Bone Marrow Progenitors Are Not the Source of Expanding Oval Cells in Injured Liver
http://www.100md.com
《干细胞学杂志》
a Marion Bessin Liver Research Center and Department of Medicine,
b Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York, USA
Key Words. Liver progenitor cells ? Oval cells ? Bone marrow progenitors ? Transdifferentiation
Correspondence: Mariana D. Dabeva, M.D., Ph.D., Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. Telephone: 718-430-3171; Fax: 718-430-8975; e-mail: dabeva@aecom.yu.edu
ABSTRACT
Over the years, substantial evidence has accumulated suggesting the existence of potential liver stem cells (LSCs) in the adult liver. In all cases, the putative LSCs were activated to proliferate and differentiate when the regenerative capacity of terminally differentiated hepatocytes was compromised. The progeny of potential LSCs, referred to as oval cells, behave like bipotential progenitors capable of differentiation into mature hepatocytes and biliary epithelial cells, thus recapitulating hepatoblast differentiation during fetal development . Oval cells also reveal some phenotypic characteristics of hematopoietic progenitor cells; they express c-kit and its ligand stem cell factor and the related flt-3 and flt-3 ligand , CD34 , Thy-1 (CD90) , and Sca1 . In addition, oval cells are phenotypically heterogeneous , and some investigators identified them in the periductular/intraportal zone, consistent with the interpretation that some may originate from an extrahepatic (bone marrow ) source .
Extensive research during the past few years has changed our view concerning stem cells. It has become evident that stem cells are very pliable and that they can change their phenotype when taken from the stem cell niche and transplanted into a new residence . These studies raised the possibility that BM stem cells may be useful for liver cell transplantation and prompted several investigators to study whether BM progenitors brought into the liver can transdifferentiate into hepatocytes. In general, the protocols for these studies used female animals, lethally irradiated and rescued with male genetically labeled male BM cells or purified hematopoietic stem cells (HSCs). In most cases, the animals were subjected to liver injury, and after different periods of time (ranging from several days to months), the appearance of Y chromsosome–positive hepatocytes in their livers was observed . Similar results were obtained with humans: appearance of Y chromosome–positive hepatocytes in livers of female patients that had received male BM transplants and also female livers grafted into male patients .
However, recent studies carefully analyzing the fate of transplanted BM cells or purified hematopoietic cell fractions showed that a single green fluorescent protein–positive HSC can reconstitute the BM of lethally irradiated nontransgenic recipients but did not contribute appreciably to the non-hematopoietic tissues, including the liver . Convincing evidence that cell fusion between donor HSC and endogenous hepatocytes accounts for the high liver repopulation by HSC in the fumaryl acetoacetate hydrolase deficient (FAH–/–) model of massive liver injury has been reported from the laboratories of Russell and colleagues and Grompe and colleagues .
If BM stem cells can engraft and differentiate into oval cells in the liver, they would be a valuable source for gene therapy. Petersen et al. transplanted syngeneic BM cells into lethally irradiated female animals treated with 2-acetyl-aminofluorene (2-AAF) and CCl4, which causes hepatic necrosis and impairment of hepatocyte proliferation, and reported the appearance of BM-derived oval cells in the liver of these animals.
To determine unambiguously whether BM cells can differentiate into oval cells and repopulate the injured liver, we have substituted the BM of lethally irradiated female DPP4-deficient F344 rats with BM cells from syngeneic normal male F344 rats and then subjected the recipients to three models of activation and expansion of oval cells. We found that endogenous liver progenitors, and not BM progenitors, are the overwhelming source of expanding oval cells in the injured liver.
MATERIALS AND METHODS
Experimental Design
To be able to determine whether BM cells differentiate into liver progenitor/oval cells, we ablated the BM of female DPP4-deficient F344 rats and reconstituted it by transplanting 5 x 107 normal male syngeneic F344 rat BM cells 24 hours after TBI (Fig. 1). After BM reconstitution was complete (4–6 weeks), recipient animals were treated with chemical agents that cause liver injury and impaired hepatocyte proliferation, resulting in activation, proliferation, and accumulation of oval cells. The recipient animals were divided into three experimental groups, and each group was subjected to one of the following liver injury protocols: D-gal injection, which causes hepatocyte necrosis and proliferation of oval cells with a peak on days 4 to 5 ; Rs/PH, which causes maximum expansion of oval cells between days 7 and 14 after PH ; and 2-AAF/PH, which results in strong proliferation of oval cells with maximum accumulation in the liver on days 10 through 12 .
In the liver of wild-type F344 rats, all epithelial cells express the enzyme DPP4, including oval cells. As shown in Figure 2, in the livers of wild-type, DPP4+ animals subjected to 2-AAF/PH (Figs. 2A–2C), Rs/PH (Figs. 2D–2F), and D-gal (Figs. 2G–2H) treatment, both oval cells (Figs. 2B, 2E, 2H) and small basophilic hepatocytes (SBHs), considered by some investigators to represent a unique liver progenitor cell population , reveal a positive histochemical reaction for DPP4 (Figs. 2C, 2F). If BM progenitors were the source of oval cells, we would expect to see numerous DPP4+ oval cells in all three models of liver injury in which hepatocyte proliferation is compromised.
Figure 2. Expression of DPP4 by epithelial cells of the liver of wild-type F 344 rats. DPP4 is expressed at the canalicular surface of hepatocytes. It is also expressed by SBHs (canalicular surface) and oval cells (more diffuse distribution). (A–C): 2-acetylaminofluorene/PH model, day 10 after PH. (A):Arrows point to oval cells, and the dotted arrows to a cluster of SBHs. (B): Higher magnification of the area with oval cells in (A). (C): Higher magnification of the cluster of SBHs presented in (A). (D–F): Retrorsine/PH model, 10 days after PH. (D):Arrows point to the oval cells and the dotted arrows to a cluster of SBHs. (E): Higher magnification of the area with oval cells in (D). (F): Higher magnification of the cluster of SBHs presented in (D). (G–H): D-galactosamine model 4 days after treatment; arrows point to oval cells. Original magnification: (A, D, and G) x100; (B, C, E, F, and H) x200. Abbreviations: PH, partial hepa-tectomy; SBH, small basophilic hepatocytes.
BM Reconstitution
To be able to determine accurately whether BM progenitor cells transdifferentiate into oval cells, we used male BM cells of normal F344 rats and transplanted them into female recipients deficient in the exopeptidase DPP4. Because we did not know in advance which fraction of BM cells might possess the capability to transdifferentiate into oval cells, we transplanted 50 x 106 unfractionated BM cells into lethally irradiated female rats. Reconstitution of the female BM with male hematopoietic cells was determined using the Y chromosome–specific sry gene. BM substitution in all experimental groups was at least 80%, determined 30 days after transplantation, at the time of PH or at the time of animal euthanasia. These results ensured that if BM cells can transdifferentiate into oval cells, we should be able to detect them as DPP4+ oval cells in the liver of BM transplant recipients.
DPP4+ Cells in the Liver of Recipient Animals
The serine exopeptidase DDP4 (CD26) was described as a cell-surface molecule expressed on the surface of resting and activated T cells, activated B cells, and activated NK cells . Upon reconstitution of the BM of DPP4-deficient animals with normal BM cells, we expected to find DPP4+ cells in the liver of the recipient rats. Indeed, 4–6 weeks after BMT, a substantial number of such cells were observed, especially as clusters in the portal region of the liver lobules of untreated rats (Fig. 3A) and at the time of partial hepatectomy (Fig. 3B). It has to be noted that after the various injury protocols for activation and proliferation of oval cells, the number of DPP4+ cells did not increase. In contrast, most cells from the DPP4+ periportal clusters dispersed throughout the liver lobule and appeared as single cells, including 2-AAF/PH (Fig. 3C), D-gal (Fig. 3D), and Rs/PH (Fig. 3E).
Figure 3. DPP4+ cells in the liver of recipient animals. The appearance of DPP4+ cells was analyzed before and after liver injury. (A): Thirty days after bone marrow transplantation. Clusters of DPP4+ cells are observed in the periportal region. (B): Liver section from a 2-AAF–treated animal at the time of PH showing numerous clusters of DPP4+ cells. (C): 2-AAF/PH model 10 days after PH. (D): D-galactosamine model 5 days after liver injury. (E): Rs/PH model 10 days after PH. Note the spreading of the DPP4+ cells throughout the lobule while the areas containing oval cells (arrows) are mostly devoid of DPP4+ cells. (F): Rs/PH model 30 days after PH. Occasional small clusters of DPP4+ cells are observed. SBHs do not express DPP4+. (G): 2-AAF/PH day 8 after PH, with dotted arrow showing a cluster of SBH. (H): Rs/PH day 20 after PH, with dotted arrow showing a cluster of SBHs. Original magnification x200. Abbreviations: 2-AAF, 2-acetylaminofluorene; PH, partial hepatectomy; Rs, retrorsine; SBH, small basophilic hepatocyte.
The 2-AAF/PH and Rs/PH models are also characterized by the appearance and expansion of foci of SBH (Figs. 3G, 3H). As could be clearly seen, the clusters of small hepatocytes that could be considered as hepatocytic progenitor or transitional cells do not contain DPP4+ small hepatocytes (compare with Figs. 2C, 2F), although scattered DPP4+ cells could be seen at the border of these clusters. Consequently, SBHs in these models have an endogenous origin and do not originate from the transplanted BM cells.
Because the DPP4+ cells by their location and appearance could be either liver progenitor/oval cells or blood cells, we compared the distribution of the oval cells and the DPP4+ cells in the liver of the three experimental models.
Expansion of Oval Cells in the Liver Injury Models
One of the phenotypic landmarks of oval cells is the expression of the intermediate filament CK-19. As shown in normal liver (Fig. 4A) or in the 2-AAF/PH liver at the time of PH (Fig. 4B), only biliary epithelial cells are positive for CK-19. Oval cells that appear in all three models are small with a large nucleus-to-cytoplasmic ratio. They are CK-19+ and form duct-like/arborizing structures that are readily distinguishable from the other small cells. In our experiments, the number of oval cells peaked in the 2-AAF model between days 9 and 13 after PH (Fig. 4C), in the D-gal model on day 5 (Fig. 4D), and in the Rs/PH model between days 7 and 14 (Fig. 4E), although some oval cells were still present 30 days after PH (Fig. 4F). In both 2-AAF/PH and Rs/PH livers, the clusters of small hepatocytes were CK-19– (Figs. 4G, 4H) as previously reported . The same results were obtained with another antibody specific for oval cells, OV-6 (data not shown). Notably, the high number of oval cells in these tissues did not coincide with the low number of scattered DPP4+ cells. These results, combined with those presented in Figure 4, show that (a) in all three models, the oval cells were activated and proliferated and accumulated, whereas the number of DPP4+ cells did not increase after the injury, and (b) oval cells divided rapidly on activation and formed groups and branching structures of CK-19+ cells, which were not evident with the DPP4+ cells, most of which appeared as single cells. Taking these data into account, we concluded that it is not likely that BM progenitors transdifferentiate into oval cells in the Rs/PH, D-gal, or 2-AAF/PH injured liver.
Figure 4. Expansion of oval cells in the liver injury models. In normal liver, only bile ducts are stained for CK-19. (A): Liver of an animal 30 days after bone marrow transplantation. (B): Liver of an animal treated with 2-AAF at the time of PH. In all three liver injury models, substantial expansion of CK-19+ oval cells, forming duct-like structures, is observed. (C): 2-AAF/PH model 10 days after PH. (D): D-galactosamine model 5 days after liver injury. (E, F): Rs/PH model 10 and 30 days after PH. The clusters of SBHs are CK-19–. (G): 2-AAF/PH day 8 after PH, with dotted arrow showing a cluster of SBHs. (H): Rs/PH day 20 after PH, with dotted arrow showing a cluster of SBHs. Original magnification x200. Abbreviations: 2-AAF, 2-acetylaminofluorene; PH, partial hepatectomy; Rs, retrorsine; SBH, small basophilic hepatocyte.
Nature of the DPP4+ Cells in the Liver
The DPP4+ Cells in the Liver Are Blood Cells ? If the DPP4+ cells in the liver were not oval cells, then the question arises as to whether they are blood cells (mainly T cells) expressing this enzyme. All leukocytes express the surface molecule CD45. We analyzed serial liver sections from animals euthanized 6 weeks after BM cell transplantation before the liver injury, when the DPP4+ cells appear in clusters in the periportal region, and found that the DPP4+ cell in these clusters coexpressed CD45, showing that these cells were in the hematopoietic lineage (Figs. 5A, 5B). However, most of these cells did not express CD90 (Fig. 5C), which is considered a marker for oval cells . The same result was obtained using double-fluorescent immunolabeling for simultaneous detection of DPP4 and CD45 on liver sections from animals subjected to Rs/PH (Fig. 6A) or D-gal (Fig. 6B) treatment and from animals subjected to the 2-AAF/PH protocol (Fig. 6C, liver tissue taken at the time of PH; and Fig. 6D, 16 days after PH). These data provide substantial evidence that the DPP4+ cells in the liver are hematopoietic cells.
Figure 5. DPP4+ cells in the liver express leukocyte common antigen CD45. Serial sections from the liver taken 6 weeks after bone marrow transplantation. (A): First section, showing immunohistochemical staining for CD45 (leukocyte common antigen). (B): Second section, showing histochemical staining for DPP4. (C): Third section, showing immunohistochemical staining for CD90 (Thy1.1). The DPP4+ cells in the cluster are also CD45+, but they are negative for CD90. Original magnification x400.
Figure 6. Double-IF labeling of CD45/DPP4 and CD45/CK-19. Double-IF labeling was performed as described in Materials and Methods. CD45 is visualized with green color; DPP4 and CK-19 are visualized with red color. (A–D): Double-IF labeling for DPP4 and CD45. (A): Rs/PH model 30 days after PH. (B): D-gal model 4 days after liver injury. (C): 2-AAF model of the time of PH. (D): 2-AAF model 16 days after PH. Upon merging the two images, all DPP4+ cells (initially red) are also CD45+ (initially green), producing a yellow color. (E–H): Double-IF labeling for CD45 and CK-19. (E): Rs/PH model at the time of PH. (F): Rs/PH model 30 days after PH. (G): D-gal model 4 days after liver injury. (H): 2-AAF/PH model 10 days after PH. In all four panels, the clusters of CD45+ cells are distinct from those of CK-19+ cells, because there are no cells coexpressing these markers (yellow color). Abbreviations: D-gal, D-galactosamine; IF, immunofluorescent; PH, partial hepatectomy; Rs, retrorsine; 2-AAF, 2-acetylaminofluorene.
Oval Cells Are CD45– ? In consecutive analyses conducted with all three models of liver injury, we found that the accumulating CK-19+ oval cells (red color) do not coexpress the leukocyte-specific marker CD45 (green color). Shown in Figure 6 is the Rs model at the time of PH (Fig. 6E), the Rs model 30 days after PH (Fig. 6F), the D-gal model 4 days after injection of the agent (Fig. 6G), and the 2-AAF model 10 days after PH (Fig. 6H). It should be noted, however, that in all three cases, substantial accumulation of CD45+ blood cells was observed. These data showed that oval cells expanding in the above models are not CD45+ and that they are distinct from the donor (DPP4+) leucocytes accumulating in the livers of injured animals.
DPP4+ Cells Are Almost Exclusively CK-19– ? To obtain final proof that the DPP4+ cells in the liver of the transplanted animals are not oval cells, we performed double labeling to detect coexpression of CK-19 and DPP4. CK-19+ cells were labeled with FITS (green color), whereas the DPP4+ cells were labeled with Cy3 (red color). The results, presented in Figures 7A and 7B, show that the label for the DPP4+ cells found in the liver 6 weeks after transplantation or at the time of PH (Rs/PH model) did not overlap with that of CK-19+ cells. Very rarely we were able to detect coexpression of CK-19 and DPP4 in single cells, as shown in Figure 7C (2-AAF model 10 days after PH) and Figure 7F (Rs model 30 days after PH). In most sections examined, such coexpression of CK-19 and DPP4 was not observed, as shown in Figure 7D (2-AAF model 16 days after PH) or Figure 7E (D-gal model 4 days after injection of the agent).
Figure 7. Double-IF labeling of DPP4 and CK-19. Double-IF labeling was performed as described in Materials and Methods. CK-19 is visualized with green color, DPP4 with red color. (A): Control animal 30 days after bone marrow transplantation. (B): Liver of Rs-treated animal at the time of PH. (C, D): 2-acetylaminofluorene/PH model 10 and 16 days after PH. (E): D-galactosamine model 4 days after liver injury. (F): Rs/PH model 30 days after PH. Very rarely (at frequency less than 1%), double-labeled DPP4+ and CK-19+ oval cells were detected (white arrows in C and F). Original magnification x400. Abbreviations: IF, immunofluorescent; PH, partial hepatectomy; Rs, retrorsine.
To determine more precisely the frequency of transdifferentiation of BM progenitors into oval cells of the liver, we counted the number of oval cells that were positive for both CK-19 and DPP4 relative to the total number of DPP4+ cells. As shown in Table 1, the number of double-labeled cells (DPP4+ and CK-19+) was less than 1% of all DPP4+ cells in the livers of the control animals before the liver injury and in the livers of rats subjected to the three models of expansion of oval cells, D-gal, Rs/PH, and 2-AAF-PH. These results showed again that very few BM cells appeared as oval cells in the liver and that transdifferentiation of BM progenitors into oval cells is a very rare event, if it occurs at all.
Table 1. Quantitation of DPP4+ cells and of double-labeled DPP4+ and CK-19+ cells in the liver of recipient animals after bone marrow transplantation in different experimental models
DISCUSSION
This research was supported by the National Institutes of Health Grants R21 DK61145 (to M.D.D.). The authors thank Ethel Hurston for technical assistance.
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b Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York, USA
Key Words. Liver progenitor cells ? Oval cells ? Bone marrow progenitors ? Transdifferentiation
Correspondence: Mariana D. Dabeva, M.D., Ph.D., Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. Telephone: 718-430-3171; Fax: 718-430-8975; e-mail: dabeva@aecom.yu.edu
ABSTRACT
Over the years, substantial evidence has accumulated suggesting the existence of potential liver stem cells (LSCs) in the adult liver. In all cases, the putative LSCs were activated to proliferate and differentiate when the regenerative capacity of terminally differentiated hepatocytes was compromised. The progeny of potential LSCs, referred to as oval cells, behave like bipotential progenitors capable of differentiation into mature hepatocytes and biliary epithelial cells, thus recapitulating hepatoblast differentiation during fetal development . Oval cells also reveal some phenotypic characteristics of hematopoietic progenitor cells; they express c-kit and its ligand stem cell factor and the related flt-3 and flt-3 ligand , CD34 , Thy-1 (CD90) , and Sca1 . In addition, oval cells are phenotypically heterogeneous , and some investigators identified them in the periductular/intraportal zone, consistent with the interpretation that some may originate from an extrahepatic (bone marrow ) source .
Extensive research during the past few years has changed our view concerning stem cells. It has become evident that stem cells are very pliable and that they can change their phenotype when taken from the stem cell niche and transplanted into a new residence . These studies raised the possibility that BM stem cells may be useful for liver cell transplantation and prompted several investigators to study whether BM progenitors brought into the liver can transdifferentiate into hepatocytes. In general, the protocols for these studies used female animals, lethally irradiated and rescued with male genetically labeled male BM cells or purified hematopoietic stem cells (HSCs). In most cases, the animals were subjected to liver injury, and after different periods of time (ranging from several days to months), the appearance of Y chromsosome–positive hepatocytes in their livers was observed . Similar results were obtained with humans: appearance of Y chromosome–positive hepatocytes in livers of female patients that had received male BM transplants and also female livers grafted into male patients .
However, recent studies carefully analyzing the fate of transplanted BM cells or purified hematopoietic cell fractions showed that a single green fluorescent protein–positive HSC can reconstitute the BM of lethally irradiated nontransgenic recipients but did not contribute appreciably to the non-hematopoietic tissues, including the liver . Convincing evidence that cell fusion between donor HSC and endogenous hepatocytes accounts for the high liver repopulation by HSC in the fumaryl acetoacetate hydrolase deficient (FAH–/–) model of massive liver injury has been reported from the laboratories of Russell and colleagues and Grompe and colleagues .
If BM stem cells can engraft and differentiate into oval cells in the liver, they would be a valuable source for gene therapy. Petersen et al. transplanted syngeneic BM cells into lethally irradiated female animals treated with 2-acetyl-aminofluorene (2-AAF) and CCl4, which causes hepatic necrosis and impairment of hepatocyte proliferation, and reported the appearance of BM-derived oval cells in the liver of these animals.
To determine unambiguously whether BM cells can differentiate into oval cells and repopulate the injured liver, we have substituted the BM of lethally irradiated female DPP4-deficient F344 rats with BM cells from syngeneic normal male F344 rats and then subjected the recipients to three models of activation and expansion of oval cells. We found that endogenous liver progenitors, and not BM progenitors, are the overwhelming source of expanding oval cells in the injured liver.
MATERIALS AND METHODS
Experimental Design
To be able to determine whether BM cells differentiate into liver progenitor/oval cells, we ablated the BM of female DPP4-deficient F344 rats and reconstituted it by transplanting 5 x 107 normal male syngeneic F344 rat BM cells 24 hours after TBI (Fig. 1). After BM reconstitution was complete (4–6 weeks), recipient animals were treated with chemical agents that cause liver injury and impaired hepatocyte proliferation, resulting in activation, proliferation, and accumulation of oval cells. The recipient animals were divided into three experimental groups, and each group was subjected to one of the following liver injury protocols: D-gal injection, which causes hepatocyte necrosis and proliferation of oval cells with a peak on days 4 to 5 ; Rs/PH, which causes maximum expansion of oval cells between days 7 and 14 after PH ; and 2-AAF/PH, which results in strong proliferation of oval cells with maximum accumulation in the liver on days 10 through 12 .
In the liver of wild-type F344 rats, all epithelial cells express the enzyme DPP4, including oval cells. As shown in Figure 2, in the livers of wild-type, DPP4+ animals subjected to 2-AAF/PH (Figs. 2A–2C), Rs/PH (Figs. 2D–2F), and D-gal (Figs. 2G–2H) treatment, both oval cells (Figs. 2B, 2E, 2H) and small basophilic hepatocytes (SBHs), considered by some investigators to represent a unique liver progenitor cell population , reveal a positive histochemical reaction for DPP4 (Figs. 2C, 2F). If BM progenitors were the source of oval cells, we would expect to see numerous DPP4+ oval cells in all three models of liver injury in which hepatocyte proliferation is compromised.
Figure 2. Expression of DPP4 by epithelial cells of the liver of wild-type F 344 rats. DPP4 is expressed at the canalicular surface of hepatocytes. It is also expressed by SBHs (canalicular surface) and oval cells (more diffuse distribution). (A–C): 2-acetylaminofluorene/PH model, day 10 after PH. (A):Arrows point to oval cells, and the dotted arrows to a cluster of SBHs. (B): Higher magnification of the area with oval cells in (A). (C): Higher magnification of the cluster of SBHs presented in (A). (D–F): Retrorsine/PH model, 10 days after PH. (D):Arrows point to the oval cells and the dotted arrows to a cluster of SBHs. (E): Higher magnification of the area with oval cells in (D). (F): Higher magnification of the cluster of SBHs presented in (D). (G–H): D-galactosamine model 4 days after treatment; arrows point to oval cells. Original magnification: (A, D, and G) x100; (B, C, E, F, and H) x200. Abbreviations: PH, partial hepa-tectomy; SBH, small basophilic hepatocytes.
BM Reconstitution
To be able to determine accurately whether BM progenitor cells transdifferentiate into oval cells, we used male BM cells of normal F344 rats and transplanted them into female recipients deficient in the exopeptidase DPP4. Because we did not know in advance which fraction of BM cells might possess the capability to transdifferentiate into oval cells, we transplanted 50 x 106 unfractionated BM cells into lethally irradiated female rats. Reconstitution of the female BM with male hematopoietic cells was determined using the Y chromosome–specific sry gene. BM substitution in all experimental groups was at least 80%, determined 30 days after transplantation, at the time of PH or at the time of animal euthanasia. These results ensured that if BM cells can transdifferentiate into oval cells, we should be able to detect them as DPP4+ oval cells in the liver of BM transplant recipients.
DPP4+ Cells in the Liver of Recipient Animals
The serine exopeptidase DDP4 (CD26) was described as a cell-surface molecule expressed on the surface of resting and activated T cells, activated B cells, and activated NK cells . Upon reconstitution of the BM of DPP4-deficient animals with normal BM cells, we expected to find DPP4+ cells in the liver of the recipient rats. Indeed, 4–6 weeks after BMT, a substantial number of such cells were observed, especially as clusters in the portal region of the liver lobules of untreated rats (Fig. 3A) and at the time of partial hepatectomy (Fig. 3B). It has to be noted that after the various injury protocols for activation and proliferation of oval cells, the number of DPP4+ cells did not increase. In contrast, most cells from the DPP4+ periportal clusters dispersed throughout the liver lobule and appeared as single cells, including 2-AAF/PH (Fig. 3C), D-gal (Fig. 3D), and Rs/PH (Fig. 3E).
Figure 3. DPP4+ cells in the liver of recipient animals. The appearance of DPP4+ cells was analyzed before and after liver injury. (A): Thirty days after bone marrow transplantation. Clusters of DPP4+ cells are observed in the periportal region. (B): Liver section from a 2-AAF–treated animal at the time of PH showing numerous clusters of DPP4+ cells. (C): 2-AAF/PH model 10 days after PH. (D): D-galactosamine model 5 days after liver injury. (E): Rs/PH model 10 days after PH. Note the spreading of the DPP4+ cells throughout the lobule while the areas containing oval cells (arrows) are mostly devoid of DPP4+ cells. (F): Rs/PH model 30 days after PH. Occasional small clusters of DPP4+ cells are observed. SBHs do not express DPP4+. (G): 2-AAF/PH day 8 after PH, with dotted arrow showing a cluster of SBH. (H): Rs/PH day 20 after PH, with dotted arrow showing a cluster of SBHs. Original magnification x200. Abbreviations: 2-AAF, 2-acetylaminofluorene; PH, partial hepatectomy; Rs, retrorsine; SBH, small basophilic hepatocyte.
The 2-AAF/PH and Rs/PH models are also characterized by the appearance and expansion of foci of SBH (Figs. 3G, 3H). As could be clearly seen, the clusters of small hepatocytes that could be considered as hepatocytic progenitor or transitional cells do not contain DPP4+ small hepatocytes (compare with Figs. 2C, 2F), although scattered DPP4+ cells could be seen at the border of these clusters. Consequently, SBHs in these models have an endogenous origin and do not originate from the transplanted BM cells.
Because the DPP4+ cells by their location and appearance could be either liver progenitor/oval cells or blood cells, we compared the distribution of the oval cells and the DPP4+ cells in the liver of the three experimental models.
Expansion of Oval Cells in the Liver Injury Models
One of the phenotypic landmarks of oval cells is the expression of the intermediate filament CK-19. As shown in normal liver (Fig. 4A) or in the 2-AAF/PH liver at the time of PH (Fig. 4B), only biliary epithelial cells are positive for CK-19. Oval cells that appear in all three models are small with a large nucleus-to-cytoplasmic ratio. They are CK-19+ and form duct-like/arborizing structures that are readily distinguishable from the other small cells. In our experiments, the number of oval cells peaked in the 2-AAF model between days 9 and 13 after PH (Fig. 4C), in the D-gal model on day 5 (Fig. 4D), and in the Rs/PH model between days 7 and 14 (Fig. 4E), although some oval cells were still present 30 days after PH (Fig. 4F). In both 2-AAF/PH and Rs/PH livers, the clusters of small hepatocytes were CK-19– (Figs. 4G, 4H) as previously reported . The same results were obtained with another antibody specific for oval cells, OV-6 (data not shown). Notably, the high number of oval cells in these tissues did not coincide with the low number of scattered DPP4+ cells. These results, combined with those presented in Figure 4, show that (a) in all three models, the oval cells were activated and proliferated and accumulated, whereas the number of DPP4+ cells did not increase after the injury, and (b) oval cells divided rapidly on activation and formed groups and branching structures of CK-19+ cells, which were not evident with the DPP4+ cells, most of which appeared as single cells. Taking these data into account, we concluded that it is not likely that BM progenitors transdifferentiate into oval cells in the Rs/PH, D-gal, or 2-AAF/PH injured liver.
Figure 4. Expansion of oval cells in the liver injury models. In normal liver, only bile ducts are stained for CK-19. (A): Liver of an animal 30 days after bone marrow transplantation. (B): Liver of an animal treated with 2-AAF at the time of PH. In all three liver injury models, substantial expansion of CK-19+ oval cells, forming duct-like structures, is observed. (C): 2-AAF/PH model 10 days after PH. (D): D-galactosamine model 5 days after liver injury. (E, F): Rs/PH model 10 and 30 days after PH. The clusters of SBHs are CK-19–. (G): 2-AAF/PH day 8 after PH, with dotted arrow showing a cluster of SBHs. (H): Rs/PH day 20 after PH, with dotted arrow showing a cluster of SBHs. Original magnification x200. Abbreviations: 2-AAF, 2-acetylaminofluorene; PH, partial hepatectomy; Rs, retrorsine; SBH, small basophilic hepatocyte.
Nature of the DPP4+ Cells in the Liver
The DPP4+ Cells in the Liver Are Blood Cells ? If the DPP4+ cells in the liver were not oval cells, then the question arises as to whether they are blood cells (mainly T cells) expressing this enzyme. All leukocytes express the surface molecule CD45. We analyzed serial liver sections from animals euthanized 6 weeks after BM cell transplantation before the liver injury, when the DPP4+ cells appear in clusters in the periportal region, and found that the DPP4+ cell in these clusters coexpressed CD45, showing that these cells were in the hematopoietic lineage (Figs. 5A, 5B). However, most of these cells did not express CD90 (Fig. 5C), which is considered a marker for oval cells . The same result was obtained using double-fluorescent immunolabeling for simultaneous detection of DPP4 and CD45 on liver sections from animals subjected to Rs/PH (Fig. 6A) or D-gal (Fig. 6B) treatment and from animals subjected to the 2-AAF/PH protocol (Fig. 6C, liver tissue taken at the time of PH; and Fig. 6D, 16 days after PH). These data provide substantial evidence that the DPP4+ cells in the liver are hematopoietic cells.
Figure 5. DPP4+ cells in the liver express leukocyte common antigen CD45. Serial sections from the liver taken 6 weeks after bone marrow transplantation. (A): First section, showing immunohistochemical staining for CD45 (leukocyte common antigen). (B): Second section, showing histochemical staining for DPP4. (C): Third section, showing immunohistochemical staining for CD90 (Thy1.1). The DPP4+ cells in the cluster are also CD45+, but they are negative for CD90. Original magnification x400.
Figure 6. Double-IF labeling of CD45/DPP4 and CD45/CK-19. Double-IF labeling was performed as described in Materials and Methods. CD45 is visualized with green color; DPP4 and CK-19 are visualized with red color. (A–D): Double-IF labeling for DPP4 and CD45. (A): Rs/PH model 30 days after PH. (B): D-gal model 4 days after liver injury. (C): 2-AAF model of the time of PH. (D): 2-AAF model 16 days after PH. Upon merging the two images, all DPP4+ cells (initially red) are also CD45+ (initially green), producing a yellow color. (E–H): Double-IF labeling for CD45 and CK-19. (E): Rs/PH model at the time of PH. (F): Rs/PH model 30 days after PH. (G): D-gal model 4 days after liver injury. (H): 2-AAF/PH model 10 days after PH. In all four panels, the clusters of CD45+ cells are distinct from those of CK-19+ cells, because there are no cells coexpressing these markers (yellow color). Abbreviations: D-gal, D-galactosamine; IF, immunofluorescent; PH, partial hepatectomy; Rs, retrorsine; 2-AAF, 2-acetylaminofluorene.
Oval Cells Are CD45– ? In consecutive analyses conducted with all three models of liver injury, we found that the accumulating CK-19+ oval cells (red color) do not coexpress the leukocyte-specific marker CD45 (green color). Shown in Figure 6 is the Rs model at the time of PH (Fig. 6E), the Rs model 30 days after PH (Fig. 6F), the D-gal model 4 days after injection of the agent (Fig. 6G), and the 2-AAF model 10 days after PH (Fig. 6H). It should be noted, however, that in all three cases, substantial accumulation of CD45+ blood cells was observed. These data showed that oval cells expanding in the above models are not CD45+ and that they are distinct from the donor (DPP4+) leucocytes accumulating in the livers of injured animals.
DPP4+ Cells Are Almost Exclusively CK-19– ? To obtain final proof that the DPP4+ cells in the liver of the transplanted animals are not oval cells, we performed double labeling to detect coexpression of CK-19 and DPP4. CK-19+ cells were labeled with FITS (green color), whereas the DPP4+ cells were labeled with Cy3 (red color). The results, presented in Figures 7A and 7B, show that the label for the DPP4+ cells found in the liver 6 weeks after transplantation or at the time of PH (Rs/PH model) did not overlap with that of CK-19+ cells. Very rarely we were able to detect coexpression of CK-19 and DPP4 in single cells, as shown in Figure 7C (2-AAF model 10 days after PH) and Figure 7F (Rs model 30 days after PH). In most sections examined, such coexpression of CK-19 and DPP4 was not observed, as shown in Figure 7D (2-AAF model 16 days after PH) or Figure 7E (D-gal model 4 days after injection of the agent).
Figure 7. Double-IF labeling of DPP4 and CK-19. Double-IF labeling was performed as described in Materials and Methods. CK-19 is visualized with green color, DPP4 with red color. (A): Control animal 30 days after bone marrow transplantation. (B): Liver of Rs-treated animal at the time of PH. (C, D): 2-acetylaminofluorene/PH model 10 and 16 days after PH. (E): D-galactosamine model 4 days after liver injury. (F): Rs/PH model 30 days after PH. Very rarely (at frequency less than 1%), double-labeled DPP4+ and CK-19+ oval cells were detected (white arrows in C and F). Original magnification x400. Abbreviations: IF, immunofluorescent; PH, partial hepatectomy; Rs, retrorsine.
To determine more precisely the frequency of transdifferentiation of BM progenitors into oval cells of the liver, we counted the number of oval cells that were positive for both CK-19 and DPP4 relative to the total number of DPP4+ cells. As shown in Table 1, the number of double-labeled cells (DPP4+ and CK-19+) was less than 1% of all DPP4+ cells in the livers of the control animals before the liver injury and in the livers of rats subjected to the three models of expansion of oval cells, D-gal, Rs/PH, and 2-AAF-PH. These results showed again that very few BM cells appeared as oval cells in the liver and that transdifferentiation of BM progenitors into oval cells is a very rare event, if it occurs at all.
Table 1. Quantitation of DPP4+ cells and of double-labeled DPP4+ and CK-19+ cells in the liver of recipient animals after bone marrow transplantation in different experimental models
DISCUSSION
This research was supported by the National Institutes of Health Grants R21 DK61145 (to M.D.D.). The authors thank Ethel Hurston for technical assistance.
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