Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities

The utilization of human hepatocytes for biomedical research, drug discovery, and treatment of liver diseases is hindered by the limited availability of donated livers and the variability of their derived hepatocytes. Human embryonic stem cells (hESCs) are pluripotent and provide a unique, unlimited resource for human hepatocytes. However, differentiation of hESCs to hepatocytes remains a challenge. We have developed a multistage procedure by which hESCs can be directly differentiated to hepatocyte-like cells without embryoid body formation and the requirement of sodium butyrate. The hESC-derived hepatocyte-like cells (HLCs) exhibited characteristic hepatocyte morphology, expressed hepatocyte markers, including alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha, and possessed hepatocyte-specific activities, such as p450 metabolism, albumin production, glycogen storage, and uptake and excretion of indocyanine green. Hepatocyte growth factor was found to play a positive role in promoting hepatocyte differentiation. Our differentiation system has shown that hESCs can be differentiated to hepatocyte-like cells capable of executing a range of hepatocyte functions. Therefore, it presents a proof-of-principle of potential applications of using the hESC-derived hepatocytes. Additionally, the hESC-derived HLCs provide a unique model to study the mechanisms involved in human hepatocyte differentiation and liver function.     

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 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.     

Trophoblast differentiation of human embryonic stem cells

Molecular mechanisms regulating human trophoblast differentiation remain poorly understood due to difficulties in obtaining primary tissues from very early developmental stages in humans. Therefore, the use of human embryonic stem cells (hESCs) as a source for generating trophoblast tissues is of significant interest. Trophoblast-like cells have been obtained through treatment of hESCs with bone morphogenetic protein (BMP) or inhibitors of activin/nodal/transforming growth factor-β signaling, or through protocols involving formation of embryoid bodies (EBs); however, there is controversy over whether hESC-derived cells are indeed analogous to true trophoblasts found in vivo. In this review, we provide an overview of previously described efforts to obtain trophoblasts from hESCs. We also discuss the merits and limitations of hESCs as a source of trophoblast derivatives.     

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.     

Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation

Human embryonic stem cells (hESCs) represent an important resource for novel cell-based regenerative medical therapies. hESCs are known to differentiate into mature cells of defined lineages through the formation of embryoid bodies (EBs) which are amenable to suspension culture for several weeks. However, EBs derived from hESCs in standard static cultures are typically non-homogeneous, leading to inefficient cellular development. Here, we systematically compare the formation, growth, and differentiation capabilities of hESC-derived EBs in stirred and static suspension cultures. A 15-fold expansion in total number of EB-derived cells cultured for 21 days in a stirred flask was observed, compared to a fourfold expansion in static (non-stirred) cultures. Additionally, stirred vessel mediated cultures have a more homogeneous EB morphology and size. Importantly, the EBs cultivated in spinner flasks retained comparable ability to produce hematopoietic progenitor cells as those grown in static culture. These results demonstrate the decoupling between EB cultivation method and EB-derived cells' ability to form hematopoietic progenitors, and will allow for improved production of scalable quantities of hematopoietic cells or other differentiated cell lineages from hESCs in a controlled environment.     

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.     

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry⁺NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.     

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.     

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.     

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.     

Sodium butyrate-treated embryonic stem cells yield hepatocyte-like cells expressing a glycolytic phenotype

Embryonic stem cells serve as a promising technology to obtain specific cell types for a number of biomedical applications. Because traditional techniques, such as embryoid body formation result in a wide array of differentiated cells such as hepatic, neuronal, and cardiac lineages, strategies have been utilized which favor cell-specific differentiation to generate more uniformity. In the present study, we have investigated the use of sodium butyrate in a monolayer culture configuration to mediate hepatocyte differentiation of murine embryonic stem cells. Several functional assays used to characterize hepatocyte function (viz. urea secretion, intracellular albumin content, cytokeratin 18, and glycogen staining) were used to analyze the differentiating cell population, suggesting the presence of an enriched population of hepatocyte-like cells. Since mature hepatocytes mediate energy metabolism predominantly through oxidative means as opposed to hepatocyte precursors, which are primarily glycolytic, we have performed a kinetic analysis of glycolytic and functional capacity to characterize the differentiated cells. In conjunction with mitochondrial mass and activity measurements, we show that Na-butyrate-mediated differentiated cells mediate energy metabolism predominantly through glycolysis. This metabolic and mitochondrial characterization can assist in evaluating stem cell differentiation and may prove useful in identifying key regulatory molecules in mediating further differentiation.     

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.     

Differentiation and Selection of Hepatocyte Precursors in Suspension Spheroid Culture of Transgenic Murine Embryonic Stem Cells

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for “live” monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.     

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.