- Email: [email protected]
Bipotential Properties and Proliferation of Fetal Liver Epithelial Progenitor Cells in Mice
Bipotential Properties and Proliferation of Fetal Liver Epithelial Progenitor Cells in Mice J.F. Zheng, L.J. Liang, C.X. Wu, J.S. Chen, and Z.S. Zhang ABSTRACT The potential of embryonal day 14 (ED) fetal liver epithelial progenitor (FLEP) cells to repopulate the normal and damaged liver was studied throughout a 3-month period in syngeneic mice. In normal liver, FLEP cells proliferated and differentiated into hepatocytes after transplantation, and liver repopulation was moderate (5%–10%) after 3 months. In diethylnitrosamine-treated livers FLEP cells continued to proliferate at 3 months after transplantation; both the number and size of clusters derived from FLEP cells gradually increased throughout time. Transplanted cells proliferated and differentiated into both hepatocytes and bile ducts to produce extensive liver repopulation (30%–50%). This report showed that isolated fetal liver epithelial cells exhibit bipotential properties of stem cells, which can engraft, proliferate, and differentiate into hepatocytes and bile duct epithelial cells with high repopulation capacity in the injured liver.
L
IVER REPOPULATION by cell transplantation has elicited considerable interest for clinical applications, as well as investigation of mechanisms concerning hepatic biology. However, hepatocyte transplantation has rarely produced therapeutic effects in human clinical trials, mainly because the numbers of transplanted hepatocytes are insufficient to achieve biologic effects.1 Moreover, hepatocytes are quiescent cells that are difficult to maintain in culture and to cryopreserve. Progenitor stem cells reside within or adjacent to the canals of Hering, comprising a quiescent compartment of dormant cells in the adult liver. They can be activated to proliferate and differentiate into hepatocytes or bile duct epithelial cells when hepatocytes are impaired persistently.2 Attempts to identify their counterpart in fetal liver have lead to the suggestion that the dormant stem-like cells originate most probably from bipotential fetal liver epithelial progenitor (FLEP) cells.3 These cells have characteristics of hepatic stem cells with multiple differentiation capabilities to repopulate the liver. This study was designed to isolate and transplant FLEP cells into the liver of syngenic mice to explore the ability of FLEP cells to proliferate and differentiate into hepatocytes and bile duct epithelial cells in an injured liver. MATERIALS AND METHODS Animals Six-to-eight week old BALB/c mice were purchased from the Animal Breeding Center of Sun Yat-Sen University (Guangzhou, China). All animal handling and experimental procedures were in
accordance with our Guidelines of the Animal Care and Use Committee.
Transplantation of FLEP Cells and DiethylnitrosamineInduced Liver Injury FLEP cells were isolated on embryonal day (ED) 14 from normal pregnant mice as described previously.3 The isolated FLEP cells were considered to be composed of 50% male cells and 50% female cells. Two-thirds hepatectomy was performed after anesthesia. After hepatectomy freshly isolated ED 14 FLEP cells (1 ⫻ 106 cells) were injected into the female liver via the superior mesenteric vein using insulin syringes.4 After FLEP cell injection, mice were allowed to recover for 1 week. Thereafter, DEN (Sigma-Aldrich, St. Louis, MO) was continuously administered in the drinking water at a final concentration of 100 g/L for 12 weeks. Female BALB/c mice were randomly divided into three groups. Mice in the normal control group were given FLEP cells only. The mice in the experimental group were From the Department of Hepatobiliary Surgery, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (J.F.Z., L.J.L.) and Department of Hepatobiliary Surgery, the People’s Hospital of Hainan Province, Haikou (C.X.W., J.S.C., Z.S.Z.), P.R. China. Supported by the Natural Science Foundation of Hainan Province, P.R. China (805107). Address reprint requests to Li-Jian Liang, MD, Professor, Department of Hepatobiliary Surgery, the First Affiliated Hospital, Sun Yat-Sen University, No. 74 Zhongshan Road II, Guangzhou, 510080, P.R. China. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.154
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1710
Transplantation Proceedings, 40, 1710 –1713 (2008)
FETAL LIVER EPITHELIAL PROGENITOR CELLS
1711
Fig 1. Characteristics of ED 14 FLEP cells in mice. A: Fetal liver tissue, HE staining; B: AFP, immunohistochemical staining for FLEP cells; C: Albumin, immunohistochemical staining; D: CK-19, immunohistochemical staining. Original magnification, ⫻200.
continuously administered DEN in their drinking water for liver injury induction. Mice were killed under anesthesia and 5 livers removed for histologic evaluation at 1, 2, and 3 months after cell transplantation.
Liver Histopathology To identify characteristics of hepatocytes in fetal mouse liver sections, immunohistochemistry was performed for alpha-fetoprotein (AFP), albumin, and CK-19 with diaminobenzidine using standard techniques. To identify the origin of cells in the liver, immunohistochemistry was performed for sex-determining region for Y chromosome (sry) protein as described previously.5 Fluorescent in situ hybridization (FISH) was also performed for Y chromosome to identify the origin of cells in the liver using the Cambio protocol.6 It was expected that transplanted cells (originating from fetal liver) would be approximately 50% male and 50% female. Several fields in each slide were randomly selected.
RESULTS Characteristics of FLEP Cells
At ED 14 mice livers contained immature epithelial cells and hematopoietic cells at various stages of differentiation (Fig 1A). Cells from ED 14 fetal liver were analyzed for expression of AFP, albumin, and CK-19. The percentage of FLEP cells was approximately 20% in the ED 14 fetal liver, determined as the number of cells expressing AFP (Fig 1B), albumin (Fig 1C), or CK-19 (Fig 1D).
Repopulation of the Liver by FLEP Cells in DEN-Treated Mice and in Normal Mice
In DEN-treated mice, sry⫹ cells were distributed in periportal regions of the liver after cell transplantation. One month after FLEP cell transplantation, we observed some sry⫹ cells (Fig 2B). Two months after transplantation, sry⫹ hepatocytes increased and were present in the liver parenchyma (Fig 2C). Three months after transplantation, numerous sry⫹ mature hepatocytes were observed, representing a substantial portion of the liver parenchyma (Fig 2D). We also observed sry⫹ cholangiocytes that formed mature sry positive bile ducts; sry⫹ bile duct structures were evident as early as 1 month after FLEP cell transplantation, but became more numerous and fully developed in portal regions at 3 months (Fig 2E). The numbers of transplanted cells increased over time: repopulation ranged from 5% to 10% at 1 month, 15% to 20% at 2 months, and 30% to 50% at 3 months. In the absence of DEN, sry⫹ cells were still observed 3 months after FLEP cell transplantation but were less numerous (5%–10% of the total number of cells) than observed in DEN-treated animals (Fig 2F). The results were consistent with those found using FISH for the Y chromosome (Fig 3). DISCUSSION
To determine whether ED 14 FLEP cells represent hepatic precursor cells, we explored the characteristic of markers for various liver cell types. Our results confirmed the
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ZHENG, LIANG, WU ET AL
existence of three subpopulations of epithelial cells expressed AFP, albumin, or CK-19 in ED 14 fetal liver. Hepatic stem cells include oval cells in canals of Hering of the adult liver and fetal liver hepatoblasts.7 Our results showed that early FLEP cells selectively proliferate in the normal liver in response to a regenerative stimulus, such as partial hepatectomy. They differentiate into mature hepatocytes being incorporated into the host liver lobule as part of normal hepatocytic cords. Transplanted FLEP cells repopulate to comprise 5% to 10% of the liver cells after 3 months. Stimulation of the regeneration is amplified by two-thirds hepatectomy in addition to DEN administration. At 1 month after administration of DEN, sry⫹ cells were observed around the portal areas. At 2 months, positive cells were detected in the liver lobes, indicative of FLEP cell migration into the hepatic parenchyma. At 3 months, there was extensive proliferation and liver repopulation with FLEP cells in DEN-treated mice. The persistent liver
Fig 3. Repopulation of FLEP cells in female recipient liver tissues by FISH for Y chromosome. A: Normal male liver, positive Y-chromosome signals appeared as green dots in the nuclei stained with DAPI, a chromosomal marker that shows up as blue fluorescence. B: One month after FLEP cells transplantation and DEN treatment, some cells showed green signals in the nuclei stained with DAPI. C: Three months after FLEP cells transplantation and DEN treatment, the number of cells showed green signals increased. D: Three months after FLEP cells transplantation without DEN treatment, scattered positive cells were observed. Original magnifications, ⫻400.
Fig 2. Proliferation and differentiation of FLEP cells in DENtreated mice and in normal mice. A: immunohistochemical positive control staining for sry protein, normal male mouse liver with nuclear positivity in the majority of hepatocytes (brown nuclei). B: One month after FLEP cells transplantation and DEN treatment; some cells stained for sry. C: Two months after FLEP cells transplantation and DEN treatment; sry⫹ cells were present in the liver parenchyma. D and E: Three months after FLEP cells transplantation and DEN treatment, sry⫹ cells formed numerous mature hepatocytes showed canalicular structures (D), or bile duct epithelial cells in the bile duct region, as evidenced by diffusely stained sry⫹ small epithelial cells in bile duct-like structures (E). F: Three months after FLEP cells transplantation without DEN treatment; scattered sry⫹ cells were observed. Original magnifications, ⫻200 (A–E) and ⫻100 (D).
damage by DEN administration is important for proliferation and differentiation of FLEP cells. More interestingly, we demonstrated that transplanted FLEP cells differentiate into mature bile duct epithelial cells. Our studies provide direct evidence for differentiation of FLEP cells with dual markers into hepatocytes and bile duct epithelial cells after transplantation into the regenerating liver of DEN-treated mice. This is consistent with other reports that bipotential FLEP cells can proliferate and differentiate into hepatocytes and bile ducts.8 In conclusion, we have reported that isolated fetal liver epithelial cells exhibit bipotential properties of stem cells, which can engraft, proliferate, and differentiate into hepatocytes and bile duct epithelial cells with high repopulation capacity in the injured liver. REFERENCES 1. Muraca M, Gerunda G, Neri D, et al: Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet 359:317, 2002 2. Yin L, Sun M, Ilic Z, et al: Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats. Hepatology 35:315, 2002 3. Dabeva MD, Petkov PM, Sandhu J, et al: Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver. Am J Pathol 156:2017, 2000
FETAL LIVER EPITHELIAL PROGENITOR CELLS 4. Kushida T, Inaba M, Hisha H, et al: Crucial role of donorderived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein. Stem Cells 19:226, 2001 5. Yannaki E, Athanasiou E, Xagorari A, et al: G-CSF-primed hematopoietic stem cells or G-CSF per se accelerate recovery and improve survival after liver injury, predominantly by promoting endogenous repair programs. Exp Hematol 33:108, 2005
1713 6. Available at www.cambio.co.uk 7. Dan YY, Riehle KJ, Lazaro C, et al: Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci USA 103:9912, 2006 8. Notenboom RG, van den Bergh Weerman MA, Dingemans KP, et al: Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage. Hepatology 38:683, 2003
L
IVER REPOPULATION by cell transplantation has elicited considerable interest for clinical applications, as well as investigation of mechanisms concerning hepatic biology. However, hepatocyte transplantation has rarely produced therapeutic effects in human clinical trials, mainly because the numbers of transplanted hepatocytes are insufficient to achieve biologic effects.1 Moreover, hepatocytes are quiescent cells that are difficult to maintain in culture and to cryopreserve. Progenitor stem cells reside within or adjacent to the canals of Hering, comprising a quiescent compartment of dormant cells in the adult liver. They can be activated to proliferate and differentiate into hepatocytes or bile duct epithelial cells when hepatocytes are impaired persistently.2 Attempts to identify their counterpart in fetal liver have lead to the suggestion that the dormant stem-like cells originate most probably from bipotential fetal liver epithelial progenitor (FLEP) cells.3 These cells have characteristics of hepatic stem cells with multiple differentiation capabilities to repopulate the liver. This study was designed to isolate and transplant FLEP cells into the liver of syngenic mice to explore the ability of FLEP cells to proliferate and differentiate into hepatocytes and bile duct epithelial cells in an injured liver. MATERIALS AND METHODS Animals Six-to-eight week old BALB/c mice were purchased from the Animal Breeding Center of Sun Yat-Sen University (Guangzhou, China). All animal handling and experimental procedures were in
accordance with our Guidelines of the Animal Care and Use Committee.
Transplantation of FLEP Cells and DiethylnitrosamineInduced Liver Injury FLEP cells were isolated on embryonal day (ED) 14 from normal pregnant mice as described previously.3 The isolated FLEP cells were considered to be composed of 50% male cells and 50% female cells. Two-thirds hepatectomy was performed after anesthesia. After hepatectomy freshly isolated ED 14 FLEP cells (1 ⫻ 106 cells) were injected into the female liver via the superior mesenteric vein using insulin syringes.4 After FLEP cell injection, mice were allowed to recover for 1 week. Thereafter, DEN (Sigma-Aldrich, St. Louis, MO) was continuously administered in the drinking water at a final concentration of 100 g/L for 12 weeks. Female BALB/c mice were randomly divided into three groups. Mice in the normal control group were given FLEP cells only. The mice in the experimental group were From the Department of Hepatobiliary Surgery, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (J.F.Z., L.J.L.) and Department of Hepatobiliary Surgery, the People’s Hospital of Hainan Province, Haikou (C.X.W., J.S.C., Z.S.Z.), P.R. China. Supported by the Natural Science Foundation of Hainan Province, P.R. China (805107). Address reprint requests to Li-Jian Liang, MD, Professor, Department of Hepatobiliary Surgery, the First Affiliated Hospital, Sun Yat-Sen University, No. 74 Zhongshan Road II, Guangzhou, 510080, P.R. China. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.154
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1710
Transplantation Proceedings, 40, 1710 –1713 (2008)
FETAL LIVER EPITHELIAL PROGENITOR CELLS
1711
Fig 1. Characteristics of ED 14 FLEP cells in mice. A: Fetal liver tissue, HE staining; B: AFP, immunohistochemical staining for FLEP cells; C: Albumin, immunohistochemical staining; D: CK-19, immunohistochemical staining. Original magnification, ⫻200.
continuously administered DEN in their drinking water for liver injury induction. Mice were killed under anesthesia and 5 livers removed for histologic evaluation at 1, 2, and 3 months after cell transplantation.
Liver Histopathology To identify characteristics of hepatocytes in fetal mouse liver sections, immunohistochemistry was performed for alpha-fetoprotein (AFP), albumin, and CK-19 with diaminobenzidine using standard techniques. To identify the origin of cells in the liver, immunohistochemistry was performed for sex-determining region for Y chromosome (sry) protein as described previously.5 Fluorescent in situ hybridization (FISH) was also performed for Y chromosome to identify the origin of cells in the liver using the Cambio protocol.6 It was expected that transplanted cells (originating from fetal liver) would be approximately 50% male and 50% female. Several fields in each slide were randomly selected.
RESULTS Characteristics of FLEP Cells
At ED 14 mice livers contained immature epithelial cells and hematopoietic cells at various stages of differentiation (Fig 1A). Cells from ED 14 fetal liver were analyzed for expression of AFP, albumin, and CK-19. The percentage of FLEP cells was approximately 20% in the ED 14 fetal liver, determined as the number of cells expressing AFP (Fig 1B), albumin (Fig 1C), or CK-19 (Fig 1D).
Repopulation of the Liver by FLEP Cells in DEN-Treated Mice and in Normal Mice
In DEN-treated mice, sry⫹ cells were distributed in periportal regions of the liver after cell transplantation. One month after FLEP cell transplantation, we observed some sry⫹ cells (Fig 2B). Two months after transplantation, sry⫹ hepatocytes increased and were present in the liver parenchyma (Fig 2C). Three months after transplantation, numerous sry⫹ mature hepatocytes were observed, representing a substantial portion of the liver parenchyma (Fig 2D). We also observed sry⫹ cholangiocytes that formed mature sry positive bile ducts; sry⫹ bile duct structures were evident as early as 1 month after FLEP cell transplantation, but became more numerous and fully developed in portal regions at 3 months (Fig 2E). The numbers of transplanted cells increased over time: repopulation ranged from 5% to 10% at 1 month, 15% to 20% at 2 months, and 30% to 50% at 3 months. In the absence of DEN, sry⫹ cells were still observed 3 months after FLEP cell transplantation but were less numerous (5%–10% of the total number of cells) than observed in DEN-treated animals (Fig 2F). The results were consistent with those found using FISH for the Y chromosome (Fig 3). DISCUSSION
To determine whether ED 14 FLEP cells represent hepatic precursor cells, we explored the characteristic of markers for various liver cell types. Our results confirmed the
1712
ZHENG, LIANG, WU ET AL
existence of three subpopulations of epithelial cells expressed AFP, albumin, or CK-19 in ED 14 fetal liver. Hepatic stem cells include oval cells in canals of Hering of the adult liver and fetal liver hepatoblasts.7 Our results showed that early FLEP cells selectively proliferate in the normal liver in response to a regenerative stimulus, such as partial hepatectomy. They differentiate into mature hepatocytes being incorporated into the host liver lobule as part of normal hepatocytic cords. Transplanted FLEP cells repopulate to comprise 5% to 10% of the liver cells after 3 months. Stimulation of the regeneration is amplified by two-thirds hepatectomy in addition to DEN administration. At 1 month after administration of DEN, sry⫹ cells were observed around the portal areas. At 2 months, positive cells were detected in the liver lobes, indicative of FLEP cell migration into the hepatic parenchyma. At 3 months, there was extensive proliferation and liver repopulation with FLEP cells in DEN-treated mice. The persistent liver
Fig 3. Repopulation of FLEP cells in female recipient liver tissues by FISH for Y chromosome. A: Normal male liver, positive Y-chromosome signals appeared as green dots in the nuclei stained with DAPI, a chromosomal marker that shows up as blue fluorescence. B: One month after FLEP cells transplantation and DEN treatment, some cells showed green signals in the nuclei stained with DAPI. C: Three months after FLEP cells transplantation and DEN treatment, the number of cells showed green signals increased. D: Three months after FLEP cells transplantation without DEN treatment, scattered positive cells were observed. Original magnifications, ⫻400.
Fig 2. Proliferation and differentiation of FLEP cells in DENtreated mice and in normal mice. A: immunohistochemical positive control staining for sry protein, normal male mouse liver with nuclear positivity in the majority of hepatocytes (brown nuclei). B: One month after FLEP cells transplantation and DEN treatment; some cells stained for sry. C: Two months after FLEP cells transplantation and DEN treatment; sry⫹ cells were present in the liver parenchyma. D and E: Three months after FLEP cells transplantation and DEN treatment, sry⫹ cells formed numerous mature hepatocytes showed canalicular structures (D), or bile duct epithelial cells in the bile duct region, as evidenced by diffusely stained sry⫹ small epithelial cells in bile duct-like structures (E). F: Three months after FLEP cells transplantation without DEN treatment; scattered sry⫹ cells were observed. Original magnifications, ⫻200 (A–E) and ⫻100 (D).
damage by DEN administration is important for proliferation and differentiation of FLEP cells. More interestingly, we demonstrated that transplanted FLEP cells differentiate into mature bile duct epithelial cells. Our studies provide direct evidence for differentiation of FLEP cells with dual markers into hepatocytes and bile duct epithelial cells after transplantation into the regenerating liver of DEN-treated mice. This is consistent with other reports that bipotential FLEP cells can proliferate and differentiate into hepatocytes and bile ducts.8 In conclusion, we have reported that isolated fetal liver epithelial cells exhibit bipotential properties of stem cells, which can engraft, proliferate, and differentiate into hepatocytes and bile duct epithelial cells with high repopulation capacity in the injured liver. REFERENCES 1. Muraca M, Gerunda G, Neri D, et al: Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet 359:317, 2002 2. Yin L, Sun M, Ilic Z, et al: Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats. Hepatology 35:315, 2002 3. Dabeva MD, Petkov PM, Sandhu J, et al: Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver. Am J Pathol 156:2017, 2000
FETAL LIVER EPITHELIAL PROGENITOR CELLS 4. Kushida T, Inaba M, Hisha H, et al: Crucial role of donorderived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein. Stem Cells 19:226, 2001 5. Yannaki E, Athanasiou E, Xagorari A, et al: G-CSF-primed hematopoietic stem cells or G-CSF per se accelerate recovery and improve survival after liver injury, predominantly by promoting endogenous repair programs. Exp Hematol 33:108, 2005
1713 6. Available at www.cambio.co.uk 7. Dan YY, Riehle KJ, Lazaro C, et al: Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci USA 103:9912, 2006 8. Notenboom RG, van den Bergh Weerman MA, Dingemans KP, et al: Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage. Hepatology 38:683, 2003