- Email: [email protected]
863 HEPATOCYTE RXRa – A KEY ELEMENT OF LIVER REGENERATION
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Poster Session − Saturday, April 25
was harvested at this time for subsequent analysis. Fibrosis was detected by picrosirius red staining. 25 randomly selected non-overlapping areas were photographed at ×20 magnification. The area of red staining was quantified by image analysis (Adobe Photoshop). Albumin measurements were performed by ELISA. Results: Cells cultured for 7 days in conditioned media possessed the morphologic and phenotypic characteristics of macrophages; over 80% were F4/80 and CD11b positive on flow cytometry. One month after cell therapy, liver fibrosis was significantly lower than controls (mean±SEM: 2.5±0.4 v 3.7±0.3%, p < 0.05). Serum albumin levels significantly increased in mice receiving macrophages (39±1 to 46±2.6g/l, p < 0.05). Conclusions: BMDM cell therapy improves clinically meaningful parameters of chronic liver disease in this murine model. Importantly, this effect is mediated by a single cell type, bringing clarity to the cause-effect relationship. These data will inform clinically applicable cell therapies. 861 CHIMERIC PHENOTYPE OF MOUSE MESENCHYMAL STEM CELLS FROM ADIPOSE TISSUE AFTER HEPATOGENIC DIFFERENTIATION L. Bachmetov1 , H. Gitman1 , R. Zemel1 , R. Tur-Kaspa1,2 . 1 Molecular Hepatology Research Laboratory, Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, 2 Medicine D and Liver Institute, Tel-Aviv University, Rabin Medical Center, Petach-Tikva, Israel E-mail: [email protected] Background: Recent studies demonstrate that several stem cells can differentiate into hepatocytes and may play a role in recovery, proliferation and restoration of liver mass following liver injury. Increasing evidence suggests that adipose tissue contains an accessible source of mesenchymal stem cells similar to BM-derived stem cells that have the ability to differentiate into tissue-specific cell types, including hepatocytes. Aims: To evaluate the efficacy of hepatic differentiation of mouse adipose derived mesenchymal stem (mADMSC) in vitro. Methods: Mesenchymal Stem cells were isolated from inguinal fat pads of C57BL mice and characterized. Hepatogenic differentiation of mADMSC was performed by sequential addition of differentiation factors to cells grown for 3 weeks on collagen or fibronectin-coated plats. Efficiency of hepatic differentiation was evaluated using RT-PCR, and cytochemistry analysis for hepatocyte markers and by functional analysis such as glycogen storage, ICG uptake and urea production. Results: Freshly isolated mADMSC demonstrated fibroblast-like morphology and expressed typical mesenchymal stem cell surface markers such as CD 105, CD 90, CD 44, and CD 29. The mesenchymal cells efficiently differentiated into mesodermal cell lineage such as osteocytes and adipocytes. Upon hepatic differentiation the cells showed constitutive expression of AFP, ALB, CK18, CK19 and G6Pase, while genes representing mature and functional hepatic activity such as CYP 450 7a1,TDO, HNF3 was expressed only after 3 weeks of differentiation. These cells also demonstrated hepatocytes functions such as glycogen storage, ICG uptake and urea production. Gene expression and cytochemistry analyses revealed that the differentiated mADMSC retained the expression of the mesenchymal markers along with the expression of the hepatic markers. Conclusions: Our results suggest that mADMSC display a potential to differentiate into hepatocyte-like cells in response to defined culture conditions. We observed that while in vitro mADMSCs acquired some phenotypic and functional liver-specific markers, they retained expression of mesenchymal markers and may represent the transitional state between MSCs and mature hepatocytes.
862 RELATION BETWEEN LIVER PROGENITOR CELL EXPANSION AND EXTRACELLULAR MATRIX DEPOSITION IN A CDE INDUCED MURINE MODEL OF CHRONIC LIVER INJURY N. Van Hul1 , J. Abarca-Quinones1 , C. Sempoux2 , Y. Horsmans1 , I. Leclercq1 . 1 Laboratory of Gastroenterology, 2 Laboratory of Pathology, Universit´e Catholique de Louvain, Brussel, Belgium E-mail: [email protected] Background and Aims: In chronic liver injury, liver progenitor cells (LPCs) proliferate in the periportal area and migrate inside the lobule where they undergo further differentiation. This process is associated with extracellular matrix (ECM) remodeling. Here we analyzed, in a time-line fashion, LPCs expansion and matrix accumulation in the choline deficient, ethionine supplemented (CDE) murine model of LPC proliferation. Methods: A CDE diet was administrated to C57Bl6 mice for 0, 3, 7, 10, 14, 18 or 21 days. Livers were retrieved and gene expression was evaluated by RT-PCR while (immuno)histochemistry was used to reveal the LPCs and ECM. Results: After three days of CDE diet, collagen deposition can already be observed in the periportal area. Expansion of LPCs, as assessed by an increase of cytokeratin 19 (CK19) positive cells, did not occur until day 7. At that time, ECM already accumulated to values ten times higher than in controls. Thereafter, LPCs and ECM increased in parallel. Further, ECM not only appears prior to the increase of LPCs, but is also found in front of LPCs along the porto-veinous gradient of lobular invasion. Double immunostaining revealed that, at all time, LPCs are embedded in ECM. Moreover, LPCs infiltrating the liver parenchyma are chaperoned by a-SMA positive cells. Gene expression analyses confirmed those observations. Albumin expression took a fall at day 3, confirming the selective damage to hepatocytes due to the CDE diet, and showed slow recover through the following days. The expression of CK19, a-fetoprotein, E-cadherin or CD49f mRNA, largely over-expressed by LPCs, significantly increased starting from day 7. By contrast, there was a rapid burst in the expression of components of the ECM, collagen I, laminin and CTGF mRNA at day 3, as well as in a-SMA expression. Conclusions: Our data demonstrate that, in the CDE model, ECM deposition and activation of matrix producing cells occurred as an initial phase, prior to LPCs expansion, and in front of LPCs along the portoveinous gradient of lobular invasion. Those observations may reveal a fundamental role for the established hepatic microenvironment or niche during the process of activation, proliferation and differentiation of liver progenitor cells. 863 HEPATOCYTE RXRa − A KEY ELEMENT OF LIVER REGENERATION Y.-J. Wan, X. Yang, M. Guo. Pharmacology, University of Kansas, Kansas City, KA, USA E-mail: [email protected] Activation of nuclear receptors including constitutive androstane receptor (CAR), pregnane x receptor (PXR), and peroxisome proliferator activated receptor a (PPARa) results in hepatomegaly, and these nuclear receptors have been implicated in the regulation of hepatocyte proliferation and liver regeneration. The current study examines the role of hepatocyte retinoid x receptor a (RXRa), an essential partner for nuclear receptors, in liver regeneration using partial hepatectomy model. The results showed that hepatocyte RXRa deficiency caused delayed DNA synthesis, mitosis, and hepatocyte proliferation in respond to particle hepatectomy. Our findings showed that there are several mechanisms accounted for this delay. Activation of IL-6/Stat3-mediated cytokine pathway as well as growth factor (HGF) signaling was impaired in mouse livers lack of RXRa. The expression and regulation of Cyclin D1/CDK4, Cyclin E1/CDK2, Cyclin A2/CDK2, and CyclinB1/CDK1 after partial hepatectomy were altered due to hepatocyte RXRa deficiency. In addition, circadian-regulated cell cycle progression was also compromised. Thus, hepatocyte RXRa has an impact
04d: MOLECULAR AND CELLULAR BIOLOGY − d) LIVER REGENERATION on G1, G2, and M check points and can be considered as a cell cycle modulator during liver regeneration. In summary, hepatocyte RXRa regulates cytokine and growth factor signaling pathways as well as PPARa-regulated circadian cell cycle progression during liver regeneration. 864 UNIMMORTALIZED HEPATIC OVAL CELLS MAINTAIN THE PROGENITOR CELL PHENOTYPES AFTER TWO-YEAR PROLONGED CULTIVATION IN VITRO P. Wang, M. Cong, T. Liu, S. Tang, Y. Xu, B. Wang, J. Jia, H. You. Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China E-mail: [email protected] Background: Hepatic oval cells have been reported to be isolated, propagated and differentiated into hepatocytes and cholangiocytes in culture, thus providing an attractive renewable source of stem cell therapy. But, after prolonged cultivation whether their proliferation ability and gene expression profile shift away from progenitor cells is not addressed. Methods: Hepatic oval cells were isolated by collagenase/proteinase perfusion and Percoll density gradient separation from rat fed with cholinedeficient diet supplemented with 0.1% ethonine. The cells were continuously propagated every 5 days to 100 passages over 2 years in DMEM/F12 supplemented with 10% fetal bovine serum, 0.5 U/ml insulin, 1 ng/ml epidermal growth factor, 0.5 ng/ml stem cell factor. The proliferation ability of hepatic oval cells at different passages was evaluated by growth curve and cell cycle analysis, and the anchorage-independent growth was determined by soft agar assay. The gene expression profiles of hepatic oval cells were analyzed by flow cytometry, real-time PCR and western blot. Results: After serial passages, hepatic oval cells sustained the typical epithelial morphology, except the cell size slightly larger after 50th passage. The results of growth curve and cell cycle analysis showed that hepatic oval cells survived after serial passages without loss of their proliferation ability and maintained diploid cells with features of chromosomal stability. Flow cytometry analysis demonstrated that hepatic oval cells maintained expression of the common marker for hepatic oval cells, OV-6, and bile ductal antigen, BD1 and BD2. Furthermore, real-time PCR and western blot results showed that hepatic oval cells continuously expressed the epithelial cell marker E-cadherin, hepatic progenitor cell marker fetal M2 isoform of pyruvate kinase, cholangiocyte marker cytokeratin 19, and hepatocyte markers alpha-fetoprotein and albumin from 2nd to 100th passage. No significant difference in colony number and colony size was found in hepatic oval cells from 2nd to 100th passage, indicating serial passage did not cause malignant transformation. Conclusion: These results suggested after long-term cultivation hepatic oval cells retain their progenitor cell phenotypes, including the proliferation ability and the expression profiles of the progenitor cell markers. Therefore, hepatic oval cells without immortalized manipulation may provide an expandable cell source for liver cell therapies. 865 DIFFERENTIATION OF MOUSE AND HUMAN EMBRYONIC STEM CELLS TO HEPATOCYTES INDUCED BY COMBINATION OF CYTOKINES AND SODIUM BUTYRATE M. Zhou1 , Q.-L. Ying2 , L. Tan1 , S. Qu1 , X. Yang3 , J. Fan3 , H. Song1 . 1 Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, China; 2 Center for Stem Cell and Regenerative Medicine, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, USA; 3 Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China E-mail: [email protected] There are increasing evidences that embryonic stem cells(ESCs) are capable of differentiating into hepatocytes in vitro. Here we presented a novel 3-step procedure to efficiently direct the differentiation of mouse and human ESCs into hepatocytes induced by combination of cytokines and sodium butyrate. Mouse and human ESCs were first differentiated into
S315
definitive endoderm cells by 3 days of Activin A treatment; next, presence of acid fibroblast growth factor(aFGF)and sodium butyrate(NaB)in the culture medium for 5 days, definitive endoderm cells efficiently differentiated to hepatocytes. After 10 days of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using light microscopy, electron microscopy, immunofluorescence, immunoblotting and RT-PCR, respectively. Furthermore, these cells were tested about the functions associated with mature hepatocytes including glycogen storage, indocyanine green uptake and release, and the ratio of hepatic differentiation was determined by counting the albumin-positive cells.Then we investigated the curative effects of transplantation of hepatocytes differentiated from ESCs on treatment of chronic liver failure induced by CCl4. In the presence of conditional medium containing cytokines and sodium butyrate, numerous epithelial cells resembling hepatocytes were observed at 18th day. The RT-PCR analysis showed that these cells expressed the adult liver cell markers tyrosine aminotransferase (TAT), albumin (ALB), alpha-1 antitrypsin (AAT), transthyretin (TTR) and glucose-6-phosphate (G-6-P). Immunofluorescence staining were shown that those ES-derived hepatocytes were a-fetoprotein, albumin, and cytokeratin-18 positive, with the ability of storing glycogen and indocyanine green uptake and release. Approximately 74% of the cells expressed the hepatic marker albumin at 18th day. The transplanted hepatocytes were incorporated well into the liver parenchymal structure and expressed albumin for at least 2 weeks. Injured mice transplanted with ES-derived hepatocytes, the symptom of chronic liver failure could be improved obviously. We have developed an efficient way to direct the differentiation of mouse and human ESCs into cells that exhibit characteristics of mature hepatocytes. It would facilitate searching the molecular mechanisms underlying liver development, and provide a source of hepatocytes for transplantation therapy and drug screening. 866 INCREASED HEPATIC GLUCOSYLCERAMIDE LEVELS IN GBA2 DEFICIENT MICE ARE BENEFICIAL DURING THE EARLY PHASE OF LIVER REGENERATION A.M. Oprisoreanu, E. Almajan, N. Popovici, T. Sauerbruch, Y. Yildiz. Internal Medicine I, University of Bonn, Bonn, Germany E-mail: [email protected] Introduction: Gba2 knockout mice accumulate the glycolipid glucosylceramide in different tissues, including the liver (Yildiz et al. JCI 2006). Glycolipids are a class of cellular membrane lipids that differ from the regular membrane phospholipids in a remarkable variation in its head group structures. This suggests that glycolipids serve specialized functions in cell signaling, differentiation and recognition processes (van Meer et al. 2003). Different studies have suggested a special role of glycolipids in cell cycle and cell proliferation. Our aim was to investigate the role of glucosylceramide in liver regeneration using the two-thirds partial hepatectomy model according to the method of Higgins and Anderson. Methods: Groups of five 10−12 week old Gba2 knockout and wild-type animals were anesthetized and subjected to 70% partial hepatectomy. At 0, 6 h, 12 h, 24 h, 48 h, 3d, 5d and 7d post-hepatectomy mice were killed and the remnant livers were snap frozen or fixed in 4% paraformaldehyde. Cell proliferation was determined by bromodeoxyuridine (BrDU) incorporation, and cell cycle-related proteins were analyzed by immunoblots. Results and Conclusions: The proliferation rate and the expression of cell cycle-related proteins, such as cyclin D, are significantly increased in GBA2 deficient mice until the second day post-hepatectomy. This difference between GBA2 deficient mice and control animals disappears after the third day of liver regeneration. At the same time, the liver regeneration index (liver to body weight ratio) starts significantly to decrease in GBA2 deficient mice as compared to wild-type animals. In conclusion, our data suggests that the accumulation of glucosylceramide may be beneficial in the early phase of liver regeneration. Later on, other factors appear to outweigh the potential advantage of glucosylceramide, which are absent in the Gba2 knockout mice.
Poster Session − Saturday, April 25
was harvested at this time for subsequent analysis. Fibrosis was detected by picrosirius red staining. 25 randomly selected non-overlapping areas were photographed at ×20 magnification. The area of red staining was quantified by image analysis (Adobe Photoshop). Albumin measurements were performed by ELISA. Results: Cells cultured for 7 days in conditioned media possessed the morphologic and phenotypic characteristics of macrophages; over 80% were F4/80 and CD11b positive on flow cytometry. One month after cell therapy, liver fibrosis was significantly lower than controls (mean±SEM: 2.5±0.4 v 3.7±0.3%, p < 0.05). Serum albumin levels significantly increased in mice receiving macrophages (39±1 to 46±2.6g/l, p < 0.05). Conclusions: BMDM cell therapy improves clinically meaningful parameters of chronic liver disease in this murine model. Importantly, this effect is mediated by a single cell type, bringing clarity to the cause-effect relationship. These data will inform clinically applicable cell therapies. 861 CHIMERIC PHENOTYPE OF MOUSE MESENCHYMAL STEM CELLS FROM ADIPOSE TISSUE AFTER HEPATOGENIC DIFFERENTIATION L. Bachmetov1 , H. Gitman1 , R. Zemel1 , R. Tur-Kaspa1,2 . 1 Molecular Hepatology Research Laboratory, Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, 2 Medicine D and Liver Institute, Tel-Aviv University, Rabin Medical Center, Petach-Tikva, Israel E-mail: [email protected] Background: Recent studies demonstrate that several stem cells can differentiate into hepatocytes and may play a role in recovery, proliferation and restoration of liver mass following liver injury. Increasing evidence suggests that adipose tissue contains an accessible source of mesenchymal stem cells similar to BM-derived stem cells that have the ability to differentiate into tissue-specific cell types, including hepatocytes. Aims: To evaluate the efficacy of hepatic differentiation of mouse adipose derived mesenchymal stem (mADMSC) in vitro. Methods: Mesenchymal Stem cells were isolated from inguinal fat pads of C57BL mice and characterized. Hepatogenic differentiation of mADMSC was performed by sequential addition of differentiation factors to cells grown for 3 weeks on collagen or fibronectin-coated plats. Efficiency of hepatic differentiation was evaluated using RT-PCR, and cytochemistry analysis for hepatocyte markers and by functional analysis such as glycogen storage, ICG uptake and urea production. Results: Freshly isolated mADMSC demonstrated fibroblast-like morphology and expressed typical mesenchymal stem cell surface markers such as CD 105, CD 90, CD 44, and CD 29. The mesenchymal cells efficiently differentiated into mesodermal cell lineage such as osteocytes and adipocytes. Upon hepatic differentiation the cells showed constitutive expression of AFP, ALB, CK18, CK19 and G6Pase, while genes representing mature and functional hepatic activity such as CYP 450 7a1,TDO, HNF3 was expressed only after 3 weeks of differentiation. These cells also demonstrated hepatocytes functions such as glycogen storage, ICG uptake and urea production. Gene expression and cytochemistry analyses revealed that the differentiated mADMSC retained the expression of the mesenchymal markers along with the expression of the hepatic markers. Conclusions: Our results suggest that mADMSC display a potential to differentiate into hepatocyte-like cells in response to defined culture conditions. We observed that while in vitro mADMSCs acquired some phenotypic and functional liver-specific markers, they retained expression of mesenchymal markers and may represent the transitional state between MSCs and mature hepatocytes.
862 RELATION BETWEEN LIVER PROGENITOR CELL EXPANSION AND EXTRACELLULAR MATRIX DEPOSITION IN A CDE INDUCED MURINE MODEL OF CHRONIC LIVER INJURY N. Van Hul1 , J. Abarca-Quinones1 , C. Sempoux2 , Y. Horsmans1 , I. Leclercq1 . 1 Laboratory of Gastroenterology, 2 Laboratory of Pathology, Universit´e Catholique de Louvain, Brussel, Belgium E-mail: [email protected] Background and Aims: In chronic liver injury, liver progenitor cells (LPCs) proliferate in the periportal area and migrate inside the lobule where they undergo further differentiation. This process is associated with extracellular matrix (ECM) remodeling. Here we analyzed, in a time-line fashion, LPCs expansion and matrix accumulation in the choline deficient, ethionine supplemented (CDE) murine model of LPC proliferation. Methods: A CDE diet was administrated to C57Bl6 mice for 0, 3, 7, 10, 14, 18 or 21 days. Livers were retrieved and gene expression was evaluated by RT-PCR while (immuno)histochemistry was used to reveal the LPCs and ECM. Results: After three days of CDE diet, collagen deposition can already be observed in the periportal area. Expansion of LPCs, as assessed by an increase of cytokeratin 19 (CK19) positive cells, did not occur until day 7. At that time, ECM already accumulated to values ten times higher than in controls. Thereafter, LPCs and ECM increased in parallel. Further, ECM not only appears prior to the increase of LPCs, but is also found in front of LPCs along the porto-veinous gradient of lobular invasion. Double immunostaining revealed that, at all time, LPCs are embedded in ECM. Moreover, LPCs infiltrating the liver parenchyma are chaperoned by a-SMA positive cells. Gene expression analyses confirmed those observations. Albumin expression took a fall at day 3, confirming the selective damage to hepatocytes due to the CDE diet, and showed slow recover through the following days. The expression of CK19, a-fetoprotein, E-cadherin or CD49f mRNA, largely over-expressed by LPCs, significantly increased starting from day 7. By contrast, there was a rapid burst in the expression of components of the ECM, collagen I, laminin and CTGF mRNA at day 3, as well as in a-SMA expression. Conclusions: Our data demonstrate that, in the CDE model, ECM deposition and activation of matrix producing cells occurred as an initial phase, prior to LPCs expansion, and in front of LPCs along the portoveinous gradient of lobular invasion. Those observations may reveal a fundamental role for the established hepatic microenvironment or niche during the process of activation, proliferation and differentiation of liver progenitor cells. 863 HEPATOCYTE RXRa − A KEY ELEMENT OF LIVER REGENERATION Y.-J. Wan, X. Yang, M. Guo. Pharmacology, University of Kansas, Kansas City, KA, USA E-mail: [email protected] Activation of nuclear receptors including constitutive androstane receptor (CAR), pregnane x receptor (PXR), and peroxisome proliferator activated receptor a (PPARa) results in hepatomegaly, and these nuclear receptors have been implicated in the regulation of hepatocyte proliferation and liver regeneration. The current study examines the role of hepatocyte retinoid x receptor a (RXRa), an essential partner for nuclear receptors, in liver regeneration using partial hepatectomy model. The results showed that hepatocyte RXRa deficiency caused delayed DNA synthesis, mitosis, and hepatocyte proliferation in respond to particle hepatectomy. Our findings showed that there are several mechanisms accounted for this delay. Activation of IL-6/Stat3-mediated cytokine pathway as well as growth factor (HGF) signaling was impaired in mouse livers lack of RXRa. The expression and regulation of Cyclin D1/CDK4, Cyclin E1/CDK2, Cyclin A2/CDK2, and CyclinB1/CDK1 after partial hepatectomy were altered due to hepatocyte RXRa deficiency. In addition, circadian-regulated cell cycle progression was also compromised. Thus, hepatocyte RXRa has an impact
04d: MOLECULAR AND CELLULAR BIOLOGY − d) LIVER REGENERATION on G1, G2, and M check points and can be considered as a cell cycle modulator during liver regeneration. In summary, hepatocyte RXRa regulates cytokine and growth factor signaling pathways as well as PPARa-regulated circadian cell cycle progression during liver regeneration. 864 UNIMMORTALIZED HEPATIC OVAL CELLS MAINTAIN THE PROGENITOR CELL PHENOTYPES AFTER TWO-YEAR PROLONGED CULTIVATION IN VITRO P. Wang, M. Cong, T. Liu, S. Tang, Y. Xu, B. Wang, J. Jia, H. You. Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China E-mail: [email protected] Background: Hepatic oval cells have been reported to be isolated, propagated and differentiated into hepatocytes and cholangiocytes in culture, thus providing an attractive renewable source of stem cell therapy. But, after prolonged cultivation whether their proliferation ability and gene expression profile shift away from progenitor cells is not addressed. Methods: Hepatic oval cells were isolated by collagenase/proteinase perfusion and Percoll density gradient separation from rat fed with cholinedeficient diet supplemented with 0.1% ethonine. The cells were continuously propagated every 5 days to 100 passages over 2 years in DMEM/F12 supplemented with 10% fetal bovine serum, 0.5 U/ml insulin, 1 ng/ml epidermal growth factor, 0.5 ng/ml stem cell factor. The proliferation ability of hepatic oval cells at different passages was evaluated by growth curve and cell cycle analysis, and the anchorage-independent growth was determined by soft agar assay. The gene expression profiles of hepatic oval cells were analyzed by flow cytometry, real-time PCR and western blot. Results: After serial passages, hepatic oval cells sustained the typical epithelial morphology, except the cell size slightly larger after 50th passage. The results of growth curve and cell cycle analysis showed that hepatic oval cells survived after serial passages without loss of their proliferation ability and maintained diploid cells with features of chromosomal stability. Flow cytometry analysis demonstrated that hepatic oval cells maintained expression of the common marker for hepatic oval cells, OV-6, and bile ductal antigen, BD1 and BD2. Furthermore, real-time PCR and western blot results showed that hepatic oval cells continuously expressed the epithelial cell marker E-cadherin, hepatic progenitor cell marker fetal M2 isoform of pyruvate kinase, cholangiocyte marker cytokeratin 19, and hepatocyte markers alpha-fetoprotein and albumin from 2nd to 100th passage. No significant difference in colony number and colony size was found in hepatic oval cells from 2nd to 100th passage, indicating serial passage did not cause malignant transformation. Conclusion: These results suggested after long-term cultivation hepatic oval cells retain their progenitor cell phenotypes, including the proliferation ability and the expression profiles of the progenitor cell markers. Therefore, hepatic oval cells without immortalized manipulation may provide an expandable cell source for liver cell therapies. 865 DIFFERENTIATION OF MOUSE AND HUMAN EMBRYONIC STEM CELLS TO HEPATOCYTES INDUCED BY COMBINATION OF CYTOKINES AND SODIUM BUTYRATE M. Zhou1 , Q.-L. Ying2 , L. Tan1 , S. Qu1 , X. Yang3 , J. Fan3 , H. Song1 . 1 Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, China; 2 Center for Stem Cell and Regenerative Medicine, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, USA; 3 Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China E-mail: [email protected] There are increasing evidences that embryonic stem cells(ESCs) are capable of differentiating into hepatocytes in vitro. Here we presented a novel 3-step procedure to efficiently direct the differentiation of mouse and human ESCs into hepatocytes induced by combination of cytokines and sodium butyrate. Mouse and human ESCs were first differentiated into
S315
definitive endoderm cells by 3 days of Activin A treatment; next, presence of acid fibroblast growth factor(aFGF)and sodium butyrate(NaB)in the culture medium for 5 days, definitive endoderm cells efficiently differentiated to hepatocytes. After 10 days of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using light microscopy, electron microscopy, immunofluorescence, immunoblotting and RT-PCR, respectively. Furthermore, these cells were tested about the functions associated with mature hepatocytes including glycogen storage, indocyanine green uptake and release, and the ratio of hepatic differentiation was determined by counting the albumin-positive cells.Then we investigated the curative effects of transplantation of hepatocytes differentiated from ESCs on treatment of chronic liver failure induced by CCl4. In the presence of conditional medium containing cytokines and sodium butyrate, numerous epithelial cells resembling hepatocytes were observed at 18th day. The RT-PCR analysis showed that these cells expressed the adult liver cell markers tyrosine aminotransferase (TAT), albumin (ALB), alpha-1 antitrypsin (AAT), transthyretin (TTR) and glucose-6-phosphate (G-6-P). Immunofluorescence staining were shown that those ES-derived hepatocytes were a-fetoprotein, albumin, and cytokeratin-18 positive, with the ability of storing glycogen and indocyanine green uptake and release. Approximately 74% of the cells expressed the hepatic marker albumin at 18th day. The transplanted hepatocytes were incorporated well into the liver parenchymal structure and expressed albumin for at least 2 weeks. Injured mice transplanted with ES-derived hepatocytes, the symptom of chronic liver failure could be improved obviously. We have developed an efficient way to direct the differentiation of mouse and human ESCs into cells that exhibit characteristics of mature hepatocytes. It would facilitate searching the molecular mechanisms underlying liver development, and provide a source of hepatocytes for transplantation therapy and drug screening. 866 INCREASED HEPATIC GLUCOSYLCERAMIDE LEVELS IN GBA2 DEFICIENT MICE ARE BENEFICIAL DURING THE EARLY PHASE OF LIVER REGENERATION A.M. Oprisoreanu, E. Almajan, N. Popovici, T. Sauerbruch, Y. Yildiz. Internal Medicine I, University of Bonn, Bonn, Germany E-mail: [email protected] Introduction: Gba2 knockout mice accumulate the glycolipid glucosylceramide in different tissues, including the liver (Yildiz et al. JCI 2006). Glycolipids are a class of cellular membrane lipids that differ from the regular membrane phospholipids in a remarkable variation in its head group structures. This suggests that glycolipids serve specialized functions in cell signaling, differentiation and recognition processes (van Meer et al. 2003). Different studies have suggested a special role of glycolipids in cell cycle and cell proliferation. Our aim was to investigate the role of glucosylceramide in liver regeneration using the two-thirds partial hepatectomy model according to the method of Higgins and Anderson. Methods: Groups of five 10−12 week old Gba2 knockout and wild-type animals were anesthetized and subjected to 70% partial hepatectomy. At 0, 6 h, 12 h, 24 h, 48 h, 3d, 5d and 7d post-hepatectomy mice were killed and the remnant livers were snap frozen or fixed in 4% paraformaldehyde. Cell proliferation was determined by bromodeoxyuridine (BrDU) incorporation, and cell cycle-related proteins were analyzed by immunoblots. Results and Conclusions: The proliferation rate and the expression of cell cycle-related proteins, such as cyclin D, are significantly increased in GBA2 deficient mice until the second day post-hepatectomy. This difference between GBA2 deficient mice and control animals disappears after the third day of liver regeneration. At the same time, the liver regeneration index (liver to body weight ratio) starts significantly to decrease in GBA2 deficient mice as compared to wild-type animals. In conclusion, our data suggests that the accumulation of glucosylceramide may be beneficial in the early phase of liver regeneration. Later on, other factors appear to outweigh the potential advantage of glucosylceramide, which are absent in the Gba2 knockout mice.