and resistance to chemotherapy in hepatocellular carcinoma. However, the mechanisms regulating activation and expansion of liver cancer stem cells are still not known enough. We have shown that KLF4, one of reprogramming genes have role in controlling CSC subset leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis in HCC. So, our research will define novel therapeutic targets for the treatment of progression and metastasis of HCC with CSC-like signatures and epithelial-mesenchymal transition phenotype. Furthermore, liver stem cells/progenitor cells can be obtained during endoderm differentiation in vitro from iPSC colonies and can be used to direct differentiation of fully functional hepatocytes. To use iPSC-driven hepatocyte strategy for cellular therapies in end-stage liver diseases still we need more improvements in protocols. The second part of our research we previously found that R-spondin, the ligand of LGR5 induces expansion of EpCAM+ cell population and amplify significantly Wnt3a induced Tcf/Lef transcriptional activity in HCC cell line. Hence, we hypothesized that R-spondin may also enhance endoderm differentiation from iPSC colonies and provide more liver stem/progenitor cells in number.