Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated “RoGel,” which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC–HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.     

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.     

Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.     

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.     

Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.     

Efficient generation of functional hepatocyte-like cells from human fetal hepatic progenitor cells in vitro

Differentiation of human hepatic progenitor cells to functional hepatocytes holds great potential to develop new therapeutic strategies for liver disease and to provide a platform for drug toxicity screens and identification of novel pharmaceuticals. We report here that human fetal hepatic progenitor cells (hFHPCs) efficiently differentiate to hepatocyte-like cells by continuous exposure to a combination of soluble factors for 7 days in vitro. We compared the effect of hepatocyte growth factor (HGF), oncostatin M (OSM), dexamethasone (DEX), or a combination on the expression of a liver-specific marker, albumin (ALB). Real-time RT-PCR analysis showed that, upon exposure to a combination of OSM, DEX, and HGF, the expression of ALB gradually increased in a time-dependent manner. In contrast, the level of the hepatic progenitor cell marker alpha-fetoprotein (AFP) decreased as differentiation progressed. Moreover, cells exposed to the combination of OSM, DEX, and HGF gradually featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and cytochrome P450 (CYP) activity. The effect of these factors on the differentiation of hFHPCs may be blocked by U0126, an inhibitor of the ERK1/2 signaling pathway. In conclusion, we demonstrate that a combination of soluble factors facilitates the efficient generation of highly differentiated hepatocyte-like cells from hFHPCs and ERK1/2 signaling pathway involved in this process. Results suggest that this system will be useful for generating functional hepatocytes and, hence, may serve as a cell source suitable for preclinical pharmacological research and testing.     

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.     

The differentiation of hepatocyte-like cells from monkey embryonic stem cells

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.     

How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

Background aimsThe protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases.MethodsAfter hepatic differentiation of umbilical cord–derived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model.ResultsMSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure.ConclusionsAfter differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases.     

Pluripotent stem cell-derived hepatocyte-like cells

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~ 0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iHLCs will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.     

Derivation and applications of human hepatocyte-like cells

Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) promise a valuable source of cells with human genetic background, physiologically relevant liver functions, and unlimited supply. With over 10 years’ efforts in this field, great achievements have been made. HLCs have been successfully derived and applied in disease modeling, toxicity testing and drug discovery. Large cohorts of induced pluripotent stem cells-derived HLCs have been recently applied in studying population genetics and functional outputs of common genetic variants in vitro. This has offered a new paradigm for genome-wide association studies and possibly in vitro pharmacogenomics in the nearly future. However, HLCs have not yet been successfully applied in bioartificial liver devices and have only displayed limited success in cell transplantation. HLCs still have an immature hepatocyte phenotype and exist as a population with great heterogeneity, and HLCs derived from different hPSC lines display variable differentiation efficiency. Therefore, continuous improvement to the quality of HLCs, deeper investigation of relevant biological processes, and proper adaptation of recent advances in cell culture platforms, genome editing technology, and bioengineering systems are required before HLCs can fulfill the needs in basic and translational research. In this review, we summarize the discoveries, achievements, and challenges in the derivation and applications of HLCs.     

Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro

Bone marrow (BM) cells originally include alpha-fetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albumin-expressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases.     

Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.     

Spheroid formation and differentiation into hepatocyte-like cells of rat mesenchymal stem cell induced by co-culture with liver cells

Mesenchymal stem cells (MSCs) derived from bone marrow have been shown to differentiate into hepatocytes, which would be an ideal resource for transplantation or artificial liver devices. Here we investigated the efficiency of co-culture system consisting of rat MSCs and adult liver cells to induce differentiation of MSCs into hepatocyte-like cells. Marked MSCs were either co-cultured with freshly isolated liver cells or treated with hepatocyte growth factor (HGF) for 21 days. In co-culture systems, MSCs formed spheroids of round-shaped cells while keeping normal proliferation and viability, strongly expressed albumin, alpha-fetoprotein, and cytokeratin-18 in mRNA and protein level from day 3 to 21. As a control, MSCs treated with HGF showed weak gene expressions in day 14 and had a few cells of protein staining in day 21. These results indicate that the co-culture microenvironment plays a decisive role for the hepatic differentiation of MSCs, and it is more efficient than HGF treatment. Insights gained from this study will be helpful to design optimal culture systems for the hepatic differentiation of human MSCs and the hepatic function maintenance of hepatocytes in vitro.     

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.