3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFβRII-SMADS pathway.

Background: Exosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture.

Objectives: For clinical applications of cell-based therapies, a large quantity of human pluripotent stem cells (hPSCs) produced in standardized and scalable culture processes is required. Currently, microcarrier-free suspension culture shows potential for large-scale expansion of hPSCs; however, hPSCs tend to aggregate during culturing leading to a negative effect on cell yield. To overcome this problem, we developed a novel protocol to effectively control the sizes of cell aggregates and enhance the cell proliferation during the expansion of hPSCs in suspension.

Salvianolic Acid B Enhances Hepatic Differentiation of Human Embryonic Stem Cells Through Upregulation of WNT Pathway and Inhibition of Notch Pathway.

Hepatocytes differentiated from human embryonic stem cells (ESCs) could provide a powerful tool for enabling cell-based therapies, studying the mechanisms underlying human liver development and disease, and testing the efficacy and safety of pharmaceuticals. However, currently most in vitro protocols yield hepatocytes with low levels of liver function. In this study, we investigated the potential of Salvianolic acid B (Sal B), an active pharmaceutical compound present in Salvia miltiorrhiza, which has been shown to have an antifibrotic effect in previous studies, to enhance hepatocyte differentiation from human ESCs.

The diversity and plasticity of adult hepatic progenitor cells and their niche.

The liver is a unique organ for homoeostasis with regenerative capacities. Hepatocytes possess a remarkable capacity to proliferate upon injury; however, in more severe scenarios liver regeneration is believed to arise from at least one, if not several facultative hepatic progenitor cell compartments. Newly identified pericentral stem/progenitor cells residing around the central vein is responsible for maintaining hepatocyte homoeostasis in the uninjured liver.

Enhancement of hepatocyte differentiation from human embryonic stem cells by Chinese medicine Fuzhenghuayu.

Chinese medicine, Fuzhenghuayu (FZHY), appears to prevent fibrosis progression and improve liver function in humans. Here we found that FZHY enhanced hepatocyte differentiation from human embryonic stem cells (hESC). After treatment with FZHY, albumin expression was consistently increased during differentiation and maturation process, and expression of metabolizing enzymes and transporter were also increased.

Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

Although hepatic progenitor cells (HPCs) are known to contribute to cholestatic liver fibrosis (CLF), how Notch signaling modulates the differentiation of HPCs to cholangiocytes in CLF is unknown. Thus, using a rat model of CLF that is induced by bile duct ligation, we inhibited Notch signaling with DAPT. In vivo, CK19, OV6, Sox9, and EpCAM expression was increased significantly.

CD34(+) Liver Cancer Stem Cells Were Formed by Fusion of Hepatobiliary Stem/Progenitor Cells with Hematopoietic Precursor-Derived Myeloid Intermediates.

A large number of cancer stem cells (CSCs) were identified and characterized; however, the origins and formation of CSCs remain elusive. In this study, we examined the origination of the newly identified CD34(+) liver CSC (LCSC). We found that CD34(+) LCSC coexpressed liver stem cell and myelomonocytic cell markers, showing a mixed phenotype, a combination of hepatobiliary stem/progenitor cells (HSPCs) and myelomonocytic cells.

Clonogenically Culturing and Expanding CD34+ Liver Cancer Stem Cells in Vitro.

A large number of cancer stem cells (CSCs) have been isolated and identified; however, none has been cultured in an unlimited manner in vitro without losing tumorigenicity and multipotency. In this study, we successfully clonogenically cultured a newly identified CD34+ liver CSC (LCSC) on feeder cells up to 22 passages (to date) without losing CSC property. Cloned CD34+ LCSC formed a round packed morphology and it could also be cryopreserved and recultured.

Identification of cancer stem cell subpopulations of CD34(+) PLC/PRF/5 that result in three types of human liver carcinomas.

CD34(+) stem cells play an important role during liver development and regeneration. Thus, we hypothesized that some human liver carcinomas (HLCs) might be derived from transformed CD34(+) stem cells. Here, we determined that a population of CD34(+) cells isolated from PLC/PRF/5 hepatoma cells (PLC) appears to function as liver cancer stem cells (LCSCs) by forming HLCs in immunodeficient mice with as few as 100 cells.

Ethanol negatively regulates hepatic differentiation of hESC by inhibition of the MAPK/ERK signaling pathway in vitro.

Background: Alcohol insult triggers complex events in the liver, promoting fibrogenic/inflammatory signals and in more advanced cases, aberrant matrix deposition. It is well accepted that the regenerative capacity of the adult liver is impaired during alcohol injury. The liver progenitor/stem cells have been shown to play an important role in liver regeneration -in response to various chronic injuries; however, the effects of alcohol on stem cell differentiation in the liver are not well understood.

Hepatoma SK Hep-1 cells exhibit characteristics of oncogenic mesenchymal stem cells with highly metastatic capacity.

Background: SK Hep-1 cells (SK cells) derived from a patient with liver adenocarcinoma have been considered a human hepatoma cell line with mesenchymal origin characteristics, however, SK cells do not express liver genes and exhibit liver function, thus, we hypothesized whether mesenchymal cells might contribute to human liver primary cancers. Here, we characterized SK cells and its tumourigenicity.

Methods And Principal Findings: We found that classical mesenchymal stem cell (MSC) markers were presented on SK cells, but endothelial marker CD31, hematopoietic markers CD34 and CD45 were negative.

Insulin and IGFs enhance hepatocyte differentiation from human embryonic stem cells via the PI3K/AKT pathway.

Human embryonic stem cells (hESCs) can be progressively differentiated into definitive endoderm (DE), hepatic progenitors, and hepatocytes, and thus provide an excellent model system for the mechanistic study of hepatocyte differentiation, which is currently poorly understood. Here, we found that insulin enhanced hepatocyte differentiation from hESC-derived DE. Insulin activated the PI3K/AKT pathway, but not the mitogen-activated protein kinase pathway in the DE cells, and inhibition of the PI3K/AKT pathways by inhibitors markedly inhibited hepatocyte differentiation.

Highly efficient differentiation of functional hepatocytes from human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters.

Hepatic differentiation of human embryonic stem cells on growth factor-containing surfaces.

Embryonic stem cells (ESCs) hold considerable promise in tissue engineering and regenerative medicine as a source of tissue-specific cells. Hepatocytes derived from ESCs will be useful for therapies, bioartificial liver assistance devices and drug discovery. In traditional stem cell cultivation/differentiation experiments, growth factors (GFs) are added in soluble form in order to provide signals for tissue-specific differentiation.

Decellularized liver matrix as a carrier for the transplantation of human fetal and primary hepatocytes in mice.

The transplantation of primary hepatocytes has been shown to augment the function of damaged livers and to bridge patients to liver transplantation. However, primary hepatocytes often have low levels of engraftment and survive for only a short time after transplantation. To explore the potential benefits of using decellularized liver matrix (DLM) as a carrier for hepatocyte transplantation, DLM from whole mouse livers was generated.

Bottom-up signaling from HGF-containing surfaces promotes hepatic differentiation of mesenchymal stem cells.

The capacity of stem cells to differentiate into specific cell types makes them very promising in tissue regeneration and repair. However, realizing this promise requires novel methods for guiding lineage-specific differentiation of stem cells. In this study, hepatocyte growth factor (HGF), an important morphogen in liver development, was co-printed with collagen I (Col) to create arrays of protein spots on glass.

New approaches in the differentiation of human embryonic stem cells and induced pluripotent stem cells toward hepatocytes.

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem.

Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations.

Background & Aims: Our previous studies showed that CD133, EpCAM, and aldehyde dehydrogenase (ALDH) are useful markers to identify cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) tissues. The present study aims to evaluate chemosensitivity and invasion capability of HCC based on CSC marker profiles, and to explore the underlying molecular mechanisms.

Methods: Hepatoma cell lines were separated into subpopulations according to CD133, EpCAM, and ALDH expression profiles.

Label-free imaging and analysis of the effects of lipolysis products on primary hepatocytes.

The increased accumulation of intracellular lipid droplets within hepatocytes is a pathologic hallmark of liver injury of various etiologies, especially non-alcoholic steatohepatitis (NASH). The dynamics, subcellular origin, and chemical composition of lipid droplets under various pathophysiologic conditions, however, remain poorly understood. We used coherent Raman microscopy and spontaneous Raman spectroscopy to monitor and analyze the formation of lipid droplets in living primary rat hepatocytes exposed to triglyceride-rich lipoprotein (TGRL) lipolysis products.

Using growth factor arrays and micropatterned co-cultures to induce hepatic differentiation of embryonic stem cells.

The success in driving embryonic stem cells towards hepatic lineage has been confounded by the complexity and cost of differentiation protocols that employ large quantities of expensive growth factors (GFs). Instead of supplementing culture media with soluble GFs, we investigated cultivation and differentiation of mouse embryonic stem cells (mESCs) on printed arrays of GFs. Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP4) were mixed in solution with fibronectin and collagen (I) and then printed onto silane-modified glass slides to form 500 μm diameter protein spots.

Immunohistochemical staining of cancer stem cell markers in hepatocellular carcinoma.

Background: Cancer stem cells (CSCs) are thought to be a critical subpopulation in tumor development, progression, metastasis and recurrence, and the identification of these cells is an initial step in understanding their role in oncogenesis and in seeking valuable markers for diagnosis or development of targeting therapeutics.

Aims: To identify CSCs in hepatocellular carcinoma (HCC) specimens and define their tissue specificity.

Methods: Immunohistochemical staining of CSC markers: CD44, CD90, CD133 and aldehyde dehydrogenase (ALDH) was performed in 25 HCC specimens, 4 hepatoblastomas, 8 peri-malignant tissues, and 19 cases of viral hepatitis.

Cultivating hepatocytes on printed arrays of HGF and BMP7 to characterize protective effects of these growth factors during in vitro alcohol injury.

The goal of the present study was to investigate hepato-protective effects of growth factor (GF) arrays during alcohol injury. Hepatocyte growth factor (HGF) and bone morphogenetic protein (BMP)7 were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 500 microm diameter spots. Primary rat hepatocytes were seeded on top of the arrays forming clusters corresponding in size to the underlying protein spots.

Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells.

Human embryonic stem cells (hESCs) may provide a cell source for functional hepatocytes for clinical applications and drug development. Initially, the hESC population was enriched to be more than 85% definitive endoderm (DE) as assessed by the expression of CXCR4, SOX17, and FOXA2. We then successfully converted DE into hepatic progenitors with 93% of the cells being positive for alpha-feto protein within 9 days.