- Email: [email protected]
PROGENITOR CELLS INCREASE HEPATIC METABOLIC FUNCTION AFTER IN VITRO DIFFERENTIATION
S308
Poster Session − Saturday, April 25
glass microscope slide. Primary screening identified 6 polymers (three polyacrylates 2AE7, 7G7, 3AA7 and three polyurethanes 134, 212, 223) that supported HLC attachment and identity. We further characterised hepatocytic function in detail on these six matrices. Using this strategy, polymer 134 was identified as the most effective cellular support associated with enhanced expression of Fibrinogen, transthyretin (TTR) and soluble fibronectin. Key cytochrome p450 (CYP) activities on the different extracellular matrices were also characterised. CYP1A2 activity was increased ~6 fold and CYP3A4 ~2 fold on polymer 134 as compared to standard matrigel conditions or the other polymers tested. Conclusion: In conclusion, the high through-put screening of a polymer microarray allowed the identification of a new polymer matrix that promotes long-term hepatocellular differentiated function before and after passaging. These attributes bypass current limitations associated adult human hepatocytes, can be manufactured to GMP standards and will play important roles in developing in vitro models of drug toxicicology, provide a resource for the construction of extra-corporeal devices and facilitate novel studies of human liver development and disease. 845 HINT1 IS A PROTEIN POTENTIALLY IMPLICATED IN LIVER REGENERATION C. Hora1 , O. Maurhofer1 , M. Ledermann1 , G. Ferrand2 , H. Li3 , I.B. Weinstein3,4 , J.-F. Dufour1 , J. Martin1 . 1 Institute of Clinical Pharmacology and Visceral Research, University of Berne, Berne, 2 Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; 3 Herbert Irving Comprehensive Cancer Center, Columbia University, 4 Department of Medicine, Columbia University, New York, NY, USA E-mail: [email protected] Introduction: Liver regeneration plays an important role in liver transplantation and liver cancer treatment. HINT1, a nucleotide binding protein belonging to the HIT protein family functions as a tumour suppressor and participates in the cellular response to DNA damage. The aim of our study was to investigate whether liver regeneration is affected by the lack of Hint1. Methods: 2/3 partial hepatectomy (PH) was performed in male Hint−/ − and C57BL/6 mice (9−10 weeks-old, n = 7−10). Animals were sacrificed at 3, 6, 24, 48, 72 and 96 hours post PH and the remaining livers weighed. Restoration of liver mass was calculated. Cell proliferation was determined by immunohistochemistry using BrdU and Ki67 as markers. Liver histology and apoptosis were evaluated by H&E and cleaved lamin A (cLA) staining. Gene and protein expression of CyclinD1, p21, IL6 and TNFa were analysed by QPCR and immunobloting. Data are means±SD, p0.05. Results: In Hint1−/ − mice, the liver mass did not increase until 24 h after surgery. Increases of 30±18%, 82±20% and 106±10% were recorded at 48 h, 72 h and 96 h after PH, respectively. In C57BL/6 the liver mass increased without delay by 19±7, 36±10, 43±3.5, 59±3%, 119±13.6% and 144±12% at 3 h, 6 h, 24 h, 48 h, 72 h and 96 h after PH, respectively. Proliferation assessed by BrdU and Ki67 and apoptosis assessed with cLA were not significantly different between the 2 groups. At the time of surgery, there was less IL6 and TNFa mRNA mRNA in Hint1−/ − mice vs C57BL6 mice. After PH, levels of IL6 and TNFa mRNA followed a similar pattern in both mice but remained lower in Hint1−/ − . Earlier and higher augmentation of p21 mRNA at 24 h was observed in Hint1−/ − . CyclinD1 mRNA expression was enhanced after 24 h and 72 h PH in Hint1−/ − whereas this expression peaked after 48 h in C57BL6 mice. A higher peak of p21 and CyclinD1 protein expression was measured after 24 h and 48 h in Hint1−/ − in comparison to C57BL6. Conclusion: Liver regeneration is impaired in the case of Hint1 ablation. Our results suggest that Hint1 plays a role in the initiation of liver regeneration by means of a mechanism that affects cytokines production.
846 ATP RELEASE AFTER PARTIAL HEPATECTOMY REGULATES LIVER REGENERATION E. Gonzales1 , B. Julien1 , V. Serri`ere-Lanneau1 , A. Nicou1 , I. Doignon1 , L. Lagoudakis1 , I. Garcin1 , D. Azoulay2 , J.-C. Duclos-Vall´ee2 , D. Castaing2 , D. Samuel2 , A. Hernandez-Garcia3 , S. Awad3 , L. Combettes1 , S. Thevananther3 , T. Tordjmann1 . 1 INSERM U757, Universit´e Paris-Sud, Orsay, 2 Centre H´epatobiliaire, Hopital Paul Brousse, Villejuif, France; 3 Baylor College of Medicine, Houston, Texas, USA E-mail: [email protected] Background and Aims: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger in epithelia, remains unclear. The physiological release of ATP and its impact on regeneration after partial hepatectomy, which have never been explored, were investigated in this study. Methods: Hepatic ATP release before and after partial hepatectomy was examined in plasma of rats and in human living donors for liver transplantation. The involvement of Kupffer cells was evaluated by pretreating rats with clodronate liposomes. In rat liver sections and isolated hepatocytes, quinacrine was used for in vivo staining of ATP-enriched intracellular compartments, and lysotracker was used to stain lysosomes. Rats were treated with an antagonist for P2 purinergic receptors [phosphate-6azo(benzene-2,4-disulfonic) acid, PPADS], and liver regeneration after hepatectomy was analyzed. Results: A robust and transient ATP release was observed immediately after hepatectomy in the rat and in humans. Experiments after clodronate liposomes treatment suggested a partial contribution of Kupffer cells to the ATP release in the rat. Quinacrine-stained vesicles, predominantly detected in periportal hepatocytes before, significantly disappeared after partial hepatectomy, in parallel with a decrease in ATP liver content. These vesicles were colabelled with lysotracker in liver sections and isolated hepatocytes. PPADS treatment dampened hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in BrdU incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early genes induction. Conclusion: Extracellular ATP is released immediately after partial hepatectomy from hepatocytes and Kupffer cells and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP may be released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration. 847 ADULT-DERIVED HUMAN LIVER MESENCHYMALLIKE STEM/PROGENITOR CELLS INCREASE HEPATIC METABOLIC FUNCTION AFTER IN VITRO DIFFERENTIATION D.N. Khuu1 , M. Najimi1 , P.B. Calderon2 , E.M. Sokal1 . 1 Laboratory of Pediatric Hepatology and Cell Therapy, 2 Pharmacocinetic, Metabolism, Nutrition and Toxicology Department, Universit´e Catholique de Louvain, Brussel, Belgium E-mail: [email protected] Background and Aims: Adult liver derived progenitor cells (LDPC) were recently isolated and characterized from healthy human livers. Their ability to differentiate into hepatocyte-like cells led to propose them as potential alternative cell to mature hepatocyte for cell therapy of human metabolic liver diseases. However, beside genotypic and phenotypic differentiation, these hepatic progenitor cells should exhibit some, if not all, metabolic liver functions. Such properties were investigated in the current in vitro study. Methods: LDPCs were isolated from 5 different healthy cadaveric donor livers and in vitro differentiated (P3−6) after sequential incubation with growth factors and cytokines. Three important functions were investigated: gluconeogenesis, urea synthesis and cytochrome 3A4 activity in differentiated LDPCs, with primary mature hepatocytes and hepG2 cells as controls.
04d: MOLECULAR AND CELLULAR BIOLOGY − d) LIVER REGENERATION Results: Differentiated LDPCs showed an increase in the basal mRNA expression of Puryvate Carboxylase, Phosphoenolpyruvate carboxikinase and Glucose 6-phosphatase. They expressed higher activity of glucose 6-phosphatase correlated to a significant de novo glucose synthesis (after 24 hours incubation with 10mM Lactate and 1mM Pyruvate) as compared to undifferentiated cells. Differentiated LDPCs also acquired a similar detoxification capacity as mature hepatocytes. mRNA expression of five different enzymes of the urea cycle were up-regulated and the cells were able to metabolize ammonium chloride and release urea. In addition, they highly expressed eight cytochrome enzyme mRNAs which play the main roles in detoxification and inactivation of exogenous or endogenous substances. We demonstrated that differentiated LDPCs express active CYP3A4 (the most important drug-metabolizing enzyme) which expression is pharmacologically regulated using activators (Phenobarbital) and inhibitors (Naringin) in a similar way as mature hepatocytes. Conclusions: In vitro differentiated LDPCs are able to acquire advanced liver metabolic functions suggesting that these cells are useful as alternatives to primary hepatocytes for both in vitro and in vivo settings.
848 THREE-DIMENSIONAL PLGA SCAFFOLDS PROMOTE DIFFERENTIATION AND FUNCTION OF BONE MARROW MESENCHYMAL STEM CELL DERIVED HEPATOCYTES J. Li1 , R. Tao1 , H. Cao1 , W. Wu1 , H. Huang1 , C. Gao2 , T. Hui3 , D. Farkas3 , L. Li1 . 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 2 Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, China; 3 Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA E-mail: [email protected] Background and Aims: Liver tissue engineering with hepatic stem cells provides a promising approach substitution for liver transplantation in patient with end-stage chronic liver diseases. The purpose of this study is to define hepatocyte-like cells differentiated from bone marrow mesenchymal stem cells (BMSCs) with three-dimensional biodegradable scaffold. Methods: For hepatocyte differentiation, second to third passage BMSCs isolated from normal adult F344 rats were seeded into 3-dimensional collagen poly (lactic-co-glycolic acid) (PLGA) scaffolds with hepatocyte differentiation medium (IMDM supplemented with HGF, dexamethasone, ITX+ premix, oncostatin, etc.) for 3 weeks. Differentiation of BMSCs in a monolayer culture system was set as a control group. Hepatogenesis of BMSCs was characterized by liver enriched functional markers (ALB, AFP, G-6-P, HNF-4, cytochrome P450, Glycogen, Cytokeratin (CK) 18 and CK19) in both RNA and protein levels using RT-PCR, Western blot and histostaining at day 7, 14 and 21 of differentiation period. The morphology and function of BMSCs derived hepatocytes were also checked by confocal laser microscopy (CLM) and ELISA at same time. Results: After three weeks of differentiation, RT-PCR detected the expression of severalhepatocyte specific genes (ALB, AFP, CK18, CK19, G-6-P and HNF-4) in both PLGA scaffolds and monolayer culture group. Some of these markers (ALB, AFP, CK18 and CK19) were expressed earlier by 1 week of differentiation in PLGA scaffold group. Western blot of ALB, P450, CK18 and CK19 showed similar protein expression pattern. Compared with control group, the differentiated hepatocytes grew stably and the cell number was no significant decrease under CLM in PLGA scaffolds during three week differentiation. The functions of differentiated hepatocytes (ALB secretion, urea synthesis and ammonia elimination) in PLGA scaffold group were significant higher than that in control group during the whole differentiation period. Interestingly, H&E staining showed that the differentiated BMSCs formed small round clusters at 2nd week and the liver like tissue was formed at 3rd week of differentiation in C-PLGA scaffolds. Conclusion: The collagen-modified three-dimensional PLGA scaffold can significantly promote the differentiation and function of hepatocytes from BMSCs. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.
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849 TRANSCRIPTIONAL PROFILING AND HEPATOGENIC POTENCY OF ACUTE HEPATIC FAILURE-DERIVED RAT BONE MARROW MESENCHYMAL STEM CELLS J. Li1 , R. Tao1 , W. Wu1 , H. Cao1 , X. Hong2 , Z. Ma1 , H. Huang1 , L. Li1 . 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 2 Zhejiang California International Nanosystems Institute, Hangzhou, China E-mail: [email protected] Background and Aims: Cell transplantation with hepatic stem cells provides a potential approach substitution for liver transplantation in patients with end-stage liver diseases. The purpose of this study is to define CD45− /CD90+ mesenchymal stem cells (BMSCs) derived from acute hepatic failure rats possess hepatic characteristics and differentiation potential. Methods: The Affymetrix GeneChip Rat Genome 230 2.0 Array were used to perform transcriptomic profile on CD45− /CD90+ BMSCs isolated from AHF and sham operation rats as well as primary hepatocytes. For hepatocyte differentiation, second to third passage BMSCs isolated from AHF and normal adult F344 rats were differentiated into hepatocyte with special differentiation medium (IMDM supplemented with HGF, dexamethasone, ITX+ premix, oncostatin, etc.) for 3 weeks. Hepatogenesis of CD45− /CD90+ BMSCs was characterized by liver enriched functional markers (Alb, AFP, G-6-P, CK18, CK19, HNF-1, HNF-4, Met, Tf, Kit and Glycogen) with RT-PCR, Western blot and PAS staining at day 7, 14 and 21 of differentiation period. The metabolic functions of CD45− /CD90+ BMSCs derived hepatocytes were also analyzed by ELISA at same time. Results: 37 probe sets were up-regulated more than 2.5-fold in AHFderived CD45− /CD90+ BMSCs compared to control BMSCs. 36 probe sets were present in both AHF-derived CD45− /CD90+ BMSCs and primary hepatocytes but absent in control CD45− /CD90+ BMSCs, including Tcf1 and Dbp. Compared to the control CD45− /CD90+ BMSCs, AHF-derived CD45− /CD90+ BMSCs shared more commonly expressed genes with hepatocytes. After three weeks of differentiation, RT-PCR and Western blot detected the expression of several hepatocyte specific genes (ALB, AFP, CK19, HNF-4, Met, Tf, Kit, etc.) in both group. Some markers (ALB, AFP, HNF-4 and Met) were expressed earlier by 1 week of differentiation in AHF group. H&E staining showed that glycogen were expressed earlier in AHF group than that in control group. The metabolism functions of differentiated hepatocytes (ALB secretion, urea synthesis and ammonia elimination) in AHF group were also significant higher. Conclusion: AHF-derived CD45− /CD90+ BMSCs displayed a hepatic transcriptional profiling compared with that cells isolated from sham operation rats; and early expression of liver enriched functional markers was observed in AHF derived CD45− /CD90+ BMSCs. 850 LIVER REGENERATION AFTER COMBINED PVE AND ADMINISTRATION OF CD34+ AUTOLOGOUS CELLS: PRELIMINARY CLINICAL EXPERIENCE M. Maestri1 , P. Quaretti2 , C. Perotti3 , G. Poggi4 , A. Vercelli5 , G. David1 , A. Gaspari1 , F. Maffeis1 , M.C. Canepa1 , A. Peloso1 , M. Carlino1 , M. Alessiani1 , P. Dionigi1 . 1 Surgical Sciences, Fondazione IRCCS Policlinico San Matteo/University of Pavia, 2 Interventional Radiology, 3 Immunoematology and Transfusion, Fondazione IRCCS Policlinico San Matteo, 4 Oncology, Fondazione IRCCS Maugeri Istituto Scientifico Pavia, 5 Radiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy E-mail: [email protected] Introduction: The gold standard to treat the tumors of the liver is a resection.When the anticipated future liver remnant volume (FLRV) is below 30% (healthy livers) or 40% (cirrhotic livers or previous chemotherapy), an increased risk of complications is expected. Transhepatic percutaneous portal vein embolization (PVE) has been proposed to gain an expansion of the FLRV. Stem cells have been presented as a potential therapy to augment and accelerate hepatic proliferation. Recently, a combined
Poster Session − Saturday, April 25
glass microscope slide. Primary screening identified 6 polymers (three polyacrylates 2AE7, 7G7, 3AA7 and three polyurethanes 134, 212, 223) that supported HLC attachment and identity. We further characterised hepatocytic function in detail on these six matrices. Using this strategy, polymer 134 was identified as the most effective cellular support associated with enhanced expression of Fibrinogen, transthyretin (TTR) and soluble fibronectin. Key cytochrome p450 (CYP) activities on the different extracellular matrices were also characterised. CYP1A2 activity was increased ~6 fold and CYP3A4 ~2 fold on polymer 134 as compared to standard matrigel conditions or the other polymers tested. Conclusion: In conclusion, the high through-put screening of a polymer microarray allowed the identification of a new polymer matrix that promotes long-term hepatocellular differentiated function before and after passaging. These attributes bypass current limitations associated adult human hepatocytes, can be manufactured to GMP standards and will play important roles in developing in vitro models of drug toxicicology, provide a resource for the construction of extra-corporeal devices and facilitate novel studies of human liver development and disease. 845 HINT1 IS A PROTEIN POTENTIALLY IMPLICATED IN LIVER REGENERATION C. Hora1 , O. Maurhofer1 , M. Ledermann1 , G. Ferrand2 , H. Li3 , I.B. Weinstein3,4 , J.-F. Dufour1 , J. Martin1 . 1 Institute of Clinical Pharmacology and Visceral Research, University of Berne, Berne, 2 Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; 3 Herbert Irving Comprehensive Cancer Center, Columbia University, 4 Department of Medicine, Columbia University, New York, NY, USA E-mail: [email protected] Introduction: Liver regeneration plays an important role in liver transplantation and liver cancer treatment. HINT1, a nucleotide binding protein belonging to the HIT protein family functions as a tumour suppressor and participates in the cellular response to DNA damage. The aim of our study was to investigate whether liver regeneration is affected by the lack of Hint1. Methods: 2/3 partial hepatectomy (PH) was performed in male Hint−/ − and C57BL/6 mice (9−10 weeks-old, n = 7−10). Animals were sacrificed at 3, 6, 24, 48, 72 and 96 hours post PH and the remaining livers weighed. Restoration of liver mass was calculated. Cell proliferation was determined by immunohistochemistry using BrdU and Ki67 as markers. Liver histology and apoptosis were evaluated by H&E and cleaved lamin A (cLA) staining. Gene and protein expression of CyclinD1, p21, IL6 and TNFa were analysed by QPCR and immunobloting. Data are means±SD, p0.05. Results: In Hint1−/ − mice, the liver mass did not increase until 24 h after surgery. Increases of 30±18%, 82±20% and 106±10% were recorded at 48 h, 72 h and 96 h after PH, respectively. In C57BL/6 the liver mass increased without delay by 19±7, 36±10, 43±3.5, 59±3%, 119±13.6% and 144±12% at 3 h, 6 h, 24 h, 48 h, 72 h and 96 h after PH, respectively. Proliferation assessed by BrdU and Ki67 and apoptosis assessed with cLA were not significantly different between the 2 groups. At the time of surgery, there was less IL6 and TNFa mRNA mRNA in Hint1−/ − mice vs C57BL6 mice. After PH, levels of IL6 and TNFa mRNA followed a similar pattern in both mice but remained lower in Hint1−/ − . Earlier and higher augmentation of p21 mRNA at 24 h was observed in Hint1−/ − . CyclinD1 mRNA expression was enhanced after 24 h and 72 h PH in Hint1−/ − whereas this expression peaked after 48 h in C57BL6 mice. A higher peak of p21 and CyclinD1 protein expression was measured after 24 h and 48 h in Hint1−/ − in comparison to C57BL6. Conclusion: Liver regeneration is impaired in the case of Hint1 ablation. Our results suggest that Hint1 plays a role in the initiation of liver regeneration by means of a mechanism that affects cytokines production.
846 ATP RELEASE AFTER PARTIAL HEPATECTOMY REGULATES LIVER REGENERATION E. Gonzales1 , B. Julien1 , V. Serri`ere-Lanneau1 , A. Nicou1 , I. Doignon1 , L. Lagoudakis1 , I. Garcin1 , D. Azoulay2 , J.-C. Duclos-Vall´ee2 , D. Castaing2 , D. Samuel2 , A. Hernandez-Garcia3 , S. Awad3 , L. Combettes1 , S. Thevananther3 , T. Tordjmann1 . 1 INSERM U757, Universit´e Paris-Sud, Orsay, 2 Centre H´epatobiliaire, Hopital Paul Brousse, Villejuif, France; 3 Baylor College of Medicine, Houston, Texas, USA E-mail: [email protected] Background and Aims: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger in epithelia, remains unclear. The physiological release of ATP and its impact on regeneration after partial hepatectomy, which have never been explored, were investigated in this study. Methods: Hepatic ATP release before and after partial hepatectomy was examined in plasma of rats and in human living donors for liver transplantation. The involvement of Kupffer cells was evaluated by pretreating rats with clodronate liposomes. In rat liver sections and isolated hepatocytes, quinacrine was used for in vivo staining of ATP-enriched intracellular compartments, and lysotracker was used to stain lysosomes. Rats were treated with an antagonist for P2 purinergic receptors [phosphate-6azo(benzene-2,4-disulfonic) acid, PPADS], and liver regeneration after hepatectomy was analyzed. Results: A robust and transient ATP release was observed immediately after hepatectomy in the rat and in humans. Experiments after clodronate liposomes treatment suggested a partial contribution of Kupffer cells to the ATP release in the rat. Quinacrine-stained vesicles, predominantly detected in periportal hepatocytes before, significantly disappeared after partial hepatectomy, in parallel with a decrease in ATP liver content. These vesicles were colabelled with lysotracker in liver sections and isolated hepatocytes. PPADS treatment dampened hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in BrdU incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early genes induction. Conclusion: Extracellular ATP is released immediately after partial hepatectomy from hepatocytes and Kupffer cells and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP may be released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration. 847 ADULT-DERIVED HUMAN LIVER MESENCHYMALLIKE STEM/PROGENITOR CELLS INCREASE HEPATIC METABOLIC FUNCTION AFTER IN VITRO DIFFERENTIATION D.N. Khuu1 , M. Najimi1 , P.B. Calderon2 , E.M. Sokal1 . 1 Laboratory of Pediatric Hepatology and Cell Therapy, 2 Pharmacocinetic, Metabolism, Nutrition and Toxicology Department, Universit´e Catholique de Louvain, Brussel, Belgium E-mail: [email protected] Background and Aims: Adult liver derived progenitor cells (LDPC) were recently isolated and characterized from healthy human livers. Their ability to differentiate into hepatocyte-like cells led to propose them as potential alternative cell to mature hepatocyte for cell therapy of human metabolic liver diseases. However, beside genotypic and phenotypic differentiation, these hepatic progenitor cells should exhibit some, if not all, metabolic liver functions. Such properties were investigated in the current in vitro study. Methods: LDPCs were isolated from 5 different healthy cadaveric donor livers and in vitro differentiated (P3−6) after sequential incubation with growth factors and cytokines. Three important functions were investigated: gluconeogenesis, urea synthesis and cytochrome 3A4 activity in differentiated LDPCs, with primary mature hepatocytes and hepG2 cells as controls.
04d: MOLECULAR AND CELLULAR BIOLOGY − d) LIVER REGENERATION Results: Differentiated LDPCs showed an increase in the basal mRNA expression of Puryvate Carboxylase, Phosphoenolpyruvate carboxikinase and Glucose 6-phosphatase. They expressed higher activity of glucose 6-phosphatase correlated to a significant de novo glucose synthesis (after 24 hours incubation with 10mM Lactate and 1mM Pyruvate) as compared to undifferentiated cells. Differentiated LDPCs also acquired a similar detoxification capacity as mature hepatocytes. mRNA expression of five different enzymes of the urea cycle were up-regulated and the cells were able to metabolize ammonium chloride and release urea. In addition, they highly expressed eight cytochrome enzyme mRNAs which play the main roles in detoxification and inactivation of exogenous or endogenous substances. We demonstrated that differentiated LDPCs express active CYP3A4 (the most important drug-metabolizing enzyme) which expression is pharmacologically regulated using activators (Phenobarbital) and inhibitors (Naringin) in a similar way as mature hepatocytes. Conclusions: In vitro differentiated LDPCs are able to acquire advanced liver metabolic functions suggesting that these cells are useful as alternatives to primary hepatocytes for both in vitro and in vivo settings.
848 THREE-DIMENSIONAL PLGA SCAFFOLDS PROMOTE DIFFERENTIATION AND FUNCTION OF BONE MARROW MESENCHYMAL STEM CELL DERIVED HEPATOCYTES J. Li1 , R. Tao1 , H. Cao1 , W. Wu1 , H. Huang1 , C. Gao2 , T. Hui3 , D. Farkas3 , L. Li1 . 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 2 Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, China; 3 Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA E-mail: [email protected] Background and Aims: Liver tissue engineering with hepatic stem cells provides a promising approach substitution for liver transplantation in patient with end-stage chronic liver diseases. The purpose of this study is to define hepatocyte-like cells differentiated from bone marrow mesenchymal stem cells (BMSCs) with three-dimensional biodegradable scaffold. Methods: For hepatocyte differentiation, second to third passage BMSCs isolated from normal adult F344 rats were seeded into 3-dimensional collagen poly (lactic-co-glycolic acid) (PLGA) scaffolds with hepatocyte differentiation medium (IMDM supplemented with HGF, dexamethasone, ITX+ premix, oncostatin, etc.) for 3 weeks. Differentiation of BMSCs in a monolayer culture system was set as a control group. Hepatogenesis of BMSCs was characterized by liver enriched functional markers (ALB, AFP, G-6-P, HNF-4, cytochrome P450, Glycogen, Cytokeratin (CK) 18 and CK19) in both RNA and protein levels using RT-PCR, Western blot and histostaining at day 7, 14 and 21 of differentiation period. The morphology and function of BMSCs derived hepatocytes were also checked by confocal laser microscopy (CLM) and ELISA at same time. Results: After three weeks of differentiation, RT-PCR detected the expression of severalhepatocyte specific genes (ALB, AFP, CK18, CK19, G-6-P and HNF-4) in both PLGA scaffolds and monolayer culture group. Some of these markers (ALB, AFP, CK18 and CK19) were expressed earlier by 1 week of differentiation in PLGA scaffold group. Western blot of ALB, P450, CK18 and CK19 showed similar protein expression pattern. Compared with control group, the differentiated hepatocytes grew stably and the cell number was no significant decrease under CLM in PLGA scaffolds during three week differentiation. The functions of differentiated hepatocytes (ALB secretion, urea synthesis and ammonia elimination) in PLGA scaffold group were significant higher than that in control group during the whole differentiation period. Interestingly, H&E staining showed that the differentiated BMSCs formed small round clusters at 2nd week and the liver like tissue was formed at 3rd week of differentiation in C-PLGA scaffolds. Conclusion: The collagen-modified three-dimensional PLGA scaffold can significantly promote the differentiation and function of hepatocytes from BMSCs. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.
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849 TRANSCRIPTIONAL PROFILING AND HEPATOGENIC POTENCY OF ACUTE HEPATIC FAILURE-DERIVED RAT BONE MARROW MESENCHYMAL STEM CELLS J. Li1 , R. Tao1 , W. Wu1 , H. Cao1 , X. Hong2 , Z. Ma1 , H. Huang1 , L. Li1 . 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 2 Zhejiang California International Nanosystems Institute, Hangzhou, China E-mail: [email protected] Background and Aims: Cell transplantation with hepatic stem cells provides a potential approach substitution for liver transplantation in patients with end-stage liver diseases. The purpose of this study is to define CD45− /CD90+ mesenchymal stem cells (BMSCs) derived from acute hepatic failure rats possess hepatic characteristics and differentiation potential. Methods: The Affymetrix GeneChip Rat Genome 230 2.0 Array were used to perform transcriptomic profile on CD45− /CD90+ BMSCs isolated from AHF and sham operation rats as well as primary hepatocytes. For hepatocyte differentiation, second to third passage BMSCs isolated from AHF and normal adult F344 rats were differentiated into hepatocyte with special differentiation medium (IMDM supplemented with HGF, dexamethasone, ITX+ premix, oncostatin, etc.) for 3 weeks. Hepatogenesis of CD45− /CD90+ BMSCs was characterized by liver enriched functional markers (Alb, AFP, G-6-P, CK18, CK19, HNF-1, HNF-4, Met, Tf, Kit and Glycogen) with RT-PCR, Western blot and PAS staining at day 7, 14 and 21 of differentiation period. The metabolic functions of CD45− /CD90+ BMSCs derived hepatocytes were also analyzed by ELISA at same time. Results: 37 probe sets were up-regulated more than 2.5-fold in AHFderived CD45− /CD90+ BMSCs compared to control BMSCs. 36 probe sets were present in both AHF-derived CD45− /CD90+ BMSCs and primary hepatocytes but absent in control CD45− /CD90+ BMSCs, including Tcf1 and Dbp. Compared to the control CD45− /CD90+ BMSCs, AHF-derived CD45− /CD90+ BMSCs shared more commonly expressed genes with hepatocytes. After three weeks of differentiation, RT-PCR and Western blot detected the expression of several hepatocyte specific genes (ALB, AFP, CK19, HNF-4, Met, Tf, Kit, etc.) in both group. Some markers (ALB, AFP, HNF-4 and Met) were expressed earlier by 1 week of differentiation in AHF group. H&E staining showed that glycogen were expressed earlier in AHF group than that in control group. The metabolism functions of differentiated hepatocytes (ALB secretion, urea synthesis and ammonia elimination) in AHF group were also significant higher. Conclusion: AHF-derived CD45− /CD90+ BMSCs displayed a hepatic transcriptional profiling compared with that cells isolated from sham operation rats; and early expression of liver enriched functional markers was observed in AHF derived CD45− /CD90+ BMSCs. 850 LIVER REGENERATION AFTER COMBINED PVE AND ADMINISTRATION OF CD34+ AUTOLOGOUS CELLS: PRELIMINARY CLINICAL EXPERIENCE M. Maestri1 , P. Quaretti2 , C. Perotti3 , G. Poggi4 , A. Vercelli5 , G. David1 , A. Gaspari1 , F. Maffeis1 , M.C. Canepa1 , A. Peloso1 , M. Carlino1 , M. Alessiani1 , P. Dionigi1 . 1 Surgical Sciences, Fondazione IRCCS Policlinico San Matteo/University of Pavia, 2 Interventional Radiology, 3 Immunoematology and Transfusion, Fondazione IRCCS Policlinico San Matteo, 4 Oncology, Fondazione IRCCS Maugeri Istituto Scientifico Pavia, 5 Radiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy E-mail: [email protected] Introduction: The gold standard to treat the tumors of the liver is a resection.When the anticipated future liver remnant volume (FLRV) is below 30% (healthy livers) or 40% (cirrhotic livers or previous chemotherapy), an increased risk of complications is expected. Transhepatic percutaneous portal vein embolization (PVE) has been proposed to gain an expansion of the FLRV. Stem cells have been presented as a potential therapy to augment and accelerate hepatic proliferation. Recently, a combined