Long-term maintenance of functional primary human hepatocytes using small molecules
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE135011 | GEO | 2019/09/01
REPOSITORIES: GEO
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Project description:Currently, much effort is directed to the development of new cell sources for clinical therapy using cell fate conversion approaches by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by two small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of fate conversion process. Based on functional evidence, activation of HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through activation of ERK pathway. hCdHs expressed hepatic progenitor marker genes and proteins, and could self-renew for at least 10 passages while retaining normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. RNASeq gene expression profiling confirmed transcriptional reprogramming of hCdHs toward a progenitor state and suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation into immunocompromised mice with acute liver injury, hCdHs effectively repopulated damaged parenchyma. Our study is a first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a nove tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and repair of diseased liver.
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Project description:Administration of soluble molecules to induce endogenous adult liver progenitor-like cells may provide a clinically and effectively applicable approach for liver damage repair. Here, we report that HGF in combination with a cocktail of small molecules Y-27632, A-83-01, and CHIR99021 (HACY), which we have previously defined to convert mature hepatocytes (MHs) to liver progenitor-like cells (HepLPCs) in vitro, could induce the replenishment of endogenous HepLPCs and relieve chronic liver dysfunction during persistent injury. The liver progenitor cells surface marker, CD24, was found to be highly expressed in HepLPCs in vitro. Similarly, injection of HACY into mice with carbon tetrachloride (CCL4) treatment also induced conversion of mature hepatocytes to CD24+ progenitor-like cells in vivo, leading to the attenuation of liver fibrosis. Compared to the liver progenitor cells (LPCs) derived from nonparenchymal cells, HACY-induced CD24+ HepLPCs retained an increased hepatic function by RNA sequencing analysis and could promote restoration of liver function by replacing CCL4-impaired liver cells during chronic hepatic damages. And CD24+ liver stem cells could also be observed in human liver fibrosis tissues and expanded in 3D hepatic spheroid in the present of HACY. Together, the results of our study indicated that injection of a cocktail of HACY might be a valuable multiple targets therapy for patients with chronic liver fibrotic pathologies.
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Project description:Mesenchymal stem/stromal cells (MSCs) are multipotent cells with favorable properties for cell therapy and regenerative medicine. Human endometrium harbors a small population of perivascular, clonogenic MSCs (eMSCs) identified by the SUSD2 marker. As for other MSCs, eMSCs require extensive in vitro expansion to generate clinically relevant numbers of cells, resulting in spontaneous differentiation, replicative senescence, and cell death, decreasing therapeutic potency. We previously demonstrated that A83-01, a TGF-b receptor inhibitor maintained eMSCs clonogenicity, promoted proliferation, prevented apoptosis and maintained MSC function in vitro. Here we compare the transcriptome of passaged eMSCs from six women cultured with and without A83-01 for 7 days. We identified 1206 differentially expressed genes using a false discovery rate cut-off at 0.01 and fold change >2. Significant enrichment of genes involved in inflammatory responses, angiogenesis, cell migration and proliferation, and reduced smooth muscle proliferation, collagen fibril and extracellular matrix organization genes were revealed. TGF-b, Wnt and Akt signaling pathways were decreased. Anti-fibrotic and anti-apoptotic genes were induced, and fibroblast proliferation and myofibroblast related genes were down-regulated. We validated the enhanced potency of A83-01-treated eMSCs and found increased MSC potency genes (TWIST1, TWIST2, JAG1, LIFR, and SLIT2) and no pluripotency gene expression. Increased angiogenic capacity was functionally demonstrated in vitro, and our angiogenic and cytokine protein arrays further confirmed the angiogenic, antifibrotic and immunomodulatory phenotype of A83-01-treated eMSCs. Overall, we showed that eMSCs culture expanded with A83-01 have improved potential for cell-therapies and regenerative medicine applications.
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