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Hepatic differentiation of mouse embryonic stem cells in a three-dimensional culture system using polyurethane foam
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 105, No. 4, 350–354. 2008 DOI: 10.1263/jbb.105.350
© 2008, The Society for Biotechnology, Japan
Hepatic Differentiation of Mouse Embryonic Stem Cells in a Three-Dimensional Culture System Using Polyurethane Foam Kinya Matsumoto,1 Hiroshi Mizumoto,1 Kohji Nakazawa,2 Hiroyuki Ijima,1 Kazumori Funatsu,1 and Toshihisa Kajiwara1* Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan1 and Department of Chemical Processes and Environments, University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0315, Japan2 Received 19 November 2007/Accepted 7 January 2008
Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0× 108 cells/cm3-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as α-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures. [Key words: hybrid artificial liver, embryonic stem cell, hepatic differentiation, spheroid, polyurethane foam, three dimensional culture]
of HALs is obtaining a cell source, and stem cells have recently received much attention as a novel cell source. Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts. They are characterized by an almost unlimited capacity for selfrenewal and potential to differentiate in vitro into multiple cell lineages (5). Recently, ES cells of mouse and human origin have been shown to differentiate into cells displaying hepatic phenotypes both in vitro and in vivo (6–14). These heptocyte-like cells derived from ES cells expressed some typical markers of mature hepatocytes and showed sufficient functional ability to rescue experimental liver injury when transplanted in vivo. These reports raised the hope of generating a transplantable cell source for the treatment of end-stage liver diseases. In this study, we focused our attention on ES cells as a potential source for HALs. For the differentiation of ES cells, it is important to form spherical multicellular aggregates. Here, we focused attention on the PUF/spheroid culture method which enables spontaneous spheroid formation and mass cultivation of cultured cells. Ultimately, we aimed
Acute liver failure (ALF) is a severe liver disease with a mortality of 60–90%, depending on the cause. The only effective therapy for ALF is liver transplantation. However, liver transplantation is hampered by the increasing shortage of organ donors, resulting in a high incidence of patients with ALF dying on the transplantation waiting list. Hybrid artificial livers (HAL) are expected to more successfully bridge patients with ALF to transplantation or even to promote regeneration of their own liver. A HAL is comprised of bioreactor-immobilized functional liver cells. We have previously developed a HAL by using polyurethane foam (PUF)/hepatocyte spheroid culture, in which the cultured cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF (1). The PUFHAL has been successfully scaled up to the clinical level (2). The curative effect of the PUF-HAL has been demonstrated in preclinical experiments on pigs (3, 4). Currently one of the most difficult problems in the clinical application * Corresponding author. e-mail: [email protected] phone: +81-(0)92-802-2746 fax: +81-(0)92-802-2796 350
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to develop a mass differentiation culture method from ES cells into functional hepatocytes by growth factor treatment along with spherical multicellular aggregates (spheroid) formation using the PUF/spheroid culture method. MATERIALS AND METHODS ES cell culture The ES cells (129 Line) (Chemicon, Pittsburgh, PA, USA) were maintained in an undifferentiated state in gelatin coated dishes. Undifferentiated ES cells were resuspended in Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, St. Louis, MO, USA) containing 2 mM L-glutamine (Sigma), 100 U/ml penicillin (WAKO Pure Chemical Industries, Osaka), 100 µg/ml streptomycin (Meiji Seika Kaisha, Tokyo), 20% fetal bovine serum and 300 µM monothioglycerol (Sigma) and immobilized in the PUF-plate. PUF was manufactured through a foaming process by blending isocyanate with polyol (Inoac, Nagoya). A PUF block was cut into a flat plate for the PUF stationary culture. Mouse ES cells were inoculated at a density of 2.0 ×106 cells in a 35-mm Easy Grip Style Bacteriological Petri Dish (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing the PUF plate in 2 ml of each culture medium. After 4 h of culture, the PUF plate with immobilized cells was transferred to a 60-mm Standard Style Bacteriological Petri Dish (Becton, Dickinson) containing 6 ml of each culture medium. The whole medium in the culture system was exchanged every 24 h of culture. As a control, mouse ES cells were cultured by the hanging drop method. ES cells were cultured for 2 d by the hanging drop method (1.0 ×103 ES cells per 30 µl in each drop). Embryoid bodies (EBs) in hanging drops were transferred to suspension culture in 60 mm collagen type I coated 60 mm dishes. To induce hepatic differentiation in each culture system, specific growth factors were added to culture medium (100 ng/ml acidic fibroblast growth factors [Invitrogen, Carlsbad, CA, USA] on days 9–12; 20 ng/ml hepatocyte growth factor [Toyobo, Osaka] on days 12–30; 10 ng/ml oncostatin M [R&D System, Minneapolis, MN, USA] on days 15–30; 10–7 M dexamethasone [Sigma] on days 15–30; and ITS [10 µg/ml insulin, 5.5 µg/ml transferrin, 6.7 ng/ml selenium acid; Invitrogen Corporation] on days 15–30) (Fig. 1). This method has been reported to differentiate mouse ES cells into mature hepatocytes by Hamazaki et al. (6). Primary mouse hepatocyte isolation Primary hepatocytes were isolated from 129 + Ter/Sv Jc1 mice (8 weeks old) (Kyudo, Kumamoto) by the liver perfusion method using collagenase (Wako) for use as a positive control. RT-PCR analysis Total RNA was extracted from the differentiating cells using NucleoSpin RNA II (Nippon Genetise, Tokyo), and cDNA synthesis was performed using the Superscript FirstStrand Synthesis System (Invitrogen) according to the manufacture’s protocol. cDNA was amplified using the Takara Ex Taq polymerase (Takarabio, Siga) kit. The following primer sets were used: transthyretin (TTR), forward 5′-CTGCTCTACCCGGGCTAT-3′,
FIG. 1. Experimental protocol of hepatic differentiation of ES cells by using PUF/spheroid culture.
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reverse 5′-TGAGCATCGGTCGCTCTT-3′, alpha fetoprotein (AFP), forward 5′-TCGTATTCCAACAGGAGG-3′, reverse 5′-AGGCTT TTGCTTCACCACG-3′, albumin (ALB), forward 5′-GCTACGG CACAGTGCTTG-3′, reverse gene 5′-CAGGATTGCAGACAGA TAGTC-3′, β-actin, forward 5′-TTCCTTCTTGGGTATGGAAT-3′, reverse 5′-GAGCAATGATCTTGATCTTC-3′, Arginase, forward 5′-GTGAAGAACCCACGGTCTGT-3′, reverse 5′-CTGGTTGTCA GGGGAGTGTT-3′, tryptophan 2,3-dioxgenase (TDO), forward 5′TGAATGCGCAAGAACTTCAG-3′ reverse 5′-TTCCAGAACCG AGAACTGCT-3′. Each cycle consisted of 45 s at 94°C, 45 s at 56°C, 45 s at 72°C (30 cycles: TTR, AFP, ALB, β-actin) and 45 s at 95°C, 45 s at 60°C, 60 s at 72°C (30 cycles: Arginase, TDO). Immunostaining After inducing differentiation using the PUF/spheroid culture, immunofluorescence analysis of the spheroids was performed by SRL (Tokyo). For the analysis, Polyclonal Rabbit Anti-Human Albumin and the DakoCytomation LSAB2 System-HRP (Dako, Glostrup, Denmark) were used. Determination of glycogen Glycogen production, which is one of the metabolic functions of hepatocytes, was analyzed by periodic acid-Schiff (PAS) staining after inducing hepatic differentiation. PAS staining were performed by SRL. Evaluation of liver-specific function To evaluate the liverspecific functions of differentiating ES cells, the culture medium was changed to fresh medium containing 1 mM NH4Cl. The decrease in ammonia concentration in the medium during a 24 h period was measured using the Wako ammonia test (Wako). The albumin concentration in the medium was measured by an enzymelinked immunosorbent assay by using the ELISA starter accessory package (Bethyl Laboratories, Montgomery, TX, USA) and the mouse albumin ELISA Quantitation Kit (Bethyl Laboratories). As a control for liver specific function, primary hepatocytes isolated from 129 +Ter/Sv Jc1 mice by the liver perfusion method using collagenase were cultured by using the PUF/spheroid culture method and analyzed in an identical manner to the ES cell-derived hepatocytes.
RESULTS Cell morphology and proliferation of mouse ES cells in PUF/spheroid culture The PUF/spheroid culture experiment was performed for 30 d. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF (Fig. 2A). The spheroids grew larger, and some spheroids were adhered to each other. However, at later stage of the culture, ES cell aggregates became irregular in form (Fig. 2B). Mouse ES cells proliferated from 5.68 ×105 to 1.07 ×108 cells/cm3, and a high cell density was achieved (Fig. 3). Gene expression of mouse ES cell-derived hepatocytes To assess hepatic differentiation, we used reverse tran-
FIG. 2. Morphology of mouse ES cells in PUF/spheroid culture. Mouse ES cells spontaneously formed spheroids in the pores of the PUF (day 7) (A). Spheroids adopted an irregular form at late stages of the culture (B). Scale bars: (A) 100 µm, (B) 200 µm.
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FIG. 3. Cell number of mouse ES cells. Mouse ES cells proliferated and high cell density was achieved (1.07 ×108 cells/cm3).
outmost area of the spheroids. Glycogen production, which is one of the metabolic functions of hepatocytes, was analyzed by periodic acid-Schiff (PAS) staining in ES cell-derived hepatocytes after differentiation. After differentiation (30 d), glycogen production was visible in the spheroids (Fig. 5B). Evaluation of liver specific functions Figure 6A shows the ammonia removal rate of differentiating mouse ES cells per unit volume in the PUF/spheroid culture. Ammonia removal activity in differentiating ES cells was detected by day 21 and increased with culture time. The ammonia removal rate of the differentiating mouse ES cells per unit cell number was one fiftieth that of primary mouse hepatocytes. However, because of the achievement of high cell density, the ammonia removal rate per unit volume was 1.28 (µmol/ cm3-PUF/d). The range for the ammonia removal rate of primary mouse hepatocytes was 0.85–2.79 (µmol/cm3-PUF/d). The activity of albumin secretion of differentiating ES cells was detected at day 30, and albumin secretion per unit volume was 67.2 (ng/cm3-PUF/d) (Fig. 6B). The range of the albumin secretion rate of primary mouse hepatocyte was 37.95–1153.31 (ng/cm3-PUF/d). DISCUSSION
FIG. 4. Comparison of liver-specific gene expression in differentiating ES cells between PUF/spheroid culture and HD/monolayer culture. Endoderm-specific gene was expressed from day 7 and hepatocytes-specific gene was expressed from day 15 in PUF spheroid culture. The expressions of both were increased by day 30. These data indicate that ES cells can differentiate toward cells of a hepatic lineage and mature hepatocytes.
scriptase (RT)-PCR at various times during the culture process to analyze endodermal and liver-specific genes. TTR represents endodermal or yolk-sac-like differentiation and is expressed throughout liver maturation. Alpha fetoprotein (AFP) is marker of endodermal differentiation and an early fetal hepatic marker. Albumin (ALB) and tryptophan 2,3-dioxygenase (TDO) are hepatocyte-specific genes. Undifferentiated ES cells did not express endodermal or hepatocytespecific genes. Figure 4 shows the expression of endodermal and hepatic differentiation markers in differentiating ES cells. TTR was expressed from day 7, and AFP was expressed from day 9. Endoderm-specific gene expression was increased during the culture time. Differentiating ES cells expressed hepatocyte-specific genes from day 18, and the expression was increased by 30 d. These data indicate that ES cells can differentiate toward the hepatic lineage and mature into hepatocytes. Immunostaining and periodic acid-Schiff (PAS) staining Figure 5A shows immunohistochemical examination of ALB in differentiating mouse ES cells at day 30. This result indicated that ES cell-derived hepatocytes were located in the
The formation of embryoid bodies (EBs) is the principal step in the differentiation of ES cells. An EB consists of ectodermal, mesodermal, and endodermal tissues, which recapitulate many aspects of cell differentiation during early mammalian embryogenesis and are able to differentiate into derivatives of all the three germ layers (15, 16). For this reason, EB formation has been utilized widely as a trigger of in vitro differentiation. In this study, mouse ES cells spontaneously formed spheroids in the pores of PUF, and a high cell density was achieved (1.07 ×108 cells/cm3) (Fig. 3). However, the spheroids became irregular in form (Fig. 2B) over the duration of the culture time. Undifferentiated ES cells express E-cadherin strongly, and differentiated ES cells reduce their E-cadherin expression (17, 18). Therefore, cellcell adhesion in PUF/spheroid cultures became weak by the end of the culture time because of a reduction in E-cadherin expression. Cell morphology results indicated that ES cells in the spheroids differentiated with culture time. A system to effect the differentiation from ES cells into mature hepatocytes by EB formation has been reported (6). Here we demonstrate that ES cells could be induced into functional hepatocytes by growth factor treatment along with spheroid formation using the PUF/spheroid culture method. As shown in Fig. 4, endodermal-specific genes and late stage markers of hepatic differentiation were expressed in cells cultured using the PUF/spheroid culture at the same time that they are expressed in traditional HD/monolayer culture differentiation systems. Ammonia removal is one of the most important functions of a HAL. Our result showed that mouse ES cells expressed the ammonia removal function in the PUF/spheroid culture method. The maximum removal level was comparable to that of primary mouse hepatocytes because of the high cell density. We have previously developed a HAL by using PUF/spheroid cultures, and this result further indicated that
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FIG. 5. Immunohistochemical analysis (A), evaluation of glycogen storage (B) and amylase digestion (C). Albumin positive cells were located at the periphery of spheroids on day 30 (allows) (A). Glycogen production was visible in the spheroids after induction of hepatic differentiation on day 30 (allows) (B). The deep purple-blue color of glycogen was disappeared by treatment of amylase digestion (C). Scale bars: 200 µm.
ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research B: 19360375 and a Grant-in-Aid for Scientific Research A2: 14205119 from the Japan Society for the Promotion of Science.
REFERENCES
FIG. 6. Ammonia removal rate (A) and albumin secretion rate per unit volume of PUF plate (B). The ammonia removal activity was detected by day 21 and increased over the remainder of the culture time (A). The ammonia removal rate per unit volume was 1.28 (µmol/cm3PUF/d). This activity was comparable to that of mouse primary hepatocytes. The ammonia removal was not detected on day 15. The albumin secretion activity was detected at day 30, and the albumin secretion per unit volume was 67.2 (ng/cm3-PUF/d) (B). The albumin secretion was not detected on day 15 and 21.
ES cells immobilized in our PUF-HAL may be useful as a biocomponent of a hybrid artificial liver. However, the albumin secretory function was lower than that of primary mouse hepatocytes. AFP is an early hepatic marker, and its expression decreases as the liver develops into the adult phenotype. However, AFP expression did not decrease at the late stage of our culture (Fig. 4). This result indicated that differentiating ES cells might not be mature enough to secrete albumin. We should improve this problem in the future. In conclusion, the method combining of PUF/spheroid culture and hepatic growth factors would be useful for differentiating ES cells into functional hepatocytes.
1. Ijima, H., Matsushita, T., Nakazawa, K., Fujii, Y., and Funatsu, K.: Hepatocyte spheroids in polyurethane foams: Functional analysis and application for a hybrid artificial liver. Tissue Eng., 4, 213–226 (1998). 2. Fukuda, J., Sakiyama, R., Nakazawa, K., Ijima, H., Yamashita, Y., Shimada, M., Shirabe, K., Tsujita, E., Sugimachi, K., and Funatsu, K.: Mass preparation of primary porcine hepatocytes and the design of a hybrid artificial liver module using spheroid culture for a clinical trial. Int. J. Artif. Organs, 24, 799–806 (2001). 3. Funatsu, K. and Nakazawa, K.: Novel hybrid artificial liver using hepatocyte organoids. Int. J. Artif. Organs, 25, 77–82 (2002). 4. Nakazawa, K., Ijima, H., Fukuda, J., Sakiyama, R., Yamashita, Y., Shimada, M., Shirabe, K., Tsujita, E., Sugimachi, K., and Funatsu, K.: Development of a hybrid artificial liver using polyurethane foam/hepatocyte spheroid culture in a preclinical pig experiment. Int. J. Artif. Organs, 25, 51–60 (2002). 5. Czyz, J., Wiese, C., Rolletschek, A., Blyszczuk, P., Cross, M., and Wobus, A. M.: Potential of embryonic and adult stem cells in vitro. Biol. Chem., 384, 1391–1409 (2003). 6. Hamazaki, T., Iiboshi, Y., Oka, M., Papst, P. J., Meacham, A. M., Zon, L. I., and Terada, N.: Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett., 497, 15–19 (2001). 7. Teratani, T., Yamamoto, H., Aoyagi, K., Sasaki, H., Asari, A., Quinn, G., Sasaki, H., Terada, M., and Ochiya, T.: Direct hepatic fate specification from mouse embryonic stem cells. Hepatology, 41, 836–846 (2005). 8. Soto-Gutie´rrez, A., Kobayashi, N., Rivas-Carrillo, J. D., Navarro-Alvarez, N., Zhao, D., Okitsu, T., Noguchi, H., Basma, H., Tabata, Y., Chen, Y., and other 9 authors: Reversal of mouse hepatic failure using an implanted liverassist device containing ES cell–derived hepatocytes. Nat. Biotechnol., 24, 1412–1419 (2006). 9. Rambhatla, L., Chiu, C. P., Kundu, P., Penq, Y., and Carpenter, M. K.: Genaration of hepatocyte-like cells from human embryonic stem cells. Cell Transplant., 12, 1–11 (2003). 10. Chinzei, R., Tanaka, Y., Shimizu-Saito, K., Hara, Y., Kakinuma, S., Watanabe, M., Teramoto, K., Arii, S., Takase, K., Sato, C., Terada, N., and Teraoka, H.: Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes. Hepatology, 36,
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22–29 (2002). 11. Yamada, T., Yoshikawa, M., Kanda, S., Kato, Y., Nakajima, Y., Ishizaka, S., and Tsunoda, Y.: Identified by cellular uptake of indocyanine green in vitro differentiation of embryonic stem cells into hepatocyte-like cells. Stem Cells, 20, 146–154 (2002). 12. Kania, G., Blyszczuk, P., Jochheim, A., Ott, M., and Wobus, A. M.: Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells. Biol. Chem., 385, 943–953 (2004). 13. Kumashiro, Y., Asahina, K., Ozeki, R., Shimizu-Saito, K., Tanaka, Y., Kida, Y., Inoue, K., Kaneko, M., Sato, T., Teramoto, K., Arii, S., and Teraoka, H.: Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source. Transplantation, 79, 550–557 (2005). 14. Asahina, K., Fujimori, H., Shimizu-Saito, K., Kumashiro, Y., Okamura, K., Tanaka, Y., Teramoto, K., Arii, S., and Teraoka, H.: Expression of the liver-specific gene Cyp7a1
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© 2008, The Society for Biotechnology, Japan
Hepatic Differentiation of Mouse Embryonic Stem Cells in a Three-Dimensional Culture System Using Polyurethane Foam Kinya Matsumoto,1 Hiroshi Mizumoto,1 Kohji Nakazawa,2 Hiroyuki Ijima,1 Kazumori Funatsu,1 and Toshihisa Kajiwara1* Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan1 and Department of Chemical Processes and Environments, University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0315, Japan2 Received 19 November 2007/Accepted 7 January 2008
Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0× 108 cells/cm3-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as α-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures. [Key words: hybrid artificial liver, embryonic stem cell, hepatic differentiation, spheroid, polyurethane foam, three dimensional culture]
of HALs is obtaining a cell source, and stem cells have recently received much attention as a novel cell source. Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts. They are characterized by an almost unlimited capacity for selfrenewal and potential to differentiate in vitro into multiple cell lineages (5). Recently, ES cells of mouse and human origin have been shown to differentiate into cells displaying hepatic phenotypes both in vitro and in vivo (6–14). These heptocyte-like cells derived from ES cells expressed some typical markers of mature hepatocytes and showed sufficient functional ability to rescue experimental liver injury when transplanted in vivo. These reports raised the hope of generating a transplantable cell source for the treatment of end-stage liver diseases. In this study, we focused our attention on ES cells as a potential source for HALs. For the differentiation of ES cells, it is important to form spherical multicellular aggregates. Here, we focused attention on the PUF/spheroid culture method which enables spontaneous spheroid formation and mass cultivation of cultured cells. Ultimately, we aimed
Acute liver failure (ALF) is a severe liver disease with a mortality of 60–90%, depending on the cause. The only effective therapy for ALF is liver transplantation. However, liver transplantation is hampered by the increasing shortage of organ donors, resulting in a high incidence of patients with ALF dying on the transplantation waiting list. Hybrid artificial livers (HAL) are expected to more successfully bridge patients with ALF to transplantation or even to promote regeneration of their own liver. A HAL is comprised of bioreactor-immobilized functional liver cells. We have previously developed a HAL by using polyurethane foam (PUF)/hepatocyte spheroid culture, in which the cultured cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF (1). The PUFHAL has been successfully scaled up to the clinical level (2). The curative effect of the PUF-HAL has been demonstrated in preclinical experiments on pigs (3, 4). Currently one of the most difficult problems in the clinical application * Corresponding author. e-mail: [email protected] phone: +81-(0)92-802-2746 fax: +81-(0)92-802-2796 350
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to develop a mass differentiation culture method from ES cells into functional hepatocytes by growth factor treatment along with spherical multicellular aggregates (spheroid) formation using the PUF/spheroid culture method. MATERIALS AND METHODS ES cell culture The ES cells (129 Line) (Chemicon, Pittsburgh, PA, USA) were maintained in an undifferentiated state in gelatin coated dishes. Undifferentiated ES cells were resuspended in Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, St. Louis, MO, USA) containing 2 mM L-glutamine (Sigma), 100 U/ml penicillin (WAKO Pure Chemical Industries, Osaka), 100 µg/ml streptomycin (Meiji Seika Kaisha, Tokyo), 20% fetal bovine serum and 300 µM monothioglycerol (Sigma) and immobilized in the PUF-plate. PUF was manufactured through a foaming process by blending isocyanate with polyol (Inoac, Nagoya). A PUF block was cut into a flat plate for the PUF stationary culture. Mouse ES cells were inoculated at a density of 2.0 ×106 cells in a 35-mm Easy Grip Style Bacteriological Petri Dish (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing the PUF plate in 2 ml of each culture medium. After 4 h of culture, the PUF plate with immobilized cells was transferred to a 60-mm Standard Style Bacteriological Petri Dish (Becton, Dickinson) containing 6 ml of each culture medium. The whole medium in the culture system was exchanged every 24 h of culture. As a control, mouse ES cells were cultured by the hanging drop method. ES cells were cultured for 2 d by the hanging drop method (1.0 ×103 ES cells per 30 µl in each drop). Embryoid bodies (EBs) in hanging drops were transferred to suspension culture in 60 mm collagen type I coated 60 mm dishes. To induce hepatic differentiation in each culture system, specific growth factors were added to culture medium (100 ng/ml acidic fibroblast growth factors [Invitrogen, Carlsbad, CA, USA] on days 9–12; 20 ng/ml hepatocyte growth factor [Toyobo, Osaka] on days 12–30; 10 ng/ml oncostatin M [R&D System, Minneapolis, MN, USA] on days 15–30; 10–7 M dexamethasone [Sigma] on days 15–30; and ITS [10 µg/ml insulin, 5.5 µg/ml transferrin, 6.7 ng/ml selenium acid; Invitrogen Corporation] on days 15–30) (Fig. 1). This method has been reported to differentiate mouse ES cells into mature hepatocytes by Hamazaki et al. (6). Primary mouse hepatocyte isolation Primary hepatocytes were isolated from 129 + Ter/Sv Jc1 mice (8 weeks old) (Kyudo, Kumamoto) by the liver perfusion method using collagenase (Wako) for use as a positive control. RT-PCR analysis Total RNA was extracted from the differentiating cells using NucleoSpin RNA II (Nippon Genetise, Tokyo), and cDNA synthesis was performed using the Superscript FirstStrand Synthesis System (Invitrogen) according to the manufacture’s protocol. cDNA was amplified using the Takara Ex Taq polymerase (Takarabio, Siga) kit. The following primer sets were used: transthyretin (TTR), forward 5′-CTGCTCTACCCGGGCTAT-3′,
FIG. 1. Experimental protocol of hepatic differentiation of ES cells by using PUF/spheroid culture.
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reverse 5′-TGAGCATCGGTCGCTCTT-3′, alpha fetoprotein (AFP), forward 5′-TCGTATTCCAACAGGAGG-3′, reverse 5′-AGGCTT TTGCTTCACCACG-3′, albumin (ALB), forward 5′-GCTACGG CACAGTGCTTG-3′, reverse gene 5′-CAGGATTGCAGACAGA TAGTC-3′, β-actin, forward 5′-TTCCTTCTTGGGTATGGAAT-3′, reverse 5′-GAGCAATGATCTTGATCTTC-3′, Arginase, forward 5′-GTGAAGAACCCACGGTCTGT-3′, reverse 5′-CTGGTTGTCA GGGGAGTGTT-3′, tryptophan 2,3-dioxgenase (TDO), forward 5′TGAATGCGCAAGAACTTCAG-3′ reverse 5′-TTCCAGAACCG AGAACTGCT-3′. Each cycle consisted of 45 s at 94°C, 45 s at 56°C, 45 s at 72°C (30 cycles: TTR, AFP, ALB, β-actin) and 45 s at 95°C, 45 s at 60°C, 60 s at 72°C (30 cycles: Arginase, TDO). Immunostaining After inducing differentiation using the PUF/spheroid culture, immunofluorescence analysis of the spheroids was performed by SRL (Tokyo). For the analysis, Polyclonal Rabbit Anti-Human Albumin and the DakoCytomation LSAB2 System-HRP (Dako, Glostrup, Denmark) were used. Determination of glycogen Glycogen production, which is one of the metabolic functions of hepatocytes, was analyzed by periodic acid-Schiff (PAS) staining after inducing hepatic differentiation. PAS staining were performed by SRL. Evaluation of liver-specific function To evaluate the liverspecific functions of differentiating ES cells, the culture medium was changed to fresh medium containing 1 mM NH4Cl. The decrease in ammonia concentration in the medium during a 24 h period was measured using the Wako ammonia test (Wako). The albumin concentration in the medium was measured by an enzymelinked immunosorbent assay by using the ELISA starter accessory package (Bethyl Laboratories, Montgomery, TX, USA) and the mouse albumin ELISA Quantitation Kit (Bethyl Laboratories). As a control for liver specific function, primary hepatocytes isolated from 129 +Ter/Sv Jc1 mice by the liver perfusion method using collagenase were cultured by using the PUF/spheroid culture method and analyzed in an identical manner to the ES cell-derived hepatocytes.
RESULTS Cell morphology and proliferation of mouse ES cells in PUF/spheroid culture The PUF/spheroid culture experiment was performed for 30 d. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF (Fig. 2A). The spheroids grew larger, and some spheroids were adhered to each other. However, at later stage of the culture, ES cell aggregates became irregular in form (Fig. 2B). Mouse ES cells proliferated from 5.68 ×105 to 1.07 ×108 cells/cm3, and a high cell density was achieved (Fig. 3). Gene expression of mouse ES cell-derived hepatocytes To assess hepatic differentiation, we used reverse tran-
FIG. 2. Morphology of mouse ES cells in PUF/spheroid culture. Mouse ES cells spontaneously formed spheroids in the pores of the PUF (day 7) (A). Spheroids adopted an irregular form at late stages of the culture (B). Scale bars: (A) 100 µm, (B) 200 µm.
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FIG. 3. Cell number of mouse ES cells. Mouse ES cells proliferated and high cell density was achieved (1.07 ×108 cells/cm3).
outmost area of the spheroids. Glycogen production, which is one of the metabolic functions of hepatocytes, was analyzed by periodic acid-Schiff (PAS) staining in ES cell-derived hepatocytes after differentiation. After differentiation (30 d), glycogen production was visible in the spheroids (Fig. 5B). Evaluation of liver specific functions Figure 6A shows the ammonia removal rate of differentiating mouse ES cells per unit volume in the PUF/spheroid culture. Ammonia removal activity in differentiating ES cells was detected by day 21 and increased with culture time. The ammonia removal rate of the differentiating mouse ES cells per unit cell number was one fiftieth that of primary mouse hepatocytes. However, because of the achievement of high cell density, the ammonia removal rate per unit volume was 1.28 (µmol/ cm3-PUF/d). The range for the ammonia removal rate of primary mouse hepatocytes was 0.85–2.79 (µmol/cm3-PUF/d). The activity of albumin secretion of differentiating ES cells was detected at day 30, and albumin secretion per unit volume was 67.2 (ng/cm3-PUF/d) (Fig. 6B). The range of the albumin secretion rate of primary mouse hepatocyte was 37.95–1153.31 (ng/cm3-PUF/d). DISCUSSION
FIG. 4. Comparison of liver-specific gene expression in differentiating ES cells between PUF/spheroid culture and HD/monolayer culture. Endoderm-specific gene was expressed from day 7 and hepatocytes-specific gene was expressed from day 15 in PUF spheroid culture. The expressions of both were increased by day 30. These data indicate that ES cells can differentiate toward cells of a hepatic lineage and mature hepatocytes.
scriptase (RT)-PCR at various times during the culture process to analyze endodermal and liver-specific genes. TTR represents endodermal or yolk-sac-like differentiation and is expressed throughout liver maturation. Alpha fetoprotein (AFP) is marker of endodermal differentiation and an early fetal hepatic marker. Albumin (ALB) and tryptophan 2,3-dioxygenase (TDO) are hepatocyte-specific genes. Undifferentiated ES cells did not express endodermal or hepatocytespecific genes. Figure 4 shows the expression of endodermal and hepatic differentiation markers in differentiating ES cells. TTR was expressed from day 7, and AFP was expressed from day 9. Endoderm-specific gene expression was increased during the culture time. Differentiating ES cells expressed hepatocyte-specific genes from day 18, and the expression was increased by 30 d. These data indicate that ES cells can differentiate toward the hepatic lineage and mature into hepatocytes. Immunostaining and periodic acid-Schiff (PAS) staining Figure 5A shows immunohistochemical examination of ALB in differentiating mouse ES cells at day 30. This result indicated that ES cell-derived hepatocytes were located in the
The formation of embryoid bodies (EBs) is the principal step in the differentiation of ES cells. An EB consists of ectodermal, mesodermal, and endodermal tissues, which recapitulate many aspects of cell differentiation during early mammalian embryogenesis and are able to differentiate into derivatives of all the three germ layers (15, 16). For this reason, EB formation has been utilized widely as a trigger of in vitro differentiation. In this study, mouse ES cells spontaneously formed spheroids in the pores of PUF, and a high cell density was achieved (1.07 ×108 cells/cm3) (Fig. 3). However, the spheroids became irregular in form (Fig. 2B) over the duration of the culture time. Undifferentiated ES cells express E-cadherin strongly, and differentiated ES cells reduce their E-cadherin expression (17, 18). Therefore, cellcell adhesion in PUF/spheroid cultures became weak by the end of the culture time because of a reduction in E-cadherin expression. Cell morphology results indicated that ES cells in the spheroids differentiated with culture time. A system to effect the differentiation from ES cells into mature hepatocytes by EB formation has been reported (6). Here we demonstrate that ES cells could be induced into functional hepatocytes by growth factor treatment along with spheroid formation using the PUF/spheroid culture method. As shown in Fig. 4, endodermal-specific genes and late stage markers of hepatic differentiation were expressed in cells cultured using the PUF/spheroid culture at the same time that they are expressed in traditional HD/monolayer culture differentiation systems. Ammonia removal is one of the most important functions of a HAL. Our result showed that mouse ES cells expressed the ammonia removal function in the PUF/spheroid culture method. The maximum removal level was comparable to that of primary mouse hepatocytes because of the high cell density. We have previously developed a HAL by using PUF/spheroid cultures, and this result further indicated that
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FIG. 5. Immunohistochemical analysis (A), evaluation of glycogen storage (B) and amylase digestion (C). Albumin positive cells were located at the periphery of spheroids on day 30 (allows) (A). Glycogen production was visible in the spheroids after induction of hepatic differentiation on day 30 (allows) (B). The deep purple-blue color of glycogen was disappeared by treatment of amylase digestion (C). Scale bars: 200 µm.
ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research B: 19360375 and a Grant-in-Aid for Scientific Research A2: 14205119 from the Japan Society for the Promotion of Science.
REFERENCES
FIG. 6. Ammonia removal rate (A) and albumin secretion rate per unit volume of PUF plate (B). The ammonia removal activity was detected by day 21 and increased over the remainder of the culture time (A). The ammonia removal rate per unit volume was 1.28 (µmol/cm3PUF/d). This activity was comparable to that of mouse primary hepatocytes. The ammonia removal was not detected on day 15. The albumin secretion activity was detected at day 30, and the albumin secretion per unit volume was 67.2 (ng/cm3-PUF/d) (B). The albumin secretion was not detected on day 15 and 21.
ES cells immobilized in our PUF-HAL may be useful as a biocomponent of a hybrid artificial liver. However, the albumin secretory function was lower than that of primary mouse hepatocytes. AFP is an early hepatic marker, and its expression decreases as the liver develops into the adult phenotype. However, AFP expression did not decrease at the late stage of our culture (Fig. 4). This result indicated that differentiating ES cells might not be mature enough to secrete albumin. We should improve this problem in the future. In conclusion, the method combining of PUF/spheroid culture and hepatic growth factors would be useful for differentiating ES cells into functional hepatocytes.
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