Evidence for the multipotential differentiation of adult liver stem cells in vivo

The ultimate proof that a rat liver epithelial cell line represents cultured stemlike cells requires the demonstration that these cells can give rise to hepatocytes or biliary epithelial cells following transplantation into appropriate sites in host animals. Such studies have been carried out by transplanting various cultured liver epithelial cell lines into livers or extrahepatic sites of syngeneic animals and nude mice.

5.1. Transplantation of Neoplastically Transformed Liver Epithelial Cells

Early studies of the differentiation of tumors produced following the subcutaneous or intraperitoneal transplantation of uncloned chemically transformed rat liver epithelial cells demonstrated that most tumors were poorly differentiated, although some tumors expressed morphologic features of hepatocellular carcinomas or biliary adenocarcinomas (200,201). Definitive evidence that rat liver epithelial cells possess a wide differentiation potential came from studies of the tumorigenicity of cloned lines of transformed cells derived from WB-F344 cells (202).

WB-F344 rat liver epithelial cells have been neoplastically transformed in vitro by infrequent passaging of cultures to induce their spontaneous transformation (203,204) and following exposure to N-methyl-N'-nitro-N-nitrosoguanidine to transform them chemically (205). The histological types of tumors that result from the subcutaneous or intraperitoneal transplantation of chemically transformed WB-F344 cells and subcloned cell lines include hepatocellular carcinomas, adenocarcinomas (biliary and intestinal types), adenocarcinomas with sarcomatous elements, epidermoid (squamous) carcinomas, hepatoblastomas (containing cartilage and osteoid elements), and poorly differentiated tumors of both epithelial and mesenchymal morphologies (202). Spontaneously transformed WB-F344 cell lines also produce wide variety of tumor cell types, including well-differentiated hepa-tocellular carcinomas, biliary adenocarcinomas, and hepatoblastomas, as well as mesenchymal tumors, including osteosarcomas (203,204). These results demonstrate that transformed WB-F344 cells are multipotent for the major differentiated epithelial cell types of the rat liver and suggest the possibility that their diploid counterpart may possess a broad differentiation potential.

5.2. Transplantation of Rat Liver Stem Cells Into Livers of Syngeneic Rats

To investigate the differentiation potential of cultured rat liver epithelial cells, WB-F344 cells have been transplanted into livers or extrahepatic sites of syngeneic animals to examine their fate in vivo. Several weeks following the transplantation of WB-F344 cells into the interscapular fat pads of syn-geneic Fischer 344 rats, small clusters of cells morphologically resembling hepatocytes were identified (178). However, whether these cells possessed functional attributes of hepatocytes was not determined. Nonetheless, this observation suggested that transplanted WB-F344 cells could acquire characteristics of hepatocytes in vivo.

To demonstrate a precursor-product relationship between transplanted cells and differentiated cell types in the liver, methods had to be established that would allow the definitive identification of the progeny of the transplanted cells among host cells in the adult liver. Three different strategies were utilized to examine the fate of WB-F344 rat liver epithelial cells fol lowing transplantation into adult rat livers: (1) introduction of a genetic tag/ marker enzyme into WB-F344 cells (178,206), (2) transplantation of normal WB-F344 cells into the livers of rats deficient for DPPIV enzyme activity (207), and (3) transplantation of normal WB-F344 cells into the livers of Nagase analbuminemic rats (208). In all of these model systems, transplanted WB-F344 cells (or BAG2-WB cells) integrated into hepatic plates and morphologically and functionally differentiated into hepatocytes. The results from these studies combined show that WB-F344 cells are multipotent and differentially responsive to the tissue microenvironment of the transplantation site.

5.2.1. Hepatocytic Differentiation by Transplanted WB-F344 Rat Liver Stem Cells

To facilitate transplantation studies, WB-F344 rat liver epithelial cells were genetically modified by infection with the CRE BAG2 retrovirus, which encodes the Escherichia coli P-galactosidase gene and the Tn5 neomycin resistance gene (209). The resulting cells, termed BAG2-WB, were transplanted into the livers of adult Fischer 344 rats; the livers of these rats were examined for the presence of P-galactosidase-positive cells at various times following transplantation (Fig. 3). In these studies, P-galactosidase-positive hepatocytelike cells were detected in the hepatic plates of recipient rats among the host hepatocytes (206). The size and morphologic appearance of these cells is indistinguishable from that of the host hepatocytes (178,206). The P-galactosidase-positive cells were observed at all times examined (Fig. 3), up to more than 1 yr following transplantation. Subsequent studies demonstrated that the P-galactosidase-positive hepatocytelike cells express functional differentiation typical of hepatocytes, including expression of albumin, transferrin, a1-antitrypsin, and tyrosine aminotransferase (178).

Several methods were utilized to demonstrate further that the P-galac-tosidase-positive cells observed in these livers were derived from the transplanted BAG2-WB cells. In some studies, cells were labeled with the lipophilic fluorescent membrane dye PKH26-GL (206). Examination of liver sections demonstrated the presence of fluorescent cells in the hepatic plates of the host livers, consistent with the observations made using the P-galac-tosidase marker enzyme (206). In addition, the neomycin resistance gene of the CRE BAG2 retroviral construct could be detected by polymerase chain reaction (PCR) in genomic DNA prepared from livers of rats that received BAG2-WB cell transplants (206).

Together, these studies demonstrate that transplanted WB-F344 rat liver epithelial cells incorporate into the hepatic plates of the host liver, morpho-

Fig. 3. Hepatocytic differentiation of WB-F344 cells following transplantation into the livers of adult rats. (A-C) Liver tissue obtained 128 d following transplantation of WB-F344 cells into syngeneic Fischer 344 rats. P-galactosidase-positive WB-F344-derived hepatocytes are identified by the presence of X-gal reaction product. (D-F) Representative liver cryosections from DPPIV-deficient rats demonstrating DPPIV-positive WB-F344-derived hepatocytes 30-60 d after transplantation. Liver sections were histochemically stained for both DPPIV and bile canalicular ATPase. (G-I) Albumin immunostaining of representative paraffin sections of liver tissue from Nagase analbuminemic rats at 28 d following transplantation of WB-F344 cells.

Fig. 3. Hepatocytic differentiation of WB-F344 cells following transplantation into the livers of adult rats. (A-C) Liver tissue obtained 128 d following transplantation of WB-F344 cells into syngeneic Fischer 344 rats. P-galactosidase-positive WB-F344-derived hepatocytes are identified by the presence of X-gal reaction product. (D-F) Representative liver cryosections from DPPIV-deficient rats demonstrating DPPIV-positive WB-F344-derived hepatocytes 30-60 d after transplantation. Liver sections were histochemically stained for both DPPIV and bile canalicular ATPase. (G-I) Albumin immunostaining of representative paraffin sections of liver tissue from Nagase analbuminemic rats at 28 d following transplantation of WB-F344 cells.

logically and functionally differentiate into hepatocytes, and remain a stable component of the hepatic parenchyma over long periods of time.

In other transplantation studies (207), normal WB-F344 cells were transplanted into the livers of German Fischer 344 rats, which are deficient for dipeptidylpeptidase IV enzyme activity (210,211), to examine the fate of these cells in a transplantation model that does not depend on the use of exogenous marker enzymes. Dipeptidylpeptidase IV is a bile canalicular enzyme expressed by mature hepatocytes in normal rats (212,213). The WB-F344 cell line was isolated from an American strain adult Fischer 344

rat (20) that expresses normal levels of dipeptidylpeptidase IV activity in hepatocytes. Following transplantation into the rats deficient in dipeptidylpeptidase IV, WB-F344 cells incorporated into hepatic plates and morphologically differentiated into hepatocytelike cells that expressed dipeptidylpeptidase IV enzyme activity (207).

These dipeptidylpeptidase IV-positive hepatocytes (Fig. 3) were easily distinguished from the host hepatocytes using a histochemical staining reaction for dipeptidylpeptidase IV activity (214). The dipeptidylpeptidase IV-positive hepatocytes in hepatic plates were comparable to adjacent host hepatocytes in size and morphology (Fig. 3). Close physical contact between the differentiated progeny of the transplanted cells and host hepatocytes was verified through colocalization of dipeptidylpeptidase IV staining and adenosine triphosphatase (ATPase) staining of hybrid bile canaliculi (Fig. 3). In addition, the localization of dipeptidylpeptidase IV staining to bile canaliculi showed that the surface membranes of differentiating WB-F344 cells acquired the polarization characteristic of fully differentiated hepatocytes (207). These results provide additional evidence that WB-F344 cells morphologically and functionally differentiate into hepatocytes following their transplantation into the liver microenvironment of adult rats.

In a third transplantation model, wild-type WB-F344 cells were transplanted into Nagase analbuminemic rats (215,216) to examine the efficacy of liver stemlike cell transplant for phenotypic correction of a genetic liver defect (208). In previous studies, transplanted WB-F344 cells (or BAG2-WB cells) gave rise to differentiated hepatocyte progeny that expressed albumin (178,207). Therefore, in this transplantation model, albumin served dual roles: (1) as a marker for detection of the progeny of transplanted WB-F344 cells and (2) as a metabolic marker for monitoring phenotypic correction of analbuminemia. Albumin-positive hepatocytes were detected in the hepatic plates among albumin-negative host hepatocytes in all rats receiving cell transplants (Fig. 3). In some cases, individual albuminpositive hepatocytes were observed, whereas in other instances, clusters of albumin-positive hepatocytes were detected (Fig. 3). The WB-F344 cells were transplanted into Nagase rats treated with cyclosporin to minimize rejection of the transplanted cells because of strain-specific differences between the Fischer 344 rat cells and the Sprague-Dawley rat hosts (208). However, once engrafted into the livers of these rats, albumin-positive WB-F344 hepatocyte progeny could be detected for up to 4 wk following the cessation of cyclosporin treatment (208).

5.2.2. Differentiation of WB-F344 Rat Liver Stem Cells Into Biliary Epithelial Cells

Until recently, studies aimed at determination of the potential for WB-F344 cells to serve as a progenitor for biliary epithelial cells and to participate in bile duct formation were lacking. However, Hixson and colleagues (217) developed techniques for introducing rat liver epithelial cells into the bile ducts. Using these methods, they showed that WB-F344 cells transplanted into the bile ducts differentiated into biliary epithelial cells and participated in bile duct formation (217).

5.3. Transplantation of Rat Liver Stem Cells Into Extrahepatic Sites

Several types of stem cells from adult tissues express a capacity for multipotential differentiation. The stem cells respond to inductive signals from the tissue microenvironment in which they engraft and differentiate into cells that express a phenotype characteristic of cells in that tissue microenvironment. To examine directly the possibility that rat liver stem cells possess the ability for multipotential differentiation in vivo, WB-F344 cells were transplanted into extrahepatic sites of nude mice. In response to signals in the various niches of the heart, transplanted WB-F344 cells differentiated into cells unique to each of these niches (218). Furthermore, early results indicated that transplanted WB-F344 cells can differentiate into cells of the hematopoietic lineage in vivo. Together, these results suggest that the WB-F344 cells have a broad differentiation potential that, depending on the tissue microenvironment at the transplantation site, can give rise to cells of several nonhepatic lineages. These studies are reviewed briefly.

5.3.1. WB-F344 Cells Differentiate Into Various Kinds of Heart Cells Following Transplantation into the Heart

To investigate the possibility that adult-derived WB-F344 rat liver stem cells can respond to signals in the microenvironment of the heart in vivo and give rise to cells of cardiac lineages, male WB-F344 cells that carry the E. coli P-galactosidase reporter gene were transplanted into the hearts of adult female nude mice (218). Six weeks following intracardiac injection, P-galactosidase-positive myocytes were identified, by light (218) and electron microscopy, in the myocardium of recipient mice among host myocytes (Fig. 4).

Engrafted cells ranged from small undifferentiated cells to long striated cells measuring up to 110 ^m in length (218). By electron microscopy, the WB-F344-derived myocytes were demonstrated to be at various stages of differentiation. The longer cells contained well-organized and differentiated

Fig. 4. Transmission electron microscopy of WB-F344-derived cardiac myocytes. P-Galactosidase-positive WB-F344-derived cardiomyocytes are identified by the presence of electron-dense crystalloid X-gal reaction product precipitate in the cytoplasm of well-differentiated (A, B, and E) and differentiating (D) cardiac myocytes. The well-differentiated myocytes contain striations (A and C) and are coupled to adjacent cells through intercalated disks and gap junctions (C).

Fig. 4. Transmission electron microscopy of WB-F344-derived cardiac myocytes. P-Galactosidase-positive WB-F344-derived cardiomyocytes are identified by the presence of electron-dense crystalloid X-gal reaction product precipitate in the cytoplasm of well-differentiated (A, B, and E) and differentiating (D) cardiac myocytes. The well-differentiated myocytes contain striations (A and C) and are coupled to adjacent cells through intercalated disks and gap junctions (C).

sarcomeres, with intercalated disks and apparent gap junctional connections to adjacent host myocytes (Fig. 4), consistent with a more differentiated (mature) cell phenotype. The presence of anatomical couplings between stem cell-derived myocytes and host myocytes suggests that the WB-F344-derived myocytes participate in the function of the cardiac syncytium (218). Smaller, less-differentiated cells, some as little as 20 ^m in length, demonstrated nascent sarcomeres, suggesting that they were immature cardiac lineage committed, WB-F344-derived myocytes (Fig. 4). These developing cells were isolated in the cardiac connective tissue and did not show any apparent contact with native well-differentiated myocytes.

Taking advantage of the male origin of the WB-F344 cells, P-galactosi-dase-positive myocytes, engrafted into the hearts of female host mice, were shown to contain a Y chromosome by in situ hybridization of tissue sections (218). Individual P-galactosidase-positive myocytes were microdissected from heart using laser capture microdissection and were used to prepare DNA. PCR analysis of these DNA samples demonstrated the presence of rat Y chromosome-specific repetitive DNA sequences (218). Furthermore, the P-galactosidase-positive myocytes expressed cardiac-specific troponin T using a monoclonal antibody that recognizes an epitope on this protein specific for the cardiac isoform and that is conserved across species (219).

It has been suggested that the capacity of many types of stem cells to differentiate into various lineages may be explained by nuclear fusion between these cells and native cells in the tissue microenvironment (220,221). To test this possibility, a mouse L1 repetitive DNA element was identified (222), and a fluorescence in situ hybridization (FISH) analysis was performed using this DNA sequence as a probe. Sections of the heart of a recipient mouse that contained a donor WB-F344-derived myocyte expressing P-galactosidase were analyzed. Unlike the nuclei of host cells, the nuclei from the donor WB-F344-derived myocytes did not demonstrate any fluorescence by confocal microscopy using this probe. Together with the presence of immature nascent myocytes isolated in the cardiac connective tissue, this observation indicates that WB-F344 cells may not have fused with adult well-differentiated recipient myocytes, but rather differentiated directly into cardiomyocytes in response to signals from the cardiac microenvironment. Further studies will be needed to exclude the possibility that some transplanted cells fuse with host cells.

In addition to the observation of P-galactosidase-positive cardiomyocytes in recipient mice, transplanted WB-F344 cells gave rise to cells of other cell lineages that participate in the cardiac structure. In one mouse, a cartilaginous mass was found in the left ventricle. This mass was lined with WB-F344-derived endocardial cells that expressed von Willebrand factor and P-galactosidase and contained a rat Y chromosome (Fig. 5). The cells in the cartilaginous mass also contained a Y chromosome (Fig. 5), indicating that they also originated from donor WB-F344 cells. We suspect that, at the time of transplantation, the WB-F344 cells had aggregated into a suspended bolus too large to pass through the aortic valve. Extrapolating from the morphologic gradient concept in development, it is tempting to speculate that local diffusible signals activated different genes of WB-F344 cells located at different points in a concentration gradient (223,224), with cells on the inside and on the outside of the aggregate expressing different phenotypes. Alternatively, it is possible that suspended WB-F344 cells behaved like ES cells, forming an "embryoid body" that differentiated along a default mesenchymal cell lineage. On the other hand, the needle tip used to inject the WB-F344 cells into the left ventricle through the rib cage might have picked up some cartilage cells from a costochondral junction, which formed the nidus of a cartilage microenvironment that dominated the phenotypic differentiation of adjacent and surrounding WB-F344 cells, driving them to differentiate into cartilage cells.

In addition to the cell fates described above, when WB-F344 cells were injected in the pericardial sac, P-galactosidase-positive progeny cells displaying a flat mesothelial-like phenotype lined the epicardium (data not shown). This result suggests that, when these cells are introduced into the tissue niche represented by the pericardial sac, they are directed to engraft into the mesothelial cell lining and acquire a cell phenotype characteristic for cells of that niche of the heart.

Together, the results of intracardiac transplantation of clonal WB-F344 liver stem cells show that they engraft and differentiate in the heart in a niche-specific manner (218). WB-F344 cells acquire a myocytic phenotype in the myocardium, an endocardial phenotype in the endocardium, and a visceral pericardial phenotype in the pericardial space.

5.3.2. WB-F344 Cells Differentiate Into Hematopoietic Cell Lineages Following Transplantation Into Bone Marrow

To determine the capacity for WB-F344 cells to engraft in the bone marrow and differentiate into hematopoietic cells, WB-F344 cells (carrying genes for E. coli P-galactosidase and green fluorescent protein) were transplanted via tail vein injection into sublethally irradiated female nude mice. Bone marrow and spleen were harvested from recipient mice 7 to 9 wk later for analysis by clonogenic assays in the presence of the antibiotic G418. These assays demonstrated that WB-F344-derived hematopoietic cells collected from the bone marrow and spleen of irradiated mice receiving cell transplants produced colonies that contained cells with typical characteristics of neutrophils, monocytes, megakaryocytes, macrophages, erythroid, and pre-B cells (225). Verification that these hematopoietic cells were derived from transplanted WB-F344 cells was accomplished by PCR amplification of the rat Y chromosome repetitive sequences. These results suggest that transplanted WB-F344 cells can home into the bone marrow of host animals and differentiate into hematopoietic progenitor cells in response to instructive signals in the microenvironment of the bone marrow.

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