The role of galanin in the differentiation of mucosal mast cells in mice.

Mast cells are generally classified into two phenotypically distinct populations: mucosal-type mast cells (MMCs) and connective tissue-type mast cells (CTMCs). However, the molecular basis determining the different characteristics of the mast cell subclasses still remains unclear. Unfortunately, the number of mast cells that can be obtained from tissues is limited, which makes it difficult to study the function of each mast cell subclass. Here, we report the generation and characterization of MMCs and CTMCs derived from mouse BM mast cells (BMMCs). We found that the expression of galanin receptor 3 was elevated in MMCs when compared to the expression in CTMCs. Moreover, intraperitoneal injection of a galanin antagonist reduced MMCs and inhibited the inflammation of dextran sodium sulfate-induced colitis in mice. Therefore, these results suggest that galanin promotes MMC differentiation in vivo, and provide important insights into the molecular mechanisms underlying the differentiation of mast cell subclasses.

Wnt-ß-Catenin Signaling Promotes the Maturation of Mast Cells.

Mast cells play an important role in the pathogenesis of allergic diseases. Immature mast cells migrate into peripheral tissues from the bone marrow and undergo complete maturation. Interestingly, mast cells have characteristics similar to hematopoietic stem cells (HSCs), such as self-renewal and c-kit expression. In HSCs, Wnt signaling is involved in their maintenance and differentiation. On the other hand, the relation between Wnt signaling and mast cell differentiation is poorly understood. To study whether Wnt signals play a role in the maturation of mast cells, we studied the effect of Wnt proteins on mast cell maturation of bone marrow-derived mast cells (BMMCs). The expression levels of CD81 protein and histidine decarboxylase mRNA and activity of mast cell-specific protease were all elevated in BMMCs treated with Wnt5a. In addition, Wnt5a induced the expression of Axin2 and TCF mRNA in BMMCs. These results showed that Wnt5a could promote the maturation of mast cells via the canonical Wnt signaling pathway and provide important insights into the molecular mechanisms underlying the differentiation of mast cells.

Selective Differentiation into Hematopoietic and Cardiac Cells from Pluripotent Stem Cells Based on the Expression of Cell Surface Markers.

Flk1-expressing (+) mesodermal cells are useful source for the generation of hematopoietic cells and cardiomyocytes from pluripotent stem cells (PSCs). However, they have been reported as a heterogenous population that includes hematopoietic and cardiac progenitors. Therefore, to provide a method for a highly efficient production of hematopoietic cells and cardiomyocytes, cell surface markers are often used for separating these progenitors in Flk1(+) cells. Our recent study has shown that the expression of coxsackievirus and adenovirus receptor (CAR), a tight junction component molecule, could divide mouse and human PSC- and mouse embryo-derived Flk1(+) cells into Flk1(+)CAR(-) and Flk1(+)CAR(+) cells. Flk1(+)CAR(-) and Flk1(+)CAR(+) cells efficiently differentiated into hematopoietic cells and cardiomyocytes, respectively. These results indicate that CAR is a novel cell surface marker for separating PSC-derived Flk1(+) mesodermal cells into hematopoietic and cardiac progenitors. We herein describe a differentiation method from PSCs into hematopoietic cells and cardiomyocytes based on CAR expression.

Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells.

The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.

Expression of coxsackievirus and adenovirus receptor separates hematopoietic and cardiac progenitor cells in fetal liver kinase 1-expressing mesoderm.

In developing embryos or in vitro differentiation cultures using pluripotent stem cells (PSCs), such as embryonic stem cells and induced pluripotent stem cells, fetal liver kinase 1 (Flk1)-expressing mesodermal cells are thought to be a heterogeneous population that includes hematopoietic progenitors, endothelial progenitors, and cardiac progenitors. However, information on cell surface markers for separating these progenitors in Flk1⁺ cells is currently limited. In the present study, we show that distinct types of progenitor cells in Flk1⁺ cells could be separated according to the expression of coxsackievirus and adenovirus receptor (CAR, also known as CXADR), a tight junction component molecule. We found that mouse and human PSC- and mouse embryo-derived Flk1⁺ cells could be subdivided into Flk1⁺CAR⁺ cells and Flk1⁺CAR⁻ cells. The progenitor cells with cardiac potential were almost entirely restricted to Flk1⁺CAR⁺ cells, and Flk1⁺CAR⁻ cells efficiently differentiated into hematopoietic cells. Endothelial differentiation potential was observed in both populations. Furthermore, from the expression of CAR, Flk1, and platelet-derived growth factor receptor-α (PDGFRα), Flk1⁺ cells could be separated into three populations (Flk1⁺PDGFRα⁻ CAR⁻ cells, Flk1⁺PDGFRα⁻CAR⁺ cells, and Flk1⁺PDGFRα⁺CAR⁺ cells). Flk1⁺PDGFRα⁺ cells and Flk1⁺PDGFRα⁻ cells have been reported as cardiac and hematopoietic progenitor cells, respectively. We identified a novel population (Flk1⁺PDGFRα⁻ CAR⁺ cells) with the potential to differentiate into not only hematopoietic cells and endothelial cells but also cardiomyocytes. Our findings indicate that CAR would be a novel and prominent marker for separating PSC- and embryo-derived Flk1⁺ mesodermal cells with distinct differentiation potentials.

Efficient Engraftment of Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cells in uPA/SCID Mice by Overexpression of FNK, a Bcl-xL Mutant Gene.

Human liver chimeric mice are expected to be applied for drug toxicity tests and human hepatitis virus research. Human induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs) are a highly attractive donor source for the generation of human liver chimeric mice because they can be produced on a large scale and established from an individual. Although these cells have been successfully used to generate human liver chimeric mice, there is still room for improvement in the repopulation efficiency. To enhance the repopulation efficacy, the human iPSC-HLCs were transduced with an adenovirus vector (Ad-FNK) expressing FNK, a hyperactive mutant gene from Bcl-xL, which was expected to inhibit apoptosis in the process of integration into liver parenchyma. We then transplanted Ad-FNK-transduced human iPSC-HLCs into urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice (FNK mice) and evaluated the repopulation efficacy. The antiapoptotic effects of the human iPSC-HLCs were enhanced by FNK overexpression in vitro. Human albumin levels in the transplanted mice were significantly increased by transplantation of Ad-FNK-transduced human iPSC-HLCs (about 24,000 ng/ml). Immunohistochemical analysis with an anti-human αAT antibody revealed greater repopulation efficacy in the livers of FNK mice than control mice. Interestingly, the expression levels of human hepatocyte-related genes in the human iPSC-HLCs of FNK mice were much higher than those in the human iPSC-HLCs before transplantation. We succeeded in improving the repopulation efficacy of human liver chimeric mice generated by transplanting the Ad-FNK-transduced human iPSC-HLCs into uPA/SCID mice. Our method using ectopic expression of FNK was useful for generating human chimeric mice with high chimerism.

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.

Development of mice exhibiting hepatic microsomal activity of human CYP3A4 comparable to that in human liver microsomes by intravenous administration of an adenovirus vector expressing human CYP3A4.

Cytochrome P450 3A4 (CYP3A4) plays a crucial role in the pharmacokinetic and safety profiles of drugs. However, it is difficult to properly predict the pharmacokinetics and hepatotoxicity of drugs in humans using data from experimental animals, because the catalytic activities of CYP3A4 and other drug-metabolizing enzymes differ between human and animal organs. In order to easily generate an animal model for proper evaluation of human CYP3A4-mediated drug metabolism, we developed a human CYP3A4-expressing adenovirus (Ad) vector based on our novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-E4-122aT-hCYP3A4). Intravenous administration of Ad-E4-122aT-hCYP3A4 at a dose of 2 × 10(11) virus particles/mouse produced a mouse exhibiting human CYP3A4 activity at a level similar to that in the human liver, as shown in the dexamethasone metabolic experiment using liver microsomes. The area under the curve (AUC) of 6ßOHD was 2.7-fold higher in the Ad-E4-122aT-hCYP3A4-administered mice, compared with the mice receiving a control Ad vector. This Ad vector-expressing human CYP3A4 would thus be a powerful tool for evaluating human CYP3A4-mediated drug metabolism in the livers of experimental animals.

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.

Plasma elevation of vascular endothelial growth factor leads to the reduction of mouse hematopoietic and mesenchymal stem/progenitor cells in the bone marrow.

Vascular endothelial growth factor (VEGF) is reported to exhibit potent hematopoietic stem/progenitor cell (HSPC) mobilization activity. However, the detailed mechanisms of HSPC mobilization by VEGF have not been examined. In this study, we investigated the effect of VEGF on bone marrow (BM) cell and the BM environment by intravenous injection of VEGF-expressing adenovirus vector (Ad-VEGF) into mice. A colony assay using peripheral blood cells revealed that plasma elevation of VEGF leads to the mobilization of HSPCs into the circulation. Granulocyte colony-stimulating factor (G-CSF) is known to mobilize HSPCs by decreasing CXC chemokine ligand 12 (CXCL12) levels in the BM. However, we found almost no changes in the CXCL12 levels in the BM after Ad-VEGF injection, suggesting that VEGF can alter the BM microenvironment by different mechanisms from G-CSF. Furthermore, flow cytometric analysis and colony forming unit-fibroblast assay showed a reduction in the number of mesenchymal progenitor cells (MPCs), which have been reported to serve as niche cells to support HSPCs, in the BM of Ad-VEGF-injected mice. Adhesion of donor cells to the recipient BM after transplantation was also impaired in mice injected with Ad-VEGF, suggesting a decrease in the niche cell number. We also observed a dose-dependent chemoattractive effect of VEGF on primary BM stromal cells in vitro. These data suggest that VEGF alters the distribution of MPCs in the BM and can also mobilize MPCs to peripheral tissues. Taken together, our results imply that VEGF-elicited egress of HSPCs would be mediated, in part, by changing the number of MPCs in the BM.

Long-term self-renewal of human ES/iPS-derived hepatoblast-like cells on human laminin 111-coated dishes.

The establishment of self-renewing hepatoblast-like cells (HBCs) from human pluripotent stem cells (PSCs) would realize a stable supply of hepatocyte-like cells for medical applications. However, the functional characterization of human PSC-derived HBCs was not enough. To purify and expand human PSC-derived HBCs, human PSC-derived HBCs were cultured on dishes coated with various types of human recombinant laminins (LN). Human PSC-derived HBCs attached to human laminin-111 (LN111)-coated dish via integrin alpha 6 and beta 1 and were purified and expanded by culturing on the LN111-coated dish, but not by culturing on dishes coated with other laminin isoforms. By culturing on the LN111-coated dish, human PSC-derived HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte-like cells and cholangiocyte-like cells. These expandable human PSC-derived HBCs would be manageable tools for drug screening, experimental platforms to elucidate mechanisms of hepatoblasts, and cell sources for hepatic regenerative therapy.

Two-step differentiation of mast cells from induced pluripotent stem cells.

Mast cells play important roles in the pathogenesis of allergic diseases. They are generally classified into 2 phenotypically distinct populations: connective tissue-type mast cells (CTMCs) and mucosal-type mast cells (MMCs). The number of mast cells that can be obtained from tissues is limited, making it difficult to study the function of mast cells. Here, we report the generation and characterization of CTMC-like mast cells derived from mouse induced pluripotent stem (iPS) cells. iPS cell-derived mast cells (iPSMCs) were generated by the OP9 coculture method or embryoid body formation method. The number of Safranin O-positive cells, expression levels of CD81 protein and histidine decarboxylase mRNA, and protease activities were elevated in the iPSMCs differentiated by both methods as compared with those in bone marrow-derived mast cells (BMMCs). Electron microscopic analysis revealed that iPSMCs contained more granules than BMMCs. Degranulation was induced in iPSMCs after stimulation with cationic secretagogues or vancomycin. In addition, iPSMCs had the ability to respond to stimulation with the IgE/antigen complex in vitro and in vivo. Moreover, when iPSMCs generated on OP9 cells were cocultured with Swiss 3T3 fibroblasts, protease activities as maturation index were more elevated, demonstrating that mature mast cells were differentiated from iPS cells. iPSMCs can be used as an in vitro model of CTMCs to investigate their functions.

3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing.

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing.

Inhibition of Lnk in mouse induced pluripotent stem cells promotes hematopoietic cell generation.

Embryonic stem (ES) cell- and induced pluripotent stem (iPS) cell-derived hematopoietic stem/progenitor cells (HSPCs) are considered as an unlimited source for HSPC transplantation. However, production of immature hematopoietic cells, especially HSPCs, from ES and iPS cells has been challenging. The adaptor protein Lnk has been shown to negatively regulate HSPC function via the inhibition of thrombopoietin (TPO) and stem cell factor signaling, and Lnk-deficient HSPCs show an enhanced self-renewal and repopulation capacity. In this study, we examined the role of Lnk on the hematopoietic differentiation from mouse ES and iPS cells by the inhibition of Lnk using a dominant-negative mutant of the Lnk (DN-Lnk) gene. We generated mouse ES and iPS cells stably expressing a DN-Lnk, and found that enforced expression of a DN-Lnk in ES and iPS cells led to an enhanced generation of Flk-1-positive mesodermal cells, thereby could increase in the expression of hematopoietic transcription factors, including Scl and Runx1. We also showed that the number of both total hematopoietic cells and immature hematopoietic cells with colony-forming potential in DN-Lnk-expressing cells was significantly increased in comparison with that in control cells. Furthermore, Lnk inhibition by the overexpression of the DN-Lnk gene augmented the TPO-induced phosphorylation of Erk1/2 and Akt, indicating the enhanced sensitivity to TPO. Adenovirus vector-mediated transient DN-Lnk gene expression in ES and iPS cells could also increase the hematopoietic cell production. Our data clearly showed that the inhibition of Lnk in ES and iPS cells could result in the efficient generation and expansion of hematopoietic cells.

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.

The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets.

Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening.

Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction.

Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy.

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.

Optimization of adenovirus vectors for transduction in embryonic stem cells and induced pluripotent stem cells.

Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/ß-actin promoter with ß-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.

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.