Fuminori Sakurai

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.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.

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.

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‐1α (EF‐1α) and the cytomegalovirus enhancer/β‐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‐1α 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 PPARγ 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. STEM CELLS 2009;27:1802–1811

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.

Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector

We have recently developed a replication-defective, recombinant adenovirus (Ad) vector composed of the whole Ad serotype 35 (Ad35), a member of subgroup B. We describe herein the in vitro and in vivo gene transfer properties of Ad35 vector in comparison with Ad serotype 5 (Ad5) and the Ad5F35 vector, which is a fiber-substituted Ad5 vector containing Ad35 fiber proteins. In vitro, Ad35 vector efficiently transduced not only human CAR-positive cells but also CAR-negative cells. Following intravenous administration into mice, both Ad5 and Ad35 vectors were rapidly cleared from the bloodstream with a half-life of approximately 3 min. Ad5 vector-mediated transgene expression predominantly occurred in liver parenchymal cells, although the Ad5 vector was delivered to both liver parenchymal and nonparenchymal cells. In contrast, Ad35 vector was efficiently taken up by liver nonparenchymal cells and mediated transduction efficiency in the liver on a level 4 log orders lower than the Ad5 vector. These findings demonstrate that Ad35 vector is an attractive vehicle for gene transfer into human cells, while the biodistribution profile of Ad35 vector in mice is much different from that of the Ad5 vector.

Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes

Significance We found that individual cytochrome P450 (CYP) metabolism capacity and drug sensitivity could be predicted by examining them in the primary human hepatocytes–human induced pluripotent stem cells–hepatocyte-like cells (PHH-iPS-HLCs). We also confirmed that interindividual differences of CYP metabolism capacity and drug responsiveness that are due to the diversity of individual single nucleotide polymorphisms in the CYP gene could also be reproduced in the PHH-iPS-HLCs. These findings suggest that interindividual differences in drug metabolism capacity and drug response could be predicted by using HLCs differentiated from human iPS cells. We believe that iPS-HLCs would be a powerful technology not only for accurate and efficient drug development, but also for personalized drug therapy. Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness.

Reduction of Natural Adenovirus Tropism to Mouse Liver by Fiber-Shaft Exchange in Combination with both CAR- and αv Integrin-Binding Ablation

ABSTRACT The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the αv integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and αv integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triple-mutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors.

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector.

Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34+ cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34+ cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34+ cells transduced with the Ad35 vectors was 12–76 and 1.4–3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34+ cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34+ cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs.

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.