Embryonic Stem Cell-derived Hepatocyte-like Cells Research Articles

Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

Enhanced antioxidant capacity of dental pulp-derived iPSC-differentiated hepatocytes and liver regeneration by injectable HGF-releasing hydrogel in fulminant hepatic failure.

Acute hepatic failure (AHF) is a severe liver injury leading to sustained damage and complications. Induced pluripotent stem cells (iPSCs) may be an alternative option for the treatment of AHF. In this study, we reprogrammed human dental pulp-derived fibroblasts into iPSCs, which exhibited pluripotency and the capacity to differentiate into tridermal lineages, including hepatocyte-like cells (iPSC-Heps). These iPSC-Heps resembled human embryonic stem cell-derived hepatocyte-like cells in gene signature and hepatic markers/functions. To improve iPSC-Heps engraftment, we next developed an injectable carboxymethyl-hexanoyl chitosan hydrogel (CHC) with sustained hepatocyte growth factor (HGF) release (HGF-CHC) and investigated the hepatoprotective activity of HGF-CHC-delivered iPSC-Heps in vitro and in an immunocompromised AHF mouse model induced by thioacetamide (TAA). Intrahepatic delivery of HGF-CHC-iPSC-Heps reduced the TAA-induced hepatic necrotic area and rescued liver function and recipient viability. Compared with PBS-delivered iPSC-Heps, the HGF-CHC-delivered iPSC-Heps exhibited higher antioxidant and antiapoptotic activities that reduced hepatic necrotic area. Importantly, these HGF-CHC-mediated responses could be abolished by administering anti-HGF neutralizing antibodies. In conclusion, our findings demonstrated that HGF mediated the enhancement of iPSC-Hep antioxidant/antiapoptotic capacities and hepatoprotection and that HGF-CHC is as an excellent vehicle for iPSC-Hep engraftment in iPSC-based therapy against AHF.

Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells

Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models

As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.

PMO-134 Basal cell adhesion molecule and b1-integrins regulate the adhesion of ES cell-derived hepatocye-like cells to extracellular matrix and hepatic sinusoidal cells

IntroductionCellular transplantation is an alternative to liver transplantation, however provision of primary human hepatocytes is limited. Human embryonic stem cell-derived hepatocyte-like cells (ES-HLCs) represent a renewable source of functional hepatocytes....

Evaluation of Human Embryonic Stem Cell-derived Hepatocyte-like Cells for Detection of CYP1A Inducers

There is a great deal of interest in differentiation of human embryonic stem cells (hESCs) into hepatocyte-like cells for application in pharmaceutical screening. Cytochrome P450 (CYP) 1A is involved in the metabolic activation of procarcinogenic compounds as well as in detoxification of drugs. We differentiated hESCs into hepatocyte-like cells (hESC-derived hepatocyte-like cells) and examined whether CYP1A was induced in these cells by typical inducers of CYP1A. hESC-derived hepatocyte-like cells expressed albumin, α-fetoprotein, CYP3A4, CYP3A7, CYP1A1, CYP1A2, and UDP-glucuronyl transferase (UGT) 1A1 mRNA. The levels of CYP1A1, CYP1A2, and UGT1A1 mRNA expression were increased by omeprazole and 3-methylcholanthrene. Furthermore, the enzyme activity of CYP1A was also increased by these compounds. In conclusion, hESC-derived hepatocyte-like cells are available for the detection of CYP1A inducers.

Establishment of Novel Detection System for Embryonic Stem Cell-Derived Hepatocyte-Like Cells Based on Nongenetic Manipulation with Indocyanine Green

Hepatocytes derived from embryonic stem cells (ESCs) are expected to be useful for basic research and clinical applications. However, in several studies, genetic methods used to detect and obtain them are difficult and pose major safety problems. Therefore, in this study, we established a novel detection system for hepatocytes by using indocyanine green (ICG), which is selectively taken up by hepatocytes, based on nongenetic manipulation. ICG has maximum light absorption near 780 nm, and it fluoresces between 800 and 900 nm. Making use of these properties, we developed flow cytometry equipped with an excitation lazer of 785 nm and specific bandpass filters and successfully detected ESC-derived ICG-positive cells that were periodic acid-Schiff positive and expressed hepatocyte phenotypic mRNAs. These results demonstrate that this detection system based on nongenetic manipulation with ICG will lead to isolate hepatocytes generated from ESCs and provide the appropriate levels of stability, quality, and safety required for cell source for cell-based therapy and pharmaceutical studies such as toxicology.

Enhanced Functions of Human Embryonic Stem Cell-derived Hepatocyte-like Cells on Three-dimensional Nanofibrillar Surfaces

Human embryonic stem cell (hESC)-derived hepatocytes provide a promising unlimited resource for the treatment of liver disease. However, current protocols for the generation of mature and functional hepatocytes are inefficient. Therefore, in order to better differentiate and maintain the function of differentiating hESCs, we have hypothesized that hESCs undergo better differentiation into hepatocyte-like cells (HLCs) when induced on three-dimensional nanofibrillar surfaces. We have demonstrated that, during stepwise differentiation of induction, the markers of hepatic lineage expressed and finally lead to the generation of functional mature cells. In the presence of an ultraweb nanofiber, HLCs produced lower AFP, greater urea, glycogen storage, metabolic PROD activity, uptake of LDL and organic anion ICG, all of which are indicative of the differentiation of HLCs. These results show that topographically treated hESCs at the nano level have a distinct hepatic functionality profile which has implications for cell therapies.