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Mak WC, Magne B, Cheung KY, Atanasova D, Griffith M. Thermo-rheological responsive microcapsules for time-dependent controlled release of human mesenchymal stromal cells. Biomater Sci 2018; 5:2241-2250. [PMID: 28972602 DOI: 10.1039/c7bm00663b] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Human mesenchymal stromal cells (hMSCs) are adult-source cells that have been extensively evaluated for cell-based therapies. hMSCs delivered by intravascular injection have been reported to accumulate at the sites of injury to promote tissue repair and can also be employed as vectors for the delivery of therapeutic genes. However, the full potential of hMSCs remains limited as the cells are lost after injection due to anoikis and the adverse pathologic environment. Encapsulation of cells has been proposed as a means of increasing cell viability. However, controlling the release of therapeutic cells over time to target tissue still remains a challenge today. Here, we report the design and development of thermo-rheological responsive hydrogels that allow for precise, time dependent controlled-release of hMSCs. The encapsulated hMSCs retained good viability from 76% to 87% dependent upon the hydrogel compositions. We demonstrated the design of different blended hydrogel composites with modulated strength (S parameter) and looseness of hydrogel networks (N parameter) to control the release of hMSCs from thermo-responsive hydrogel capsules. We further showed the feasibility for controlled-release of encapsulated hMSCs within 3D matrix scaffolds. We reported for the first time by a systematic analysis that there is a direct correlation between the thermo-rheological properties associated with the degradation of the hydrogel composite and the cell release kinetics. This work therefore provides new insights into the further development of smart carrier systems for stem cell therapy.
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Affiliation(s)
- W C Mak
- Department of Clinical and Experimental Medicine, Linköping University, SE58185, Linköping, Sweden.
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Cellular Mechanisms of Liver Regeneration and Cell-Based Therapies of Liver Diseases. BIOMED RESEARCH INTERNATIONAL 2017; 2017:8910821. [PMID: 28210629 PMCID: PMC5292184 DOI: 10.1155/2017/8910821] [Citation(s) in RCA: 69] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Revised: 11/29/2016] [Accepted: 12/27/2016] [Indexed: 12/14/2022]
Abstract
The emerging field of regenerative medicine offers innovative methods of cell therapy and tissue/organ engineering as a novel approach to liver disease treatment. The ultimate scientific foundation of both cell therapy of liver diseases and liver tissue and organ engineering is delivered by the in-depth studies of the cellular and molecular mechanisms of liver regeneration. The cellular mechanisms of the homeostatic and injury-induced liver regeneration are unique. Restoration of the mass of liver parenchyma is achieved by compensatory hypertrophy and hyperplasia of the differentiated parenchymal cells, hepatocytes, while expansion and differentiation of the resident stem/progenitor cells play a minor or negligible role. Participation of blood-borne cells of the bone marrow origin in liver parenchyma regeneration has been proven but does not exceed 1-2% of newly formed hepatocytes. Liver regeneration is activated spontaneously after injury and can be further stimulated by cell therapy with hepatocytes, hematopoietic stem cells, or mesenchymal stem cells. Further studies aimed at improving the outcomes of cell therapy of liver diseases are underway. In case of liver failure, transplantation of engineered liver can become the best option in the foreseeable future. Engineering of a transplantable liver or its major part is an enormous challenge, but rapid progress in induced pluripotency, tissue engineering, and bioprinting research shows that it may be doable.
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Sun YL, Yin SY, Zhou L, Xie HY, Zhang F, Wu LM, Zheng SS. Hepatocyte differentiation of human fibroblasts from cirrhotic liver in vitro and in vivo. Hepatobiliary Pancreat Dis Int 2011; 10:55-63. [PMID: 21269936 DOI: 10.1016/s1499-3872(11)60008-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
BACKGROUND Mesenchymal stem cells (MSCs) and fibroblasts have intimate relationships, and the phenotypic homology between fibroblasts and MSCs has been recently described. The aim of this study was to investigate the hepatic differentiating potential of human fibroblasts in cirrhotic liver. METHODS The phenotypes of fibroblasts in cirrhotic liver were labeled by biological methods. After that, the differentiation potential of these fibroblasts in vitro was characterized in terms of liver-specific gene and protein expression. Finally, an animal model of hepatocyte regeneration in severe combined immunodeficient (SCID) mice was created by retrorsine injection and partial hepatectomy, and the expression of human hepatocyte proteins in SCID mouse livers was checked by immunohistochemical analysis after fibroblast administration. RESULTS Surface immunophenotyping revealed that a minority of fibroblasts expressed markers of MSCs and hepatic epithelial cytokeratins as well as alpha-smooth muscle actin, but homogeneously expressed vimentin, desmin, prolyl 4-hydroxylase and fibronectin. These fibroblasts presented the characteristics of hepatocytes in vitro and differentiated directly into functional hepatocytes in the liver of hepatectomized SCID mice. CONCLUSIONS This study demonstrated that fibroblasts in cirrhotic liver have the potential to differentiate into hepatocyte-like cells in vitro and in vivo. Our findings infer that hepatic differentiation of fibroblasts may serve as a new target for reversion of liver fibrosis and a cell source for tissue engineering.
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Affiliation(s)
- Yu-Ling Sun
- Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
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Khurana S, Mukhopadhyay A. In vitro transdifferentiation of adult hematopoietic stem cells: an alternative source of engraftable hepatocytes. J Hepatol 2008; 49:998-1007. [PMID: 18657875 DOI: 10.1016/j.jhep.2008.05.019] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/05/2008] [Revised: 04/11/2008] [Accepted: 05/06/2008] [Indexed: 02/06/2023]
Abstract
BACKGROUND/AIMS We attempted to establish an ex vivo model for transdifferentiation of hematopoietic stem cells (HSCs) into functional hepatocytes for transplantation into healthy liver. METHODS We mimicked the liver regenerating microenvironment in culture by incorporating extracellular matrix components and sera obtained from mice with liver damaged by a hepatotoxic chemical. The differentiated hepatic cells were characterized in terms of liver-specific gene and protein expression. Cellular changes were determined by examining ultrastructure, and the functional activity was confirmed by cytochrome p450 enzyme assay. The engraftability of these hepatic cells in healthy liver tissue was checked by immunohistochemical analysis. RESULTS A specific sub-population of bone marrow-derived cells transdifferentiated into hepatic cells, confirmed by the expression of genes and proteins. The differentiated cells were found functionally active and ultrastructurally similar to primary hepatocytes in terms of the formation of microvilli and other cellular organelles. In healthy liver, these cells engrafted into hepatocyte plates and maintained the expression of albumin and cytokeratin-18. CONCLUSIONS Hepatic culture system differentiated HSCs into functional hepatocytes, which were engraftable in healthy liver. This finding offers an alternative strategy for treating many liver ailments using autologous bone marrow cells, hence avoiding immuno-suppressive drugs.
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Affiliation(s)
- Satish Khurana
- Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
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Lysy PA, Smets F, Sibille C, Najimi M, Sokal EM. Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation. Hepatology 2007; 46:1574-85. [PMID: 17969047 DOI: 10.1002/hep.21839] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The phenotypic homology of fibroblasts and mesenchymal stem cells (MSCs) has been recently described. Our study investigated the in vitro potential of human skin fibroblasts to differentiate into mesodermal (osteocyte and adipocyte) and endodermal (hepatocyte) cell lineages by comparison with human bone marrow (hBM) MSCs. The endodermal potential of fibroblasts was then explored in vivo in a mouse model of liver injury. Fibroblasts were able to acquire osteocyte and adipocyte phenotypes as assessed by cytochemistry and gene expression analyses. After exposure to a specific differentiation cocktail, these cells presented hepatocyte-like morphology and acquired liver-specific markers on protein and gene expression levels. Furthermore, these fibroblast-derived hepatocyte-like cells (FDHLCs) displayed the ability to store glycogen and synthesize small amounts of urea. By gene expression analysis, we observed that fibroblasts remained in a mesenchymal-epithelial transition state after hepatocyte differentiation. Moreover, FDHLCs lost their hepatocyte-like phenotype after dedifferentiation. In vivo, human fibroblasts infused directly into the liver of hepatectomized severe combined immunodeficient (SCID) mice engrafted in situ and expressed hepatocyte markers (albumin, alpha-fetoprotein, and cytokeratin 18) together with the mesodermal marker fibronectin. Despite lower liver-specific marker expression, the in vitro and in vivo differentiation profile of fibroblasts was comparable to that of mesenchymal-derived hepatocyte-like cells (MDHLCs). In conclusion, our work demonstrates that human skin fibroblasts are able to display mesodermal and endodermal differentiation capacities and provides arguments that these cells share MSCs features both on the phenotypic and functional levels.
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Affiliation(s)
- Philippe A Lysy
- Université Catholique de Louvain et Cliniques Universitaires Saint Luc, HPED Department, PEDI Unit, Laboratory of Pediatric Hepatology and Cell Therapy, 1200 Brussels, Belgium
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Cerec V, Glaise D, Garnier D, Morosan S, Turlin B, Drenou B, Gripon P, Kremsdorf D, Guguen-Guillouzo C, Corlu A. Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor. Hepatology 2007; 45:957-67. [PMID: 17393521 DOI: 10.1002/hep.21536] [Citation(s) in RCA: 281] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
UNLABELLED Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21(CIP1) and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of beta-catenin localization and HNF3beta expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. CONCLUSION Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors.
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Kiyasov AP, Gumerova AA, Titova MA. Mesenchymal-epithelial transformation of ito cells in vitro. Bull Exp Biol Med 2007; 142:133-6. [PMID: 17369923 DOI: 10.1007/s10517-006-0311-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
Cultured pure population of Ito cells isolated from adult rat liver expressed epithelial markers cytokeratin-8, alpha-fetoprotein, and gamma-glutamyl transpeptidase after forming a dense monolayer. Mesenchymal-epithelial transformation of these cells is possible, which suggests them as candidates of hepatic stem cells.
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Lavazais E, Pogu S, Saï P, Martignat L. Cytokine mobilization of bone marrow cells and pancreatic lesion do not improve streptozotocin-induced diabetes in mice by transdifferentiation of bone marrow cells into insulin-producing cells. DIABETES & METABOLISM 2007; 33:68-78. [PMID: 17270481 DOI: 10.1016/j.diabet.2006.11.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/24/2006] [Accepted: 10/13/2006] [Indexed: 01/21/2023]
Abstract
OBJECTIVE Transdifferentiation of bone marrow cells (BMC) into insulin-producing cells might provide a new cellular therapy for type I diabetes, but its existence is controversial. Our aim was to determine if those cells could transdifferentiate, even at low frequency, into insulin-producing cells, in testing optimized experimental conditions. METHODS We grafted mice with total BMC, genetically labeled either ubiquitarily, or with a marker conditionally expressed under the control of the insulin beta-cell specific promoter. We treated some of the recipients with an agent toxic to beta-cells (streptozotocin) and with cytokines stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF). RESULTS The contribution of grafted cells could be detected neither for natural turnover (n=6), nor for beta-cell regeneration after pancreatic lesion (n=7), 90 days post-transplantation. Cytokine mobilization of BMC in the blood stream, reported to favor their transdifferentiation into cardiac and neural cells, had never been tested before for beta-cell generation. Here, we showed that injection of SCF and G-CSF did not lead to a detectable level of transdifferentiation (n=7). CONCLUSIONS We conclude that BMC cannot spontaneously transdifferentiate into insulin-producing cells in vivo, even after beta-cell lesion and mobilization induced by cytokines. Interestingly, however, treatment by cytokines may have beneficial indirect effects on STZ-induced hyperglycaemia.
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Affiliation(s)
- E Lavazais
- Cellular and Molecular Immuno-Endocrinology, Inra/ENVN/University, Atlanpole, La Chantrerie, BP 40706, 44307 Nantes cedex 03, France
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Tang XP, Zhang M, Yang X, Chen LM, Zeng Y. Differentiation of human umbilical cord blood stem cells into hepatocytes in vivo and in vitro. World J Gastroenterol 2006; 12:4014-9. [PMID: 16810750 PMCID: PMC4087712 DOI: 10.3748/wjg.v12.i25.4014] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the condition and potentiality of human umbilical cord blood stem cells (HUCBSC) to differentiate into hepatocytes in vivo or in vitro.
METHODS: In a cell culture study of human umbilical cord blood stem cell (HUCBSC) differentiation, human umbilical cord blood mononuclear cells (HUCBMNC) were separated by density gradient centrifugation. Fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) and the supernatant of fetal liver were added in the inducing groups. Only FGF was added in the control group. The expansion and differentiation of HUCBMNC in each group were observed. Human alpha fetoprotein (AFP) and albumin (ALB) were detected by immunohistochemistry. In the animal experiments, the survival SD rats with acute hepatic injury after carbon tetrachloride (CCL4) injection 48 h were randomly divided into three groups. The rats in group A were treated with human umbilical cord blood serum. The rats in group B were treated with HUCBMNC transplantation. The rats in group C were treated with HUCBMNC transplantation followed by intraperitoneal cyclophosphamide for 7 d. The rats were killed at different time points after the treatment and the liver tissue was histopathologically studied and human AFP and ALB detected by immunohistochemistry. The human X inactive-specific transcript gene fragment in the liver tissue was amplified by PCR to find human DNA.
RESULTS: The results of cell culture showed that adherent cells were stained negative for AFP or ALB in control group. However, the adherent cells in the inducing groups stained positive for AFP or ALB. The result of animal experiment showed that no human AFP or ALB positive cells present in the liver tissue of group A (control group). However, many human AFP or ALB positive cells were scattered around sinus hepaticus and the central veins of hepatic lobules and in the portal area in group B and group C after one month. The fragment of human X chromagene could be detected in the liver tissue of groups B and C, but not in group A.
CONCLUSION: Under certain conditions HUCBSC can differentiate into liver cells in vivo and in vitro.
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Affiliation(s)
- Xiao-Peng Tang
- Research Center of Liver Diseases, the Second Xiangya Hospital, Central South University, Changsha 410011, Hunan Province, China.
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Pahlavan PS, Feldmann RE, Zavos C, Kountouras J. Prometheus' challenge: molecular, cellular and systemic aspects of liver regeneration. J Surg Res 2006; 134:238-51. [PMID: 16458925 DOI: 10.1016/j.jss.2005.12.011] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2005] [Revised: 10/25/2005] [Accepted: 12/15/2005] [Indexed: 02/08/2023]
Abstract
The fascinating aspect of the liver is the capacity to regenerate after injury or resection. A variety of genes, cytokines, growth factors, and cells are involved in liver regeneration. The exact mechanism of regeneration and the interaction between cells and cytokines are not fully understood. There seems to exist a sequence of stages that result in liver regeneration, while at the same time inhibitors control the size of the regenerated liver. It has been proven that hepatocyte growth factor, transforming growth factor, epidermal growth factor, tumor necrosis factor-alpha, interleukins -1 and -6 are the main growth and promoter factors secreted after hepatic injury, partial hepatectomy and after a sequence of different and complex reactions to activate transcription factors, mainly nuclear factor kappaB and signal transduction and activator of transcription-3, affects specific genes to promote liver regeneration. Unraveling the complex processes of liver regeneration may provide novel strategies in the management of patients with end-stage liver disease. In particular, inducing liver regeneration should reduce morbidity for the donor and increase faster recovery for the liver transplantation recipient.
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Affiliation(s)
- Payam Samareh Pahlavan
- Department of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany.
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Abstract
Recent studies using animal models have elucidated a growing number of evolutionarily conserved genes and pathways that control liver development from the embryonic endoderm. It is increasingly clear that the genetic programs active in embryogenesis are often deregulated or reactivated in disease, cancer, and tissue repair. Understanding the molecular control of liver development should impact diagnosis and treatment of pediatric and adult liver diseases and aid in efforts to differentiate liver tissue in vitro for stem cell-based therapies.
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Affiliation(s)
- Valérie A McLin
- Baylor College of Medicine, Texas Childrens' Liver Center, 1102 Bates Street, Houston, TX 77006, USA
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Kucia M, Reca R, Jala VR, Dawn B, Ratajczak J, Ratajczak MZ. Bone marrow as a home of heterogenous populations of nonhematopoietic stem cells. Leukemia 2005; 19:1118-1127. [PMID: 15902288 DOI: 10.1038/sj.leu.2403796] [Citation(s) in RCA: 131] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2005] [Accepted: 04/04/2005] [Indexed: 12/17/2022]
Abstract
Evidence is presented that bone marrow (BM) in addition to CD45(positive) hematopoietic stem cells contains a rare population of heterogenous CD45(negative) nonhematopoietic tissue committed stem cells (TCSC). These nonhematopoietic TCSC (i) are enriched in population of CXCR4(+) CD34(+) AC133(+) lin(-) CD45(-) and CXCR4(+) Sca-1(+) lin(-) CD45(-) in humans and mice, respectively, (ii) display several markers of pluripotent stem cells (PSC) and (iii) as we envision are deposited in BM early in development. Thus, since BM contains versatile nonhematopoietic stem cells, previous studies on plasticity trans-dedifferentiation of BM-derived hematopoietic stem cells (HSC) that did not include proper controls to exclude this possibility could lead to wrong interpretations. Therefore, in this spotlight review we present this alternative explanation of 'plasticity' of BM-derived stem cells based on the assumption that BM stem cells are heterogenous. We also discuss a potential relationship of TCSC/PSC identified by us with other BM-derived CD45(negative) nonhematopoietic stem cells that were recently identified by other investigators (eg MSC, MAPC, USSC and MIAMI cells). Finally, we discuss perspectives and pitfalls in potential application of these cells in regenerative medicine.
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Affiliation(s)
- M Kucia
- Stem Cell Biology Program at James Graham Brown Cancer Center, University of Louisville, KY 40202, USA
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Hengstler JG, Brulport M, Schormann W, Bauer A, Hermes M, Nussler AK, Fandrich F, Ruhnke M, Ungefroren H, Griffin L, Bockamp E, Oesch F, von Mach MA. Generation of human hepatocytes by stem cell technology: definition of the hepatocyte. Expert Opin Drug Metab Toxicol 2005; 1:61-74. [PMID: 16922653 DOI: 10.1517/17425255.1.1.61] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, alpha-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte does not yet exist there is a good chance that this aim may be achieved in the future.
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Affiliation(s)
- Jan G Hengstler
- University of Leipzig, Center for Toxicology, Institute of Legal Medicine and Rudolf-Boehm Institute of Pharmacology and Toxicology, Haertelstr. 16-18, 04107 Leipzig, Germany.
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