CCAAT/enhancer binding protein-mediated regulation of TGFß receptor 2 expression determines the hepatoblast fate decision.

Human embryonic stem cells (hESCs) and their derivatives are expected to be used in drug discovery, regenerative medicine and the study of human embryogenesis. Because hepatocyte differentiation from hESCs has the potential to recapitulate human liver development in vivo, we employed this differentiation method to investigate the molecular mechanisms underlying human hepatocyte differentiation. A previous study has shown that a gradient of transforming growth factor beta (TGFß) signaling is required to segregate hepatocyte and cholangiocyte lineages from hepatoblasts. Although CCAAT/enhancer binding proteins (c/EBPs) are known to be important transcription factors in liver development, the relationship between TGFß signaling and c/EBP-mediated transcriptional regulation in the hepatoblast fate decision is not well known. To clarify this relationship, we examined whether c/EBPs could determine the hepatoblast fate decision via regulation of TGFß receptor 2 (TGFBR2) expression in the hepatoblast-like cells differentiated from hESCs. We found that TGFBR2 promoter activity was negatively regulated by c/EBPα and positively regulated by c/EBPß. Moreover, c/EBPα overexpression could promote hepatocyte differentiation by suppressing TGFBR2 expression, whereas c/EBPß overexpression could promote cholangiocyte differentiation by enhancing TGFBR2 expression. Our findings demonstrated that c/EBPα and c/EBPß determine the lineage commitment of hepatoblasts by negatively and positively regulating the expression of a common target gene, TGFBR2, respectively.

Efficient generation of functional hepatocytes from human embryonic stem cells and induced pluripotent stem cells by HNF4α transduction.

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity.

Generation of metabolically functioning hepatocytes from human pluripotent stem cells by FOXA2 and HNF1α transduction.

BACKGROUND & AIMS: Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. We have recently reported that hepatic differentiation is promoted by sequential transduction of SOX17, HEX, and HNF4α into hESC- or hiPSC-derived cells, but further maturation of hepatocyte-like cells is required for widespread use of drug screening. METHODS: To screen for hepatic differentiation-promoting factors, we tested the seven candidate genes related to liver development. RESULTS: The combination of two transcription factors, FOXA2 and HNF1α, promoted efficient hepatic differentiation from hESCs and hiPSCs. The expression profile of hepatocyte-related genes (such as genes encoding cytochrome P450 enzymes, conjugating enzymes, hepatic transporters, and hepatic nuclear receptors) achieved with FOXA2 and HNF1α transduction was comparable to that obtained in primary human hepatocytes. The hepatocyte-like cells generated by FOXA2 and HNF1α transduction exerted various hepatocyte functions including albumin and urea secretion, and the uptake of indocyanine green and low density lipoprotein. Moreover, these cells had the capacity to metabolize all nine tested drugs and were successfully employed to evaluate drug-induced cytotoxicity. CONCLUSIONS: Our method employing the transduction of FOXA2 and HNF1α represents a useful tool for the efficient generation of metabolically functional hepatocytes from hESCs and hiPSCs, and the screening of drug-induced cytotoxicity.

Efficient generation of hepatoblasts from human ES cells and iPS cells by transient overexpression of homeobox gene HEX.

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Efficient adipocyte and osteoblast differentiation from mouse induced pluripotent stem cells by adenoviral transduction.

Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. Previously, by using an adenovirus (Ad) vector containing the elongation factor-1alpha (EF-1alpha) and the cytomegalovirus enhancer/beta-actin (CA) promoters, we developed an efficient transduction system for mouse embryonic stem (ES) cells and their aggregate form, embryoid bodies (EBs). In this study, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector containing EF-1alpha and the CA promoter could efficiently transduce transgenes into mouse iPS cells. At 3,000 vector particles/cell, 80%-90% of iPS cells expressed transgenes by treatment with an Ad vector containing the CA promoter, without a decrease in pluripotency or viability. We also found that the CA promoter had potent transduction ability in iPS cell-derived EBs. Moreover, exogenous expression of a PPARgamma gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to increase cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.

Comparative study between lactose-silk fibroin conjugates and extracellular matrices as a substrate for the culture of human induced pluripotent stem cell-derived hepatocytes.

BACKGROUND: Human induced pluripotent stem cell (hiPSC)-derived hepatocytes are an attractive alternative cell source to primary human hepatocytes for tissue regeneration. OBJECTIVES: This study presents an application of lactose-silk fibroin conjugates (Lac-CY-SF) bearing 𝛽-galactose residues as a substrate for culture of hiPSC-derived hepatocytes. A comparison of hiPSC-derived hepatocytes cultured on three different substrates; Lac-CY-SF conjugates, Matrigel and type I collagen was performed. METHODS: Cell morphology, viability, maturation and albumin secretory function were assessed by phase-contrast microscopy, tetrazolium-based colorimetric assay, immunofluorescence staining and enzyme-linked immunosorbent assay. RESULTS: Morphological characteristics of the cells cultured on the conjugates resembled those on Matrigel throughout the 6-day culture period. The number of viable cells cultured on the conjugates was comparable to that on Matrigel at day 2 and 6. The protein expression of mature hepatocyte markers, asialoglycoprotein receptor 1 and albumin, by the cells cultured on the conjugates resembled that by the cells cultured on collagen at day 2 and 6. Albumin secretory function per cell cultured on the conjugates was higher than that on collagen and comparable to that on Matrigel. CONCLUSIONS: These limited results suggest that Lac-CY-SF conjugates may be as useful as Matrigel and collagen for cultivation of hiPSC-derived hepatocytes.

HHEX promotes hepatic-lineage specification through the negative regulation of eomesodermin.

Human embryonic stem cells (hESCs) could provide a major window into human developmental biology, because the differentiation methods from hESCs mimic human embryogenesis. We previously reported that the overexpression of hematopoietically expressed homeobox (HHEX) in the hESC-derived definitive endoderm (DE) cells markedly promotes hepatic specification. However, it remains unclear how HHEX functions in this process. To reveal the molecular mechanisms of hepatic specification by HHEX, we tried to identify the genes directly targeted by HHEX. We found that HHEX knockdown considerably enhanced the expression level of eomesodermin (EOMES). In addition, HHEX bound to the HHEX response element located in the first intron of EOMES. Loss-of-function assays of EOMES showed that the gene expression levels of hepatoblast markers were significantly upregulated, suggesting that EOMES has a negative role in hepatic specification from the DE cells. Furthermore, EOMES exerts its effects downstream of HHEX in hepatic specification from the DE cells. In conclusion, the present results suggest that HHEX promotes hepatic specification by repressing EOMES expression.

Efficient hepatic differentiation from human iPS cells by gene transfer.

Establishment of protocols for the differentiation of hepatic cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells could contribute to regenerative cell therapies or drug discovery and development. However, the differentiation efficiency of endoderm-derived cells, such as hepatic cells, from human ES and iPS cells is poor because hepatic cells are differentiated via multiple lineages including endodermal cells, hepatic progenitor cells, and mature hepatocytes. We show here the protocols for efficient hepatic differentiation from human ES and iPS cells by adenovirus vector-mediated gene transfer.

Efficient and directive generation of two distinct endoderm lineages from human ESCs and iPSCs by differentiation stage-specific SOX17 transduction.

The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively.

Adenovirus vector-mediated efficient transduction into human embryonic and induced pluripotent stem cells.

We examined the transduction efficiency in human embryonic stem (ES) and induced pluripotent stem (iPS) cells using an adenovirus (Ad) vector. RT-PCR analysis revealed the expression of the coxsackievirus and adenovirus receptor, a receptor for Ad, in these cells. However, gene expression after the transduction with an Ad vector was observed only in the periphery of ES and iPS cell colonies, when human ES and iPS cells were passaged as small colonies. This suggests that the Ad vector could not enter inside the ES and iPS cell colonies by their tight connection. We thus attempted to transduce foreign genes into the dissociated form of human ES and iPS cells, which were passaged using Rho-associated kinase inhibitor. In this condition, transduction efficiency in human ES and iPS cells was markedly increased and transgene expression was observed even inside the colonies by using Ad vectors. Furthermore, Ad vector-mediated transduction did not alter the expression of undifferentiated markers such as Oct-3/4, Nanog, and SSEA-4. Our results indicate that Ad vectors are effective tools for transduction into human ES and iPS cells.