小鼠成体肝脏祖细胞（AHPC）体外培养模型的建立和生物学特性研究
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摘要
目的
研究正常成体小鼠肝细胞的增殖能力和分化潜能,分离正常成体小鼠肝脏内可能存在的干细胞或祖细胞并建立体外培养的细胞模型,研究其基本的生物学特性。
方法
应用改良的Seglen二步法灌注和离心分离肝脏细胞,将肝细胞初步分为肝脏实质细胞(parenchymal hepatocytes,PH)部分和富含AHPC(adult hepaticprogenitor cells,AHPC)部分,对两部分的细胞用添加胎牛血清(FBS)的改良DMEM培养基进行培养,持续观察超过60天,分析两部分中肝细胞的形态学差异,以及通过细胞增殖和克隆的形成情况分析两部分中肝细胞增殖能力的差异。应用免疫荧光技术对具有高增殖能力细胞及其形成的克隆进行Albumin、AFP、CK19、c-kit、CD45、CD34、Oct-4、Desmin、CD16、Thy-1和nestin等染色,分析细胞标记物的表达和克隆内细胞的成熟分化情况。利用形态学观察、记录细胞增殖状况,以及免疫荧光染色技术初步分析肝脏非实质细胞(nonparenchymalcell,NPC)的生长对于AHPC活化、增殖和分化的影响。另外,尝试了AHPC克隆的传代培养。
结果
本研究中两部分肝细胞均获得较高的产量和活性(＞90%),完全满足实验的需要。PH部分和富含AHPC部分的肝细胞大小分别为(37.03±6.65)μm和(22.63±2.04)μm,存在明显的统计学差异(p＜0.05),但在大小分布比例上存在小部分的重叠。两部分的贴壁细胞中均含有NPC污染,其中富含AHPC部分较多,占贴壁细胞总数的70.3%,NPC增殖后肝细胞活化并开始增殖,所有的肝细胞克隆均表达肝星状细胞标记物Desmin。富含AHPC部分的肝细胞增殖能力明显较PH部分高,两部分的克隆形成率分别为21.45%±1.25%和0.28%±0.09%(p＜0.001)。富含AHPC部分中,约13.5%的贴壁肝细胞在接种后第2～3天活化并迅速增殖,第4～5天形成小的细胞克隆,极少数细胞(0.5%～1%)在接种后第3天即可形成克隆;培养30天后克隆内出现类似成熟的肝细胞,细胞克隆可持续扩增超过60天,最大克隆面积达到0.64mm~2,细胞平均增殖超过10个周期。贴壁后24小时,所有的肝细胞均强阳性表达肝细胞标记物Albumin,不表达AFP和CK19,培养第5天细胞克隆开始表达Albumin和AFP,第30天克履诓糠窒赴泶锏ü芟赴昙俏顲K19,同时发现Albumin阴性细胞。通过免疫荧光双染发现,培养第30天,AHPC克隆内同时存在Albumin阳性和AFP阳性、Albumin阳性和AFP阴性、Albumin阳性和CK19阴性、CK19阳性和AFP阴性、CK19阳性和AFP阳性的细胞。另外,AHPC可以传代培养超过60天,传代培养中,贴壁的AHPC克隆内细胞较小,核浆比率大,呈上皮样细胞的形态,部分细胞仍然具有独立形成克隆的能力,但观察发现,AHPC克隆解离为细胞团进行传代培养中细胞的增殖比解离为单细胞传代好。
结论
1.在正常成体小鼠肝脏内存在一种肝脏组织特异性的成体肝脏祖细胞(AHPC),并已成功建立了体外培养的细胞模型。
2.小鼠AHPC体外培养活化后可持续克隆性增殖超过60天,并可连续传代培养,具有向肝细胞和胆管细胞分化的双向分化潜能。
3.肝脏非实质细胞(NPC)的生长可以促进AHPC的增殖、成熟和分化。
4.与国外文献报导的肝脏祖细胞相比,小鼠AHPC体外培养中细胞表型不同,具有较高的增殖能力和明显的双向分化潜能,为肝细胞移植、肝脏发育和肝病等研究提供了一种新的肝脏干细胞模型和研究工具。
Objective
This study is aimed to isolate and culture candidate cells of liver stem cells or progenitor cells from normal adult mouse liver,by investigating their abilitys to proliferate and potentials to differentiate in vitro,and to establish a stable cell model cultured in vitro,followed by characterizing the fundamental properties of those cells.
Methods
Mouse hepatocytes,isolated by two-step collagenase perfusion and mechanical centrifugation,were separated into two fractions as fraction of PH(parenchymal hepatocytes,PH) and fraction with enriched AHPC(adult hepatic progenitor cells, AHPC).Two fraction cells were cultured in modified DMEM more than 60 days,and observed by phase contrast microscopy.It was clarified that the morphological and proliferating difference between those hepatocytes from two fractions.The expression of cell markers,such as Albumin,AFP,CK19,c-kit,CD45,CD34,Oct-4,Desmin, CD 16,Thy-1 and nestin in high proliferating cells and their colonies,was illuminated by immunofluorescence at different culture days,to analyze the origin of those cells and their potentials to differentiate.Besides,it was primarily investigated that the impact of NPC(nonparenchymal cells,NPC) contamination and growth on the activation,proliferation and differentiation of AHPC during primary culture,by immunofluorescence and noticing the morphological changes and growing state of NPC and AHPC.In addition,efforts were made to investigate the subculture possilbity of AHPC colonies.
Results
The total cell quantity and cell vitality of hepatocytes were relatively high in two fractions,with cell vitality more than 90%.The cell size of fraction with enriched AHPC was(22.63±2.04)μm,and PH fraction was(37.03±6.65)μm,with a significant statistical difference(p＜0.05).The percentage of different cell size, however,showed two fractions shared some cells with certain sizes.There was a contamination of HPC in both fractions,and the one of enriched AHPC had more HPC(about 73%of attached cells).In the primary culture,HPC firstly began to divide and proliferate,then attached hepatocytes showed being activated,and all formed colonies expressed Desmin,a relatively specific cell marker of hepatic stellate cell(HSC).The fraction with enriched AttPC manifested a definitely larger colony-forming rate than PH ones,respectively 21.45%±1.25%and 0.28%±0.09%(p＜0.001).It was found that a subpopulation of hepatocytes in the fraction with enriched AHPC(about 13.5%) began to proliferate significantly and to form colonies on being activated after 2～3 days in culture.The colonies could expand continually more than 2 months,with the occupied area of some reaching 0.64mm~2 and cell dividing more than 10 rounds.Some larger cells,morphologically like mature hepatocytes,appeared in colonies around day30.At 24 hours after attachment,all hepatocytes expressed Albumin,but were negative for AFP,CK19,Oct-4,CD34, CD45,Thy-1,nestin and c-kit.After being activated,the proliferating cells expressed AFP and Albumin at day5,no CK19 expression.But at day30,the expanding colonies expressed CK19,and some cells within colonies manifested Albumin negative. Immunofluorescence double staining showed some cells with specific pattern of expression in colonies at day30,such as Albumin~+AFP~-,Albumin~+AFP~+, Albumin~+CK19~+,CK19~+AFP~+,CK19~+AFP~-.All formed colonies didn't express Oct-4, CD34,CD45,Thy-1,nestin and c-kit during being cultured.Besides,AHPC colonies could be continuously subcultured for more than 60 days without losing proliferating ability.Attachment of colony aggregates showed a better proliferating performance than those of detached single cells of colonies.Attached AHPC colonies off-spring cells were small with epithelial cell-like appearance and a relatively larger nucleus plasma ratio.
Conclusion
1.There exists a kind of liver specific progenitor cells in adult normal mouse, named adult hepatic progenitor cells(AHPC),and mouse AHPC cell model cultured in vitro has been successfully established.
2.Mouse AHPC is capable of proliferation,and holds bipotentials to differentiate into hepatocytes and duct cells.In addition,it can be subcultured for more than 60 days without losing proliferating ability.
3.The contamination and growth of NPC can promote AHPC to activate and proliferate at early culture days,and however NPC may play a part role in inducing progeny cells in AHPC colonies to differentiate at later days.
4.By comparing with other reported hepatic progenitor cells isolated from normal rats and mouse,the mouse AHPC isolated in this study shows a relatively higher ability to proliferate with differently expressing characters,and holds definite two differentiating potentials.Mouse AHPC may be useful tools as new in vitro cell models to investigate liver diseases,liver development and regeneration,and can facilitate the study of liver stem cells that can be used in cell transplantation.
研究正常成体小鼠肝细胞的增殖能力和分化潜能,分离正常成体小鼠肝脏内可能存在的干细胞或祖细胞并建立体外培养的细胞模型,研究其基本的生物学特性。
方法
应用改良的Seglen二步法灌注和离心分离肝脏细胞,将肝细胞初步分为肝脏实质细胞(parenchymal hepatocytes,PH)部分和富含AHPC(adult hepaticprogenitor cells,AHPC)部分,对两部分的细胞用添加胎牛血清(FBS)的改良DMEM培养基进行培养,持续观察超过60天,分析两部分中肝细胞的形态学差异,以及通过细胞增殖和克隆的形成情况分析两部分中肝细胞增殖能力的差异。应用免疫荧光技术对具有高增殖能力细胞及其形成的克隆进行Albumin、AFP、CK19、c-kit、CD45、CD34、Oct-4、Desmin、CD16、Thy-1和nestin等染色,分析细胞标记物的表达和克隆内细胞的成熟分化情况。利用形态学观察、记录细胞增殖状况,以及免疫荧光染色技术初步分析肝脏非实质细胞(nonparenchymalcell,NPC)的生长对于AHPC活化、增殖和分化的影响。另外,尝试了AHPC克隆的传代培养。
结果
本研究中两部分肝细胞均获得较高的产量和活性(＞90%),完全满足实验的需要。PH部分和富含AHPC部分的肝细胞大小分别为(37.03±6.65)μm和(22.63±2.04)μm,存在明显的统计学差异(p＜0.05),但在大小分布比例上存在小部分的重叠。两部分的贴壁细胞中均含有NPC污染,其中富含AHPC部分较多,占贴壁细胞总数的70.3%,NPC增殖后肝细胞活化并开始增殖,所有的肝细胞克隆均表达肝星状细胞标记物Desmin。富含AHPC部分的肝细胞增殖能力明显较PH部分高,两部分的克隆形成率分别为21.45%±1.25%和0.28%±0.09%(p＜0.001)。富含AHPC部分中,约13.5%的贴壁肝细胞在接种后第2～3天活化并迅速增殖,第4～5天形成小的细胞克隆,极少数细胞(0.5%～1%)在接种后第3天即可形成克隆;培养30天后克隆内出现类似成熟的肝细胞,细胞克隆可持续扩增超过60天,最大克隆面积达到0.64mm~2,细胞平均增殖超过10个周期。贴壁后24小时,所有的肝细胞均强阳性表达肝细胞标记物Albumin,不表达AFP和CK19,培养第5天细胞克隆开始表达Albumin和AFP,第30天克履诓糠窒赴泶锏ü芟赴昙俏顲K19,同时发现Albumin阴性细胞。通过免疫荧光双染发现,培养第30天,AHPC克隆内同时存在Albumin阳性和AFP阳性、Albumin阳性和AFP阴性、Albumin阳性和CK19阴性、CK19阳性和AFP阴性、CK19阳性和AFP阳性的细胞。另外,AHPC可以传代培养超过60天,传代培养中,贴壁的AHPC克隆内细胞较小,核浆比率大,呈上皮样细胞的形态,部分细胞仍然具有独立形成克隆的能力,但观察发现,AHPC克隆解离为细胞团进行传代培养中细胞的增殖比解离为单细胞传代好。
结论
1.在正常成体小鼠肝脏内存在一种肝脏组织特异性的成体肝脏祖细胞(AHPC),并已成功建立了体外培养的细胞模型。
2.小鼠AHPC体外培养活化后可持续克隆性增殖超过60天,并可连续传代培养,具有向肝细胞和胆管细胞分化的双向分化潜能。
3.肝脏非实质细胞(NPC)的生长可以促进AHPC的增殖、成熟和分化。
4.与国外文献报导的肝脏祖细胞相比,小鼠AHPC体外培养中细胞表型不同,具有较高的增殖能力和明显的双向分化潜能,为肝细胞移植、肝脏发育和肝病等研究提供了一种新的肝脏干细胞模型和研究工具。
Objective
This study is aimed to isolate and culture candidate cells of liver stem cells or progenitor cells from normal adult mouse liver,by investigating their abilitys to proliferate and potentials to differentiate in vitro,and to establish a stable cell model cultured in vitro,followed by characterizing the fundamental properties of those cells.
Methods
Mouse hepatocytes,isolated by two-step collagenase perfusion and mechanical centrifugation,were separated into two fractions as fraction of PH(parenchymal hepatocytes,PH) and fraction with enriched AHPC(adult hepatic progenitor cells, AHPC).Two fraction cells were cultured in modified DMEM more than 60 days,and observed by phase contrast microscopy.It was clarified that the morphological and proliferating difference between those hepatocytes from two fractions.The expression of cell markers,such as Albumin,AFP,CK19,c-kit,CD45,CD34,Oct-4,Desmin, CD 16,Thy-1 and nestin in high proliferating cells and their colonies,was illuminated by immunofluorescence at different culture days,to analyze the origin of those cells and their potentials to differentiate.Besides,it was primarily investigated that the impact of NPC(nonparenchymal cells,NPC) contamination and growth on the activation,proliferation and differentiation of AHPC during primary culture,by immunofluorescence and noticing the morphological changes and growing state of NPC and AHPC.In addition,efforts were made to investigate the subculture possilbity of AHPC colonies.
Results
The total cell quantity and cell vitality of hepatocytes were relatively high in two fractions,with cell vitality more than 90%.The cell size of fraction with enriched AHPC was(22.63±2.04)μm,and PH fraction was(37.03±6.65)μm,with a significant statistical difference(p＜0.05).The percentage of different cell size, however,showed two fractions shared some cells with certain sizes.There was a contamination of HPC in both fractions,and the one of enriched AHPC had more HPC(about 73%of attached cells).In the primary culture,HPC firstly began to divide and proliferate,then attached hepatocytes showed being activated,and all formed colonies expressed Desmin,a relatively specific cell marker of hepatic stellate cell(HSC).The fraction with enriched AttPC manifested a definitely larger colony-forming rate than PH ones,respectively 21.45%±1.25%and 0.28%±0.09%(p＜0.001).It was found that a subpopulation of hepatocytes in the fraction with enriched AHPC(about 13.5%) began to proliferate significantly and to form colonies on being activated after 2～3 days in culture.The colonies could expand continually more than 2 months,with the occupied area of some reaching 0.64mm~2 and cell dividing more than 10 rounds.Some larger cells,morphologically like mature hepatocytes,appeared in colonies around day30.At 24 hours after attachment,all hepatocytes expressed Albumin,but were negative for AFP,CK19,Oct-4,CD34, CD45,Thy-1,nestin and c-kit.After being activated,the proliferating cells expressed AFP and Albumin at day5,no CK19 expression.But at day30,the expanding colonies expressed CK19,and some cells within colonies manifested Albumin negative. Immunofluorescence double staining showed some cells with specific pattern of expression in colonies at day30,such as Albumin~+AFP~-,Albumin~+AFP~+, Albumin~+CK19~+,CK19~+AFP~+,CK19~+AFP~-.All formed colonies didn't express Oct-4, CD34,CD45,Thy-1,nestin and c-kit during being cultured.Besides,AHPC colonies could be continuously subcultured for more than 60 days without losing proliferating ability.Attachment of colony aggregates showed a better proliferating performance than those of detached single cells of colonies.Attached AHPC colonies off-spring cells were small with epithelial cell-like appearance and a relatively larger nucleus plasma ratio.
Conclusion
1.There exists a kind of liver specific progenitor cells in adult normal mouse, named adult hepatic progenitor cells(AHPC),and mouse AHPC cell model cultured in vitro has been successfully established.
2.Mouse AHPC is capable of proliferation,and holds bipotentials to differentiate into hepatocytes and duct cells.In addition,it can be subcultured for more than 60 days without losing proliferating ability.
3.The contamination and growth of NPC can promote AHPC to activate and proliferate at early culture days,and however NPC may play a part role in inducing progeny cells in AHPC colonies to differentiate at later days.
4.By comparing with other reported hepatic progenitor cells isolated from normal rats and mouse,the mouse AHPC isolated in this study shows a relatively higher ability to proliferate with differently expressing characters,and holds definite two differentiating potentials.Mouse AHPC may be useful tools as new in vitro cell models to investigate liver diseases,liver development and regeneration,and can facilitate the study of liver stem cells that can be used in cell transplantation.
引文
1. Steindler D A. Stem cells, regenerative medicine, and animal models of disease [J]. ILAR J. 2007. 48(4): 323-38.
2. Battey J F. Stem cells: Current challenges and future promise [J]. Dev Dyn. 2007. 236(12): 3193-8.
3. 裴雪涛. 干细胞生物学 [M].2003: 科学出版社.
4. Steer C J. Liver regeneration [J]. FASEB J. 1995. 9(14): 1396-400.
5. Higgins G M and Anderson R M. Experimental pathology of the liver. I. restoration of the liver of the white rat following partial surgical removal. [J]. Arch Pathol. 1931.12: 186-202.
6. Michalopoulos G K and DeFrances M C. Liver regeneration [J]. Science. 1997. 276(5309): 60-66.
7. Wilson J W and Leduc E H. Role of cholangioles in restoration of the liver of the mouse after dietary injury [J]. J Pathol Bacteriol. 1958. 76(2): 441-9.
8. Conigliaro A, Colletti M, Cicchini C, et al. Isolation and characterization of a murine resident liver stem cell [J]. Cell Death Differ. 2008. 15(1): 123-133.
9. Duret C, Gerbal-Chaloin S, Ramos J, et al. Isolation, characterization, and differentiation to hepatocyte-like cells of nonparenchymal epithelial cells from adult human liver [J]. Stem Cells. 2007. 25(7): 1779-90.
10. Shackel N A and Warner F J. Identification of resident hepatic stem cell populations [J]. Hepatology. 2007. 46(6): 2042-2044.
11. Wright N, Samuelson L, Walkup M H, et al. Enrichment of a bipotent hepatic progenitor cell from naive adult liver tissue [J]. Biochem Biophys Res Commun. 2008. 366(2): 367-72.
12. Aleem Khan A, Parveen N, Habeeb M A, et al. Journey from hepatocyte transplantation to hepatic stem cells: a novel treatment strategy for liver diseases [J]. Indian J Med Res. 2006. 123(5): 601-14.
13. Alison M R, Choong C, and Lim S. Application of liver stem cells for cell therapy [J]. Semin Cell Dev Biol. 2007. 18(6): 819-26.
14. Petersen B E, Bowen W C, Patrene K D, et al. Bone marrow as a potential source of hepatic oval cells [J]. Science. 1999. 284(5417): 1168-1170.
15. Theise N D, Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation [J]. Hepatology. 2000. 31(1): 235-40.
16. Strick-Marchand H, Morosan S, Charneau P, et al. Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes [J]. PNAS. 2004. 101(22): 8360-8365.
17. Strom S C, Bruzzone P, Cai H, et al. Hepatocyte transplantation: clinical experience and potential for future use [J]. Cell Transplant. 2006. 15 Suppl 1: S105-10.
18. Mustapha N, Francoise S, and Etienne S. Hepatocyte transplantation: current and future developments [J]. Curr Opin Organ Transplant. 2007. 12(5): 503-508.
19. Machimoto T, Yasuchika K, Komori J, et al. Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model [J]. Transplantation. 2007. 84(10): 1233-9.
20. Nussler A, Konig S, Ott M, et al. Present status and perspectives of cell-based therapies for liver diseases [J]. J Hepatol. 2006. 45(1): 144-59.
21. Sen S and Jalan R. Ideal hepatocyte: quest for the Holy Grail [J]. J Gastroenterol Hepatol. 2005. 20(1): 1-4.
22. Newsome P N, Hussain M A, and Theise N D. Hepatic oval cells: helping redefine a paradigm in stem cell biology [J]. Curr Top Dev Biol. 2004. 61: 1-28.
23. Jelnes P, Santoni-Rugiu E, Rasmussen M, et al. Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration [J]. Hepatology. 2007. 45(6): 1462-70.
24. Lyra A C, Soares M B P, dos Santos R R, et al. Bone marrow stem cells and liver disease [J]. Gut. 2007. 56(11): 1640.
25. Thorgeirsson S S and Grisham J W. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence [J]. Hepatology. 2006. 43(1): 2-8.
26. Fiegel H C, Bruns H, Hoper C, et al. Cell growth and differentiation of different hepatic cells isolated from fetal rat liver in vitro [J]. Tissue Engineering. 2006. 12(1): 123-130.
27. Schmelzer E, Zhang L, Bruce A, et al. Human hepatic stem cells from fetal and postnatal donors [J]. J Exp Med. 2007.
28. Dan Y Y, Riehle K J, Lazaro C, et al. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages [J]. PNAS. 2006. 103(26): 9912-9917.
29. Tang Y, Kitisin K, Jogunoori W, et al. Progenitor/stem cells give rise to liver cancer due to aberrant TGF-{beta} and IL-6 signaling [J]. PNAS. 2008. 105(7): 2445-2450.
30. Zhang F, Chen X P, Zhang W, et al. Combined hepatocellular cholangiocarcinoma originating from hepatic progenitor cells: immunohistochemical and double-fluorescence immunostaining evidence [J]. Histopathology. 2008. 52(2): 224-32.
31. Mitaka T, Mizuguchi T, Sato F, et al. Growth and maturation of small hepatocytes [J]. J Gastroenterol Hepatol. 1998. 13 Suppl: S70-7.
32. Tateno C and Yoshizato K. Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes [J]. Am J Pathol. 1996. 148(2): 383-392.
33. Sahin M B, Schwartz R E, Buckley S M, et al. Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver [J]. Liver Transpl. 2008. 14(3): 333-345.
34. Akhurst B, Croager E J, Farley-Roche C A, et al. A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver [J]. Hepatology. 2001. 34(3): 519-22.
35. Collins F S, Rossant J, and Wurst W. A mouse for all reasons [J]. Cell. 2007. 128(1): 9-13.
36. Azuma H, Hirose T, Fujii H, et al. Enrichment of hepatic progenitor cells from adult mouse liver [J]. Hepatology. 2003. 37(6): 1385-94.
37. Waterston R H, Lindblad-Toh K, Birney E, et al. Initial sequencing and comparative analysis of the mouse genome [J]. Nature. 2002. 420(6915): 520-62.
38. Berry M N and Friend D S. High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study [J]. J. Cell Biol. 1969. 43(3): 506-520.
39. Seglen P O. Preparation of isolated rat liver cells [J]. Methods Cell Biol. 1976. 13: 29-83.
40. Pogson C I, Elliott K R F, and Lomax M A, Variations in hepatic metabolism between species., in Isolation, characterization and use of hepatocytes., R A Harris and N W Cornell, Editors. 1983, Elsevier Biomedical: New York. p. 21-30.
41. Kon J, Ooe H, Oshima H, et al. Expression of CD44 in rat hepatic progenitor cells [J]. J Hepatol. 2006. 45(1): 90-8.
42. Kreamer B L, Staecker J L, Sawada N, et al. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations [J]. In Vitro Cell Dev Biol. 1986. 22(4): 201-11.
43. Tateno C, Takai-Kajihara K, Yamasaki C, et al. Heterogeneity of growth potential of adult rat hepatocytes in vitro [J]. Hepatology. 2000. 31(1): 65-74.
44. Yamada S, Yamamoto Y, Nagasawa M, et al. In Vitro Transdifferentiation of Mature Hepatocytes into Insulin-Producing Cells [J]. Endocr J. 2006.
45. Gomez-Lechon M J, Lopez P, Donato T, et al. Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo [J]. In Vitro Cell Dev Biol. 1990. 26(1): 67-74.
46. Mitaka T, Sattler G L, and Pitot H C. Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum [J]. J Cell Physiol. 1991. 147(3): 495-504.
47. Tsuchiya A, Heike T, Fujino H, et al. Long-term Extensive Expansion of Mouse Hepatic Stem/Progenitor Cells in a Novel Serum-Free Culture System [J]. Gastroenterology. 2005. 128(7): 2089-2104.
48. Ismail T, Howl J, Wheatley M, et al. Growth of normal human hepatocytes in primary culture: effect of hormones and growth factors on DNA synthesis [J]. Hepatology. 1991. 14(6): 1076-82.
49. Kim S H and Akaike T. Epidermal growth factor signaling for matrix-dependent cell proliferation and differentiation in primary cultured hepatocytes [J]. Tissue Eng. 2007. 13(3): 601-9.
50. Eckl P M, Whitcomb W R, Michalopoulos G, et al. Effects of EGF and calcium on adult parenchymal hepatocyte proliferation [J]. J Cell Physiol. 1987. 132(2): 363-6.
51. Mitaka T, Kojima T, Mizuguchi T, et al. Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF [J]. In Vitro Cell Dev Biol Anim. 1996. 32(8): 469-77.
52. Sakai Y, Jiang J, Kojima N, et al. Enhanced in vitro maturation of fetal mouse liver cells with oncostatin M, nicotinamide, and dimethyl sulfoxide [J]. Cell Transplant. 2002. 11(5): 435-41.
53. Nagaya N, Kubota S, Suzuki N, et al. Thermoreversible gelation polymer induces the emergence of hepatic stem cells in the partially injured rat liver [J]. Hepatology. 2006. 43(5): 1053-1062.
54. Hattori N, Nishino K, Ko Y G, et al. Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells [J]. J Biol Chem. 2004.279(17): 17063-9.
55. Lowes K N, Croager E J, Olynyk J K, et al. Oval cell-mediated liver regeneration: Role of cytokines and growth factors [J]. J Gastroenterol Hepatol. 2003. 18(1): 4-12.
56. Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells [J]. Proc Natl Acad Sci U S A. 2003. 100(3): 998-1003.
57. Michalopoulos G K. Liver regeneration: molecular mechanisms of growth control [J].FASEB J. 1990. 4(2): 176-87.
58. Sawada N, Lee G H, Mochizuki Y, et al. Active proliferation of mouse hepatocytes in primary culture under defined conditions as compared to rat hepatocytes [J]. Jpn J Cancer Res. 1988. 79(9): 983-8.
59. Rhim J A, Sandgren E P, Degen J L, et al. Replacement of diseased mouse liver by hepatic cell transplantation [J]. Science. 1994. 263(5150): 1149-1152.
60. Overturf K, al-Dhalimy M, Ou C N, et al. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes [J]. Am J Pathol. 1997. 151(5): 1273-80.
61. Brawley C and Matunis E. Regeneration of male germline stem cells by spermatogonial dedifferentiation in vivo [J]. Science. 2004. 304(5675): 1331-4.
62. Kai T and Spradling A. Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries [J]. Nature. 2004. 428(6982): 564-9.
63. Nakagawa T, Nabeshima Y, and Yoshida S. Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis [J]. Dev Cell. 2007. 12(2): 195-206.
64. Simon A and Frisen J. From stem cell to progenitor and back again [J]. Cell. 2007. 128(5): 825-826.
65. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell [J]. Blood Cells. 1978. 4(1-2): 7-25.
66. Moore K A and Lemischka I R. Stem cells and their niches [J]. Science. 2006. 311(5769): 1880-1885.
67. Block G D, Locker J, Bowen W C, et al. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium [J]. J Cell Biol. 1996. 132(6): 1133-49.
68. Plescia C P, Rogler C E, and Rogler L E. Genomic expression analysis implicates Wnt signaling pathway and extracellular matrix alterations in hepatic specification and differentiation of murine hepatic stem cells [J]. Differentiation. 2001. 68(4-5): 254-269.
69. Yin L, Lynch D, Ilic Z, et al. Proliferation and differentiation of ductular progenitor cells and littoral cells during the regeneration of the rat liver to CCl4/2-AAF injury [J]. Histology and Histopathology. 2002. 17(1): 65-81.
70. Friedman S L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver [J]. Physiol Rev. 2008. 88(1): 125-72.
71. Toshihiro Mitaka, Michihide Mikami, Gerald L. Sattler, et al. Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor [J]. Hepatology. 1992. 16(2): 440-447.
72. Mitaka T. The current status of primary hepatocyte culture [J]. International Journal of Experimental Pathology. 1998. 79(6): 393-409.
73. Mitaka T, Sato F, Mizuguchi T, et al. Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells [J]. Hepatology. 1999. 29(1): 111-25.
74. Tateno C and Yoshizato K. Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells[J].Am J Pathol. 1996. 149(5): 1593-1605.
75. Wang J, Clark J B, Rhee G S, et al. Proliferation and hepatic differentiation of adult-derived progenitor cells [J]. Cells Tissues Organs. 2003. 173(4): 193-203.
1.裴雪涛.干细胞生物学[M]:科学出版社,2003.
2.Simon A,Frisen J.From stem cell to progenitor and back again[J].Cell 2007;128:825-826.
3.Schofield R.The relationship between the spleen colony-forming cell and the haemopoietic stem cell[J].Blood Cells 1978;4:7-25.
4.Moore KA,Lemischka IR.Stem cells and their niches[J].Science 2006;311:1880-1885.
5.Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J].Proc Natl Acad Sci U S A 1981;78:7634-7638.
6.Terskikh AV,Bryant P J,Schwartz PH.Mammalian stem cells[J].Pediatr Res 2006;59:13R-20R.
7.Arnhold S,Klein H,Semkova I,Addicks K,et al.Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space [J]. Invest Ophthalmol Vis Sci 2004;45:4251 -4255.
8. Vogel G. STEM CELLS: Scientists derive line from single embryo cell [J]. Science 2006;313:1031a.
9. Pearson H, Abbott A. Stem cells derived from 'dead' human embryo [J]. Nature 2006;443:376-377.
10. Smith AJ. Human embryonic stem cell research and its regulation [J]. J Dent Res 2007;86:197.
11. Koide Y, Morikawa S, Mabuchi Y, et al. Two distinct stem cell lineages in murine bone marrow [J]. Stem Cells 2007:2006-0325.
12. Preston SL, Alison MR, Forbes SJ, et al. The new stem cell biology: something for everyone [J]. Mol Pathol 2003;56:86-96.
13. Berenson RJ, Andrews RG, Bensinger WI, et al. Antigen CD34+ marrow cells engraft lethally irradiated baboons [J]. J Clin Invest 1988;81:951-955.
14. Danet GH, Luongo JL, Butler G, Lu MM, Tenner AJ, Simon MC, Bonnet DA. C1qRp defines a new human stem cell population with hematopoietic and hepatic potential [J]. Proc Natl Acad Sci U S A2002;99:10441-10445.
15. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science 1999;284:143-147.
16. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta [J]. Nat Med 1999;5:309-313.
17. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature 2002;418:41-49.
18. Holden C. STEM CELLS: Controversial marrow cells coming into their own? [J] Science 2007;315:760-761.
19. Miller RH. The promise of stem cells for neural repair [J]. Brain Research 2006;1091:258-264.
20. Levesque MF, T. N. Autologous transplantation of adult human neural stem cells and differentiated dopaminergic neurons for Parkinson's disease: one year post-operative clinical and functional metabolic results. In: American Association of Neurological Surgeons meeting. Chicago; 2002.
21. Martino G, Pluchino S. The therapeutic potential of neural stem cells [J]. Nat Rev Neurosci 2006;7:395-406.
22.Krupczak-Hollis K,Wang X,Dennewitz MB,et al.Growth hormone stimulates proliferation of old-aged regenerating liver through forkhead box mlb[J].Hepatology 2003;38:1552-1562.
23.Petersen BE,Bowen WC,Patrene KD,et al.Bone marrow as a potential source of hepatic oval cells[J].Science 1999;284:1168-1170.
24.Kallis YN,Alison MR,Forbes SJ.Bone marrow stem cells and liver disease[J].Gut 2007;56:716-724.
25.Thorgeirsson SS,Grisham JW.Hematopoietic cells as hepatocyte stem cells:a critical review of the evidence[J].Hepatology 2006;43:2-8.
26.Tateno C,Yoshizato K.Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells[J].Am J Pathol 1996;149:1593-1605.
27.Bonner-Weir S,Taneja M,Weir GC,et al.In vitro cultivation of human islets from expanded ductal tissue[J].Proc Natl Acad Sci U S A 2000;97:7999-8004.
28.Ramiya VK,Maraist M,Arfors KE,et al.Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells[J].Nat Med 2000;6:278-282.
29.Gu G,Dubauskaite J,Melton DA.Direct evidence for the pancreatic lineage:NGN3+ cells are islet progenitors and are distinct from duct progenitors[J].Development 2002;129:2447-2457.
30.Hunziker E,Stein M.Nestin-expressing cells in the pancreatic islets of langerhans[J].Biochemical and Biophysical Research Communications 2000;271:116-119.
31.宋陆军,秦新裕,牛伟新,et al.源于体外培养胰岛中表达Ngn3的细胞是胰腺内分泌前体细胞的新证据[J].中华外科杂志2005:42-45.
32.Dabeva MD,Hwang SG,Vasa SR,et al.Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver[J].Proc Natl Acad Sci U S A 1997;94:7356-7361.
33.Dor Y,Brown J,Martinez OI,et al.Adult pancreatic[beta]-cells are formed by self-duplication rather than stem-cell differentiation[J].Nature 2004;429:41-46.
34.Stanger BZ,Tanaka A J,Melton DA.Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver[J].Nature 2007;445:886-891.
35.Couzin J.Developmental biology:In embryos,pancreas and liver reach full size in different ways [J]. Science 2007;315:587.
36. Baeyens L, De Breuck S, Lardon J, et al. In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells [J]. Diabetologia 2005;48:49-57.
37. Minami K, Okuno M, Miyawaki K, et al. Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells [J]. Proc Natl Acad Sci U S A 2005;102:15116-15121.
38. Lardon J, Huyens N, Rooman I, et al. Exocrine cell transdifferentiation in dexamethasone-treated rat pancreas [J]. Virchows Arch 2004;444:61-65.
39. Desai BM, Oliver-Krasinski J, De Leon DD, et al. Preexisting pancreatic acinar cells contribute to acinar cell, but not islet beta cell, regeneration [J]. J Clin Invest 2007;117:971-977.
40. Leaf C. Why we're losing the war on cancer (and how to win it). Fortune 2004; 149:76-82, 84-76, 88 passim.
41. Karhadkar SS, Bova GS, Abdallah N, et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis [J]. Nature 2004;431:707-712.
42. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells [J]. Proc Natl Acad Sci U S A 2003;100:3983-3988.
1.Suzuki A,Nakauchi H,Taniguchi H.In vitro production of functionally mature hepatocytes from prospectively isolated hepatic stem cells[J].Cell Transplant 2003;12:469-473.
2.Parent R,Marion M J,Furio L,et al.Origin and characterization of a human bipotent liver progenitor cell line[J].Gastroenterology 2004;126:1147-1156.
3.Aurich I,Mueller LP,Aurich H,et al.Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers[J].Gut 2007;56:405-415.
4.Jang Y-Y,Collector MI,Baylin SB,et al.Hematopoietic stem cells convert into liver cells within days without fusion[J].Nat Cell Biol 2004;6:532-539.
5.Rountree CB,Barsky L,Ge S,et al.A CDl33 expressing murine liver oval cell population with bi-lineage potential[J].Stem Cells 2007.
6.Mitaka T,Mikami M,Gerald L.Sattler,Henry C.Pitot,Mochizuki.Y.Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor[J].Hepatology 1992;16:440-447.
7.NusslerA,Konig S,Ott M,et al.Present status and perspectives of cell-based therapies for liver diseases[J].J Hepatol 2006;45:144-159.
8.Strom SC,Bruzzone P,Cai H,et al.Hepatocyte transplantation:clinical experience and potential for future use[J].Cell Transplant 2006;15 Suppl 1:S105-110.
9. Habibullah CM, Ayesha Q, Khan AA, et al. Xenotransplantation of UV-B-irradiated hepatocytes. Survival and immune response [J]. Transplantation 1995;59:1495-1497.
10. Strom S, Fisher R. Hepatocyte transplantation: new possibilities for therapy [J]. Gastroenterology 2003;124:568-571.
11. Mustapha N, Francoise S, Etienne S. Hepatocyte transplantation: current and future developments [J]. Curr Opin Organ Transplant 2007; 12:503-508.
12. Machimoto T, Yasuchika K, Komori J, et al. Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model [J]. Transplantation 2007;84:1233-1239.
13. Sen S, Jalan R. Ideal hepatocyte: quest for the Holy Grail [J]. J Gastroenterol Hepatol 2005;20:1-4.
14. Wang X, Foster M, Al-Dhalimy M, et al. The origin and liver repopulating capacity of murine oval cells [J]. Proc Natl Acad Sci U S A 2003; 100 Suppl 1:11881-11888.
15. Wu XZ. Origin of cancer stem cells: the role of self-renewal and differentiation [J]. Ann Surg Oncol 2008; 15:407-414.
16. Kim H, Park C, Han KH, et al. Primary liver carcinoma of intermediate (hepatocyte-cholangiocyte) phenotype [J]. J Hepatol 2004;40:298-304.
17. Newsome PN, Hussain MA, Theise ND. Hepatic oval cells: helping redefine a paradigm in stem cell biology [J]. Curr Top Dev Biol 2004;61:l-28.
18. Wilson JW, Leduc EH. Role of cholangioles in restoration of the liver of the mouse after dietary injury [J]. J Pathol Bacteriol 1958;76:441-449.
19. Aleem Khan A, Parveen N, Habeeb MA, et al. Journey from hepatocyte transplantation to hepatic stem cells: a novel treatment strategy for liver diseases [J]. Indian J Med Res 2006;123:601-614.
20. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells [J]. Science 1999;284:1168-1170.
21. Lyra AC, Soares MBP, dos Santos RR, et al. Bone marrow stem cells and liver disease [J]. Gut 2007;56:1640-.
22. Thorgeirsson SS, Grisham JW. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence [J]. Hepatology 2006;43:2-8.
23. Tateno C, Yoshizato K. Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes [J]. Am J Pathol 1996; 148:383-392.
24. Mitaka T, Sattler GL, Pitot HC, et al. Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes [J]. Virchows Archiv. B, Cell Pathology Including Molecular Pathology 1992;62:329-335.
25. Mitaka T, Mizuguchi T, Sato F, et al. Growth and maturation of small hepatocytes [J]. J Gastroenterol Hepatol 1998; 13 Suppl:S70-77.
26. Mitaka T, Sato F, Mizuguchi T, et al. Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells [J]. Hepatology 1999;29:111-125.
27. Meier PJ, Stieger B. Bile salt transporters [J]. Annu Rev Physiol 2002;64:635-661.
28. Sidler Pfandler MA, Hochli M, Inderbitzin D, et al. Small hepatocytes in culture develop polarized transporter expression and differentiation [J]. J Cell Sci 2004; 117:4077-4087.
29. Shiojiri N, Lemire JM, Fausto N. Cell lineages and oval cell progenitors in rat liver development [J]. Cancer Res 1991;51:2611 -2620.
30. Mitaka T. The current status of primary hepatocyte culture [J]. International Journal of Experimental Pathology 1998;79:393-409.
31. Kon J, Ooe H, Oshima H, et al. Expression of CD44 in rat hepatic progenitor cells [J]. Journal of Hepatology 2006;45(1):90-8.
32. Paku S, Schnur J, Nagy P, et al. Origin and Structural Evolution of the Early Proliferating Oval Cells in Rat Liver [J]. Am J Pathol 2001;158:1313-1323.
33. Theise ND, Saxena R, Portmann BC, et al. The canals of Hering and hepatic stem cells in humans [J]. Hepatology 1999;30:1425-1433.
34. Grisham JW. Cell types in long-term propagable cultures of rat liver [J]. Ann N Y Acad Sci 1980;349:128-137.
35. Germain L, Blouin MJ, Marceau N. Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components [J]. Cancer Res 1988;48:4909-4918.
36. Allain JE, Dagher I, Mahieu-Caputo D, et al. Immortalization of a primate bipotent epithelial liver stem cell [J]. Proc Natl Acad Sci U S A 2002;99:3639-3644.
37. Duret C, Gerbal-Chaloin S, Ramos J, et al. Isolation, characterization, and differentiation to hepatocyte-like cells of nonparenchymal epithelial cells from adult human liver [J]. Stem Cells 2007;25:1779-1790.
38. Khuu DN, Najimi M, Sokal EM. Epithelial cells with hepatobiliary phenotype: is it another stem cell candidate for healthy adult human liver? [J] World J Gastroenterol 2007;13:1554-1560.
39. Braun KM, Sandgren EP. Cellular origin of regenerating parenchyma in a mouse model of severe hepatic injury [J]. Am J Pathol 2000;l 57:561-569.
40. Block GD, Locker J, Bowen WC, et al. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium [J]. J Cell Biol 1996;132:1133-1149.
41. Wang J, Clark JB, Rhee GS, et al. Proliferation and hepatic differentiation of adult-derived progenitor cells [J]. Cells Tissues Organs 2003;173:193-203.
42. Wright N, Samuelson L, Walkup MH, et al. Enrichment of a bipotent hepatic progenitor cell from naive adult liver tissue [J]. Biochem Biophys Res Commun 2008;366:367-372.
43. Azuma H, Hirose T, Fujii H, et al. Enrichment of hepatic progenitor cells from adult mouse liver [J]. Hepatology 2003;37:1385-1394.
44. Sahin MB, Schwartz RE, Buckley SM, et al. Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver [J]. Liver Transpl2008;14:333-345.
2. Battey J F. Stem cells: Current challenges and future promise [J]. Dev Dyn. 2007. 236(12): 3193-8.
3. 裴雪涛. 干细胞生物学 [M].2003: 科学出版社.
4. Steer C J. Liver regeneration [J]. FASEB J. 1995. 9(14): 1396-400.
5. Higgins G M and Anderson R M. Experimental pathology of the liver. I. restoration of the liver of the white rat following partial surgical removal. [J]. Arch Pathol. 1931.12: 186-202.
6. Michalopoulos G K and DeFrances M C. Liver regeneration [J]. Science. 1997. 276(5309): 60-66.
7. Wilson J W and Leduc E H. Role of cholangioles in restoration of the liver of the mouse after dietary injury [J]. J Pathol Bacteriol. 1958. 76(2): 441-9.
8. Conigliaro A, Colletti M, Cicchini C, et al. Isolation and characterization of a murine resident liver stem cell [J]. Cell Death Differ. 2008. 15(1): 123-133.
9. Duret C, Gerbal-Chaloin S, Ramos J, et al. Isolation, characterization, and differentiation to hepatocyte-like cells of nonparenchymal epithelial cells from adult human liver [J]. Stem Cells. 2007. 25(7): 1779-90.
10. Shackel N A and Warner F J. Identification of resident hepatic stem cell populations [J]. Hepatology. 2007. 46(6): 2042-2044.
11. Wright N, Samuelson L, Walkup M H, et al. Enrichment of a bipotent hepatic progenitor cell from naive adult liver tissue [J]. Biochem Biophys Res Commun. 2008. 366(2): 367-72.
12. Aleem Khan A, Parveen N, Habeeb M A, et al. Journey from hepatocyte transplantation to hepatic stem cells: a novel treatment strategy for liver diseases [J]. Indian J Med Res. 2006. 123(5): 601-14.
13. Alison M R, Choong C, and Lim S. Application of liver stem cells for cell therapy [J]. Semin Cell Dev Biol. 2007. 18(6): 819-26.
14. Petersen B E, Bowen W C, Patrene K D, et al. Bone marrow as a potential source of hepatic oval cells [J]. Science. 1999. 284(5417): 1168-1170.
15. Theise N D, Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation [J]. Hepatology. 2000. 31(1): 235-40.
16. Strick-Marchand H, Morosan S, Charneau P, et al. Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes [J]. PNAS. 2004. 101(22): 8360-8365.
17. Strom S C, Bruzzone P, Cai H, et al. Hepatocyte transplantation: clinical experience and potential for future use [J]. Cell Transplant. 2006. 15 Suppl 1: S105-10.
18. Mustapha N, Francoise S, and Etienne S. Hepatocyte transplantation: current and future developments [J]. Curr Opin Organ Transplant. 2007. 12(5): 503-508.
19. Machimoto T, Yasuchika K, Komori J, et al. Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model [J]. Transplantation. 2007. 84(10): 1233-9.
20. Nussler A, Konig S, Ott M, et al. Present status and perspectives of cell-based therapies for liver diseases [J]. J Hepatol. 2006. 45(1): 144-59.
21. Sen S and Jalan R. Ideal hepatocyte: quest for the Holy Grail [J]. J Gastroenterol Hepatol. 2005. 20(1): 1-4.
22. Newsome P N, Hussain M A, and Theise N D. Hepatic oval cells: helping redefine a paradigm in stem cell biology [J]. Curr Top Dev Biol. 2004. 61: 1-28.
23. Jelnes P, Santoni-Rugiu E, Rasmussen M, et al. Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration [J]. Hepatology. 2007. 45(6): 1462-70.
24. Lyra A C, Soares M B P, dos Santos R R, et al. Bone marrow stem cells and liver disease [J]. Gut. 2007. 56(11): 1640.
25. Thorgeirsson S S and Grisham J W. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence [J]. Hepatology. 2006. 43(1): 2-8.
26. Fiegel H C, Bruns H, Hoper C, et al. Cell growth and differentiation of different hepatic cells isolated from fetal rat liver in vitro [J]. Tissue Engineering. 2006. 12(1): 123-130.
27. Schmelzer E, Zhang L, Bruce A, et al. Human hepatic stem cells from fetal and postnatal donors [J]. J Exp Med. 2007.
28. Dan Y Y, Riehle K J, Lazaro C, et al. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages [J]. PNAS. 2006. 103(26): 9912-9917.
29. Tang Y, Kitisin K, Jogunoori W, et al. Progenitor/stem cells give rise to liver cancer due to aberrant TGF-{beta} and IL-6 signaling [J]. PNAS. 2008. 105(7): 2445-2450.
30. Zhang F, Chen X P, Zhang W, et al. Combined hepatocellular cholangiocarcinoma originating from hepatic progenitor cells: immunohistochemical and double-fluorescence immunostaining evidence [J]. Histopathology. 2008. 52(2): 224-32.
31. Mitaka T, Mizuguchi T, Sato F, et al. Growth and maturation of small hepatocytes [J]. J Gastroenterol Hepatol. 1998. 13 Suppl: S70-7.
32. Tateno C and Yoshizato K. Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes [J]. Am J Pathol. 1996. 148(2): 383-392.
33. Sahin M B, Schwartz R E, Buckley S M, et al. Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver [J]. Liver Transpl. 2008. 14(3): 333-345.
34. Akhurst B, Croager E J, Farley-Roche C A, et al. A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver [J]. Hepatology. 2001. 34(3): 519-22.
35. Collins F S, Rossant J, and Wurst W. A mouse for all reasons [J]. Cell. 2007. 128(1): 9-13.
36. Azuma H, Hirose T, Fujii H, et al. Enrichment of hepatic progenitor cells from adult mouse liver [J]. Hepatology. 2003. 37(6): 1385-94.
37. Waterston R H, Lindblad-Toh K, Birney E, et al. Initial sequencing and comparative analysis of the mouse genome [J]. Nature. 2002. 420(6915): 520-62.
38. Berry M N and Friend D S. High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study [J]. J. Cell Biol. 1969. 43(3): 506-520.
39. Seglen P O. Preparation of isolated rat liver cells [J]. Methods Cell Biol. 1976. 13: 29-83.
40. Pogson C I, Elliott K R F, and Lomax M A, Variations in hepatic metabolism between species., in Isolation, characterization and use of hepatocytes., R A Harris and N W Cornell, Editors. 1983, Elsevier Biomedical: New York. p. 21-30.
41. Kon J, Ooe H, Oshima H, et al. Expression of CD44 in rat hepatic progenitor cells [J]. J Hepatol. 2006. 45(1): 90-8.
42. Kreamer B L, Staecker J L, Sawada N, et al. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations [J]. In Vitro Cell Dev Biol. 1986. 22(4): 201-11.
43. Tateno C, Takai-Kajihara K, Yamasaki C, et al. Heterogeneity of growth potential of adult rat hepatocytes in vitro [J]. Hepatology. 2000. 31(1): 65-74.
44. Yamada S, Yamamoto Y, Nagasawa M, et al. In Vitro Transdifferentiation of Mature Hepatocytes into Insulin-Producing Cells [J]. Endocr J. 2006.
45. Gomez-Lechon M J, Lopez P, Donato T, et al. Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo [J]. In Vitro Cell Dev Biol. 1990. 26(1): 67-74.
46. Mitaka T, Sattler G L, and Pitot H C. Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum [J]. J Cell Physiol. 1991. 147(3): 495-504.
47. Tsuchiya A, Heike T, Fujino H, et al. Long-term Extensive Expansion of Mouse Hepatic Stem/Progenitor Cells in a Novel Serum-Free Culture System [J]. Gastroenterology. 2005. 128(7): 2089-2104.
48. Ismail T, Howl J, Wheatley M, et al. Growth of normal human hepatocytes in primary culture: effect of hormones and growth factors on DNA synthesis [J]. Hepatology. 1991. 14(6): 1076-82.
49. Kim S H and Akaike T. Epidermal growth factor signaling for matrix-dependent cell proliferation and differentiation in primary cultured hepatocytes [J]. Tissue Eng. 2007. 13(3): 601-9.
50. Eckl P M, Whitcomb W R, Michalopoulos G, et al. Effects of EGF and calcium on adult parenchymal hepatocyte proliferation [J]. J Cell Physiol. 1987. 132(2): 363-6.
51. Mitaka T, Kojima T, Mizuguchi T, et al. Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF [J]. In Vitro Cell Dev Biol Anim. 1996. 32(8): 469-77.
52. Sakai Y, Jiang J, Kojima N, et al. Enhanced in vitro maturation of fetal mouse liver cells with oncostatin M, nicotinamide, and dimethyl sulfoxide [J]. Cell Transplant. 2002. 11(5): 435-41.
53. Nagaya N, Kubota S, Suzuki N, et al. Thermoreversible gelation polymer induces the emergence of hepatic stem cells in the partially injured rat liver [J]. Hepatology. 2006. 43(5): 1053-1062.
54. Hattori N, Nishino K, Ko Y G, et al. Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells [J]. J Biol Chem. 2004.279(17): 17063-9.
55. Lowes K N, Croager E J, Olynyk J K, et al. Oval cell-mediated liver regeneration: Role of cytokines and growth factors [J]. J Gastroenterol Hepatol. 2003. 18(1): 4-12.
56. Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells [J]. Proc Natl Acad Sci U S A. 2003. 100(3): 998-1003.
57. Michalopoulos G K. Liver regeneration: molecular mechanisms of growth control [J].FASEB J. 1990. 4(2): 176-87.
58. Sawada N, Lee G H, Mochizuki Y, et al. Active proliferation of mouse hepatocytes in primary culture under defined conditions as compared to rat hepatocytes [J]. Jpn J Cancer Res. 1988. 79(9): 983-8.
59. Rhim J A, Sandgren E P, Degen J L, et al. Replacement of diseased mouse liver by hepatic cell transplantation [J]. Science. 1994. 263(5150): 1149-1152.
60. Overturf K, al-Dhalimy M, Ou C N, et al. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes [J]. Am J Pathol. 1997. 151(5): 1273-80.
61. Brawley C and Matunis E. Regeneration of male germline stem cells by spermatogonial dedifferentiation in vivo [J]. Science. 2004. 304(5675): 1331-4.
62. Kai T and Spradling A. Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries [J]. Nature. 2004. 428(6982): 564-9.
63. Nakagawa T, Nabeshima Y, and Yoshida S. Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis [J]. Dev Cell. 2007. 12(2): 195-206.
64. Simon A and Frisen J. From stem cell to progenitor and back again [J]. Cell. 2007. 128(5): 825-826.
65. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell [J]. Blood Cells. 1978. 4(1-2): 7-25.
66. Moore K A and Lemischka I R. Stem cells and their niches [J]. Science. 2006. 311(5769): 1880-1885.
67. Block G D, Locker J, Bowen W C, et al. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium [J]. J Cell Biol. 1996. 132(6): 1133-49.
68. Plescia C P, Rogler C E, and Rogler L E. Genomic expression analysis implicates Wnt signaling pathway and extracellular matrix alterations in hepatic specification and differentiation of murine hepatic stem cells [J]. Differentiation. 2001. 68(4-5): 254-269.
69. Yin L, Lynch D, Ilic Z, et al. Proliferation and differentiation of ductular progenitor cells and littoral cells during the regeneration of the rat liver to CCl4/2-AAF injury [J]. Histology and Histopathology. 2002. 17(1): 65-81.
70. Friedman S L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver [J]. Physiol Rev. 2008. 88(1): 125-72.
71. Toshihiro Mitaka, Michihide Mikami, Gerald L. Sattler, et al. Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor [J]. Hepatology. 1992. 16(2): 440-447.
72. Mitaka T. The current status of primary hepatocyte culture [J]. International Journal of Experimental Pathology. 1998. 79(6): 393-409.
73. Mitaka T, Sato F, Mizuguchi T, et al. Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells [J]. Hepatology. 1999. 29(1): 111-25.
74. Tateno C and Yoshizato K. Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells[J].Am J Pathol. 1996. 149(5): 1593-1605.
75. Wang J, Clark J B, Rhee G S, et al. Proliferation and hepatic differentiation of adult-derived progenitor cells [J]. Cells Tissues Organs. 2003. 173(4): 193-203.
1.裴雪涛.干细胞生物学[M]:科学出版社,2003.
2.Simon A,Frisen J.From stem cell to progenitor and back again[J].Cell 2007;128:825-826.
3.Schofield R.The relationship between the spleen colony-forming cell and the haemopoietic stem cell[J].Blood Cells 1978;4:7-25.
4.Moore KA,Lemischka IR.Stem cells and their niches[J].Science 2006;311:1880-1885.
5.Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J].Proc Natl Acad Sci U S A 1981;78:7634-7638.
6.Terskikh AV,Bryant P J,Schwartz PH.Mammalian stem cells[J].Pediatr Res 2006;59:13R-20R.
7.Arnhold S,Klein H,Semkova I,Addicks K,et al.Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space [J]. Invest Ophthalmol Vis Sci 2004;45:4251 -4255.
8. Vogel G. STEM CELLS: Scientists derive line from single embryo cell [J]. Science 2006;313:1031a.
9. Pearson H, Abbott A. Stem cells derived from 'dead' human embryo [J]. Nature 2006;443:376-377.
10. Smith AJ. Human embryonic stem cell research and its regulation [J]. J Dent Res 2007;86:197.
11. Koide Y, Morikawa S, Mabuchi Y, et al. Two distinct stem cell lineages in murine bone marrow [J]. Stem Cells 2007:2006-0325.
12. Preston SL, Alison MR, Forbes SJ, et al. The new stem cell biology: something for everyone [J]. Mol Pathol 2003;56:86-96.
13. Berenson RJ, Andrews RG, Bensinger WI, et al. Antigen CD34+ marrow cells engraft lethally irradiated baboons [J]. J Clin Invest 1988;81:951-955.
14. Danet GH, Luongo JL, Butler G, Lu MM, Tenner AJ, Simon MC, Bonnet DA. C1qRp defines a new human stem cell population with hematopoietic and hepatic potential [J]. Proc Natl Acad Sci U S A2002;99:10441-10445.
15. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science 1999;284:143-147.
16. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta [J]. Nat Med 1999;5:309-313.
17. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature 2002;418:41-49.
18. Holden C. STEM CELLS: Controversial marrow cells coming into their own? [J] Science 2007;315:760-761.
19. Miller RH. The promise of stem cells for neural repair [J]. Brain Research 2006;1091:258-264.
20. Levesque MF, T. N. Autologous transplantation of adult human neural stem cells and differentiated dopaminergic neurons for Parkinson's disease: one year post-operative clinical and functional metabolic results. In: American Association of Neurological Surgeons meeting. Chicago; 2002.
21. Martino G, Pluchino S. The therapeutic potential of neural stem cells [J]. Nat Rev Neurosci 2006;7:395-406.
22.Krupczak-Hollis K,Wang X,Dennewitz MB,et al.Growth hormone stimulates proliferation of old-aged regenerating liver through forkhead box mlb[J].Hepatology 2003;38:1552-1562.
23.Petersen BE,Bowen WC,Patrene KD,et al.Bone marrow as a potential source of hepatic oval cells[J].Science 1999;284:1168-1170.
24.Kallis YN,Alison MR,Forbes SJ.Bone marrow stem cells and liver disease[J].Gut 2007;56:716-724.
25.Thorgeirsson SS,Grisham JW.Hematopoietic cells as hepatocyte stem cells:a critical review of the evidence[J].Hepatology 2006;43:2-8.
26.Tateno C,Yoshizato K.Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells[J].Am J Pathol 1996;149:1593-1605.
27.Bonner-Weir S,Taneja M,Weir GC,et al.In vitro cultivation of human islets from expanded ductal tissue[J].Proc Natl Acad Sci U S A 2000;97:7999-8004.
28.Ramiya VK,Maraist M,Arfors KE,et al.Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells[J].Nat Med 2000;6:278-282.
29.Gu G,Dubauskaite J,Melton DA.Direct evidence for the pancreatic lineage:NGN3+ cells are islet progenitors and are distinct from duct progenitors[J].Development 2002;129:2447-2457.
30.Hunziker E,Stein M.Nestin-expressing cells in the pancreatic islets of langerhans[J].Biochemical and Biophysical Research Communications 2000;271:116-119.
31.宋陆军,秦新裕,牛伟新,et al.源于体外培养胰岛中表达Ngn3的细胞是胰腺内分泌前体细胞的新证据[J].中华外科杂志2005:42-45.
32.Dabeva MD,Hwang SG,Vasa SR,et al.Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver[J].Proc Natl Acad Sci U S A 1997;94:7356-7361.
33.Dor Y,Brown J,Martinez OI,et al.Adult pancreatic[beta]-cells are formed by self-duplication rather than stem-cell differentiation[J].Nature 2004;429:41-46.
34.Stanger BZ,Tanaka A J,Melton DA.Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver[J].Nature 2007;445:886-891.
35.Couzin J.Developmental biology:In embryos,pancreas and liver reach full size in different ways [J]. Science 2007;315:587.
36. Baeyens L, De Breuck S, Lardon J, et al. In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells [J]. Diabetologia 2005;48:49-57.
37. Minami K, Okuno M, Miyawaki K, et al. Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells [J]. Proc Natl Acad Sci U S A 2005;102:15116-15121.
38. Lardon J, Huyens N, Rooman I, et al. Exocrine cell transdifferentiation in dexamethasone-treated rat pancreas [J]. Virchows Arch 2004;444:61-65.
39. Desai BM, Oliver-Krasinski J, De Leon DD, et al. Preexisting pancreatic acinar cells contribute to acinar cell, but not islet beta cell, regeneration [J]. J Clin Invest 2007;117:971-977.
40. Leaf C. Why we're losing the war on cancer (and how to win it). Fortune 2004; 149:76-82, 84-76, 88 passim.
41. Karhadkar SS, Bova GS, Abdallah N, et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis [J]. Nature 2004;431:707-712.
42. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells [J]. Proc Natl Acad Sci U S A 2003;100:3983-3988.
1.Suzuki A,Nakauchi H,Taniguchi H.In vitro production of functionally mature hepatocytes from prospectively isolated hepatic stem cells[J].Cell Transplant 2003;12:469-473.
2.Parent R,Marion M J,Furio L,et al.Origin and characterization of a human bipotent liver progenitor cell line[J].Gastroenterology 2004;126:1147-1156.
3.Aurich I,Mueller LP,Aurich H,et al.Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers[J].Gut 2007;56:405-415.
4.Jang Y-Y,Collector MI,Baylin SB,et al.Hematopoietic stem cells convert into liver cells within days without fusion[J].Nat Cell Biol 2004;6:532-539.
5.Rountree CB,Barsky L,Ge S,et al.A CDl33 expressing murine liver oval cell population with bi-lineage potential[J].Stem Cells 2007.
6.Mitaka T,Mikami M,Gerald L.Sattler,Henry C.Pitot,Mochizuki.Y.Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor[J].Hepatology 1992;16:440-447.
7.NusslerA,Konig S,Ott M,et al.Present status and perspectives of cell-based therapies for liver diseases[J].J Hepatol 2006;45:144-159.
8.Strom SC,Bruzzone P,Cai H,et al.Hepatocyte transplantation:clinical experience and potential for future use[J].Cell Transplant 2006;15 Suppl 1:S105-110.
9. Habibullah CM, Ayesha Q, Khan AA, et al. Xenotransplantation of UV-B-irradiated hepatocytes. Survival and immune response [J]. Transplantation 1995;59:1495-1497.
10. Strom S, Fisher R. Hepatocyte transplantation: new possibilities for therapy [J]. Gastroenterology 2003;124:568-571.
11. Mustapha N, Francoise S, Etienne S. Hepatocyte transplantation: current and future developments [J]. Curr Opin Organ Transplant 2007; 12:503-508.
12. Machimoto T, Yasuchika K, Komori J, et al. Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model [J]. Transplantation 2007;84:1233-1239.
13. Sen S, Jalan R. Ideal hepatocyte: quest for the Holy Grail [J]. J Gastroenterol Hepatol 2005;20:1-4.
14. Wang X, Foster M, Al-Dhalimy M, et al. The origin and liver repopulating capacity of murine oval cells [J]. Proc Natl Acad Sci U S A 2003; 100 Suppl 1:11881-11888.
15. Wu XZ. Origin of cancer stem cells: the role of self-renewal and differentiation [J]. Ann Surg Oncol 2008; 15:407-414.
16. Kim H, Park C, Han KH, et al. Primary liver carcinoma of intermediate (hepatocyte-cholangiocyte) phenotype [J]. J Hepatol 2004;40:298-304.
17. Newsome PN, Hussain MA, Theise ND. Hepatic oval cells: helping redefine a paradigm in stem cell biology [J]. Curr Top Dev Biol 2004;61:l-28.
18. Wilson JW, Leduc EH. Role of cholangioles in restoration of the liver of the mouse after dietary injury [J]. J Pathol Bacteriol 1958;76:441-449.
19. Aleem Khan A, Parveen N, Habeeb MA, et al. Journey from hepatocyte transplantation to hepatic stem cells: a novel treatment strategy for liver diseases [J]. Indian J Med Res 2006;123:601-614.
20. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells [J]. Science 1999;284:1168-1170.
21. Lyra AC, Soares MBP, dos Santos RR, et al. Bone marrow stem cells and liver disease [J]. Gut 2007;56:1640-.
22. Thorgeirsson SS, Grisham JW. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence [J]. Hepatology 2006;43:2-8.
23. Tateno C, Yoshizato K. Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes [J]. Am J Pathol 1996; 148:383-392.
24. Mitaka T, Sattler GL, Pitot HC, et al. Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes [J]. Virchows Archiv. B, Cell Pathology Including Molecular Pathology 1992;62:329-335.
25. Mitaka T, Mizuguchi T, Sato F, et al. Growth and maturation of small hepatocytes [J]. J Gastroenterol Hepatol 1998; 13 Suppl:S70-77.
26. Mitaka T, Sato F, Mizuguchi T, et al. Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells [J]. Hepatology 1999;29:111-125.
27. Meier PJ, Stieger B. Bile salt transporters [J]. Annu Rev Physiol 2002;64:635-661.
28. Sidler Pfandler MA, Hochli M, Inderbitzin D, et al. Small hepatocytes in culture develop polarized transporter expression and differentiation [J]. J Cell Sci 2004; 117:4077-4087.
29. Shiojiri N, Lemire JM, Fausto N. Cell lineages and oval cell progenitors in rat liver development [J]. Cancer Res 1991;51:2611 -2620.
30. Mitaka T. The current status of primary hepatocyte culture [J]. International Journal of Experimental Pathology 1998;79:393-409.
31. Kon J, Ooe H, Oshima H, et al. Expression of CD44 in rat hepatic progenitor cells [J]. Journal of Hepatology 2006;45(1):90-8.
32. Paku S, Schnur J, Nagy P, et al. Origin and Structural Evolution of the Early Proliferating Oval Cells in Rat Liver [J]. Am J Pathol 2001;158:1313-1323.
33. Theise ND, Saxena R, Portmann BC, et al. The canals of Hering and hepatic stem cells in humans [J]. Hepatology 1999;30:1425-1433.
34. Grisham JW. Cell types in long-term propagable cultures of rat liver [J]. Ann N Y Acad Sci 1980;349:128-137.
35. Germain L, Blouin MJ, Marceau N. Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components [J]. Cancer Res 1988;48:4909-4918.
36. Allain JE, Dagher I, Mahieu-Caputo D, et al. Immortalization of a primate bipotent epithelial liver stem cell [J]. Proc Natl Acad Sci U S A 2002;99:3639-3644.
37. Duret C, Gerbal-Chaloin S, Ramos J, et al. Isolation, characterization, and differentiation to hepatocyte-like cells of nonparenchymal epithelial cells from adult human liver [J]. Stem Cells 2007;25:1779-1790.
38. Khuu DN, Najimi M, Sokal EM. Epithelial cells with hepatobiliary phenotype: is it another stem cell candidate for healthy adult human liver? [J] World J Gastroenterol 2007;13:1554-1560.
39. Braun KM, Sandgren EP. Cellular origin of regenerating parenchyma in a mouse model of severe hepatic injury [J]. Am J Pathol 2000;l 57:561-569.
40. Block GD, Locker J, Bowen WC, et al. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium [J]. J Cell Biol 1996;132:1133-1149.
41. Wang J, Clark JB, Rhee GS, et al. Proliferation and hepatic differentiation of adult-derived progenitor cells [J]. Cells Tissues Organs 2003;173:193-203.
42. Wright N, Samuelson L, Walkup MH, et al. Enrichment of a bipotent hepatic progenitor cell from naive adult liver tissue [J]. Biochem Biophys Res Commun 2008;366:367-372.
43. Azuma H, Hirose T, Fujii H, et al. Enrichment of hepatic progenitor cells from adult mouse liver [J]. Hepatology 2003;37:1385-1394.
44. Sahin MB, Schwartz RE, Buckley SM, et al. Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver [J]. Liver Transpl2008;14:333-345.