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Direct in vivo cell lineage analysis in the retrorsine and 2AAF models of liver injury after genetic labeling in adult and newborn rats.

Pichard V, Aubert D, Ferry N - PLoS ONE (2009)

Bottom Line: Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly.In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes.Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin.

View Article: PubMed Central - PubMed

Affiliation: INSERM U948, Biothérapies Hépatiques, CHU Hotel Dieu, Nantes, France.

ABSTRACT

Backgrounds and aims: When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.

Methodology/principal findings: We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.

Conclusions: Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.

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Histological analysis of the retrorsin model after genetic labeling of liver cells in newborn rats.Retrorsin model. (A) Cluster of SHPCs positive for β-galactosidase. (B) Hematoxylin and eosin staining showing large number of merging clusters 30 days post hepatectomy. (C) Immunohistochemical detection of CKs day 30 post hepatectomy. Oval cells are not detected. Hematoxylin counterstain. Magnification: ×200 A–B: ×400 C.
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pone-0007267-g004: Histological analysis of the retrorsin model after genetic labeling of liver cells in newborn rats.Retrorsin model. (A) Cluster of SHPCs positive for β-galactosidase. (B) Hematoxylin and eosin staining showing large number of merging clusters 30 days post hepatectomy. (C) Immunohistochemical detection of CKs day 30 post hepatectomy. Oval cells are not detected. Hematoxylin counterstain. Magnification: ×200 A–B: ×400 C.

Mentions: In the retrorsine group (n = 7) the proportion of β-galactosidase positive hepatocytes was evaluated in the liver lobes removed at partial hepatectomy. The mean proportion of β-galactosidase hepatocytes was 1.57±1% (Table 2). In the labeled cells population, 1.83% of the cells were neither hepatocytes nor oval cellsThe presence of β-galactosidase positive cells and the enzyme activity were analysed at sacrifice in the same animals. We observed a robust so-called “SHPC response” with the presence of numerous clusters of proliferating hepatocytes, either isolated or merging in large patches, some of which were also positive for β-galactosidase (Figure 4A–B). As shown in Table 2 there were slightly more β-galactosidase positive hepatocytes at sacrifice (2.07±1.4%) than at the time of hepatectomy. However this difference was not statistically significant (p = 0.79 by Student's t test) At the time of sacrifice it was not possible to quantify the proportion of non-hepatocyte labeled cells. It is noteworthy that we never detected oval cells in any section either by morphological examination or by immunohistological staining (Figure 4C). This confirm that SHPCs do not arise from oval cells. Although we never detected a clear proliferation of other non parenchymal cells, we cannot exclude in this setting that some labeled hepatocytes derived from labeled progenitors.


Direct in vivo cell lineage analysis in the retrorsine and 2AAF models of liver injury after genetic labeling in adult and newborn rats.

Pichard V, Aubert D, Ferry N - PLoS ONE (2009)

Histological analysis of the retrorsin model after genetic labeling of liver cells in newborn rats.Retrorsin model. (A) Cluster of SHPCs positive for β-galactosidase. (B) Hematoxylin and eosin staining showing large number of merging clusters 30 days post hepatectomy. (C) Immunohistochemical detection of CKs day 30 post hepatectomy. Oval cells are not detected. Hematoxylin counterstain. Magnification: ×200 A–B: ×400 C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747281&req=5

pone-0007267-g004: Histological analysis of the retrorsin model after genetic labeling of liver cells in newborn rats.Retrorsin model. (A) Cluster of SHPCs positive for β-galactosidase. (B) Hematoxylin and eosin staining showing large number of merging clusters 30 days post hepatectomy. (C) Immunohistochemical detection of CKs day 30 post hepatectomy. Oval cells are not detected. Hematoxylin counterstain. Magnification: ×200 A–B: ×400 C.
Mentions: In the retrorsine group (n = 7) the proportion of β-galactosidase positive hepatocytes was evaluated in the liver lobes removed at partial hepatectomy. The mean proportion of β-galactosidase hepatocytes was 1.57±1% (Table 2). In the labeled cells population, 1.83% of the cells were neither hepatocytes nor oval cellsThe presence of β-galactosidase positive cells and the enzyme activity were analysed at sacrifice in the same animals. We observed a robust so-called “SHPC response” with the presence of numerous clusters of proliferating hepatocytes, either isolated or merging in large patches, some of which were also positive for β-galactosidase (Figure 4A–B). As shown in Table 2 there were slightly more β-galactosidase positive hepatocytes at sacrifice (2.07±1.4%) than at the time of hepatectomy. However this difference was not statistically significant (p = 0.79 by Student's t test) At the time of sacrifice it was not possible to quantify the proportion of non-hepatocyte labeled cells. It is noteworthy that we never detected oval cells in any section either by morphological examination or by immunohistological staining (Figure 4C). This confirm that SHPCs do not arise from oval cells. Although we never detected a clear proliferation of other non parenchymal cells, we cannot exclude in this setting that some labeled hepatocytes derived from labeled progenitors.

Bottom Line: Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly.In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes.Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin.

View Article: PubMed Central - PubMed

Affiliation: INSERM U948, Biothérapies Hépatiques, CHU Hotel Dieu, Nantes, France.

ABSTRACT

Backgrounds and aims: When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.

Methodology/principal findings: We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.

Conclusions: Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.

Show MeSH
Related in: MedlinePlus