Or Exogenous Reporter Genes

Transplantation of donor cells that can be distinguished from host liver cells by endogenous markers or exogeneous reporter genes is another widely used method for generating chimeric livers suitable for lineage analysis and cell fate determinations. However, interpretation of events in chimeric livers generated by transplantation can be difficult as there are several confounding variables that can greatly influence the results. One issue is the stability of marker gene expression. If the marker is an endogenous gene unique to the donor cells, expression will be regulated in a normal manner. This is usually advantageous but can become a problem if expression of the marker gene is developmentally regulated, a characteristic that may preclude the analysis of early developmental stages. This is exemplified by dipeptidyl peptidase IV (DPPIV), a surface protein that appears late in fetal development (Petell et al., 1990), and is usually not expressed by progenitor cells in vitro. In vivo, expression levels can vary significantly depending on the origin of the promoter driving the marker gene, with tissue-specific promoters providing more stable expression than those of viral origin (CMV, retrovirus LTR).

Assessment of differentiation potential by transplantation also requires the development of protocols for isolating subpopulations of putative progenitors. Purity thus becomes an important consideration because the presence of contaminating cells in significant concentrations complicates interpretation by raising questions about the origin of the engrafted donor cells. In a number of studies, this issue has been resolved by showing that at low doses, the number of contaminating cells cannot account for all of the donor-derived colonies. Purified donor cells must also be able to stably integrate into hepatic cords or ducts, an ability that seems to be inherent in most liver cell types, both normal and malignant. The efficiency of engraftment, however, can vary significantly depending on the origin of the donor cells, the status of the host liver, and the mode of transplantation. Transplantation of donor cells into the liver via the spleen, e.g., increases overall viability relative to portal vein infusion but delivers only 50% of the injected cells to the liver (Rajvanshi et al., 1996). Analysis in the liver can be further complicated by lobular differences in the distribution of donor cells and variation in density related to the distance from the liver hilum.

In spite of these caveats, transplantation continues to be the method of choice for analyzing the differentiation capacity of hepatic progenitor cells. Details of a number of current transplantation models are reviewed in Chapter 36. All of these models, by necessity, share common features. In most models, the donor cells express a gene product that distinguishes them from host cells. Markers used successfully include MHC/alloantigens (Hunt et al., 1982), Y-chromosome (Theise, N. D. et al., 2000; Petersen, 2001), a1 antitrypsin (Hafenrichter et al., 1994a), and DPPIV (Thompson et al., 1991). Models relying on alloantigens, an approach pioneered by Hunt et al. (1982), utilize F1 progeny as hosts for donor cells from either of two inbred strains differing in their MHC haplotypes (ACI and Long-Evans or Wistar Furth and Fischer F344) (Hunt et al., 1982; Faris and Hixson, 1989). Donor cells are subsequently identified using alloantisera produced by immunizing ACI rats or F344 rats with Long-Evans or Wistar Furth spleen cells, respectively. In the past, we have used this model system to demonstrate that oval cells induced by 2-AAF and a CD diet formed GGT+ colonies of hepatocytes following transplantation into partially hepatectomized F1 hosts (Faris and Hixson, 1989) (Fig. 3). One drawback to tracing donor cell fates using alloantisera is that it is the host cells, not the donor cells, that show positive staining with the alloantisera. Consequently, unstained donor cells have to be discerned against a positive background of host hepatocytes, making it difficult to detect small, single donor cells or even small donor cell clusters.

In a number of recent studies, the fate of donor cells from male rats or mice following transplantation into female hosts has been determined by using flourescent in situ hybridization to detect cells bearing a Y-chromosome (Petersen, 2001). In studies by Thiese, N. et al. (2000), this approach was used to demonstrate that circulating stem cells, most likely of bone marrow origin, can integrate into the liver and differentiate into hepatocytes and cholangiocytes, an observation that challenges current concepts of differentiation and commitment. However, a recent report suggesting that bone marrow cells can fuse spontaneously with other cell types and assume their identity (Terada et al., 2002) implies that the presence of the Y-chromosome in hepatocytes of female hosts could represent a fusion event rather than differentiation of bone marrow stem cells into hepatocytes.

Transplantation models with Wt donors and mutant hosts that lack or express an inactive form of a normal liver protein have been used extensively for lineage analysis. Transplantation of Wt hepa-tocytes or progenitors capable of hepatocyte differentiation has been shown to restore serum albumin levels in Nagase analbuminemic rats (Oren et al., 1999). Engrafted donor cells were readily detected in tissue sections by in situ hybridization with albumin cDNA probes or immunohistochemically with anti-rat albumin antibodies. One of the advantages of this model system is the ability to quantitate engraftment efficiency, the extent and rate of expansion of donor cells, and the duration of engraftment by measuring changes with time in the serum albumin levels. The DPPIV transplantation model developed by Thompson et al. (Thompson, 1991) offers similar advantages. In this case, the DPPIV-negative Fischer 344 rats used as hosts produce an enzy-matically inactive form of DPPIV. This allows the localization of transplanted donor cells from Wt Fischer rats by a simple his-tochemical procedure for active DPPIV or by immunocytochemi-cal staining methods with MAbs that recognize only the active form of DPPIV (Fig. 6). Although DPPIV is a type II transmembrane protein, it is susceptible to cleavage by extracellular proteases that release a soluble 200-kDa enzymatically active fragment into the serum (Shibuya-Saruta et al., 1996). Serum levels of DPPIV can thus be used to measure the same parameters noted above for albumin (Hanski et al., 1986). One limitation of the DPPIV model system is the late expression of the enzyme during fetal development (Petell et al., 1990) and its absence on most hepatic progenitors, a temporal pattern that limits its usefulness for analyzing early time points in liver development or progenitor cell differentiation. This is not an issue for albumin because it is one of the earliest development markers detected in fetal liver.

There are increasing numbers of excellent transplantation models that make use of transgenic mice carrying marker genes as a source of donor cells. These genetically marked cells are transplanted into hosts treated with chemicals that inhibit the proliferation of hepatocytes or into transgenic or mutant strains carrying genetic defects that severely compromise hepatocyte/ ductal cell-mediated regeneration and repair. Mignon et al. (1998) transplanted donor cells from transgenic mice expressing human Bcl-2 into immunosuppressed host mice treated with a nonlethal dose of anti-fas/CD95 antibodies to induce apoptosis. Since the donor cells were protected from apoptosis by Bcl-2, they selectively expanded and gradually replaced as much as 16% of the host liver. Rhim et al. (1994, 1995) have used liver cells from P-gal mice to demonstrate the expansion of hepato-cytes transplanted into the livers of transgenic mice expressing the urokinase-type plasminogen activator (uPA) under control of the albumin promoter. Overex-pression of uPA is cytotoxic to hepatocytes and greatly compromises their regenerative capacity, giving a large growth advantage to donor P-gal+ hepatocytes or host hepatocytes that lose the uPA transgene. The end result

Exogenous Cell Transplantation

Fig. 6. Colony of DPPIV-positive hepatocytes in liver of DPPIV-deficient rat. Frozen liver sections prepared 2 mo after transplantation of E12-13 fetal liver cells depleted of TuAgl-positive hepatoblasts were stained histochemically for DPPIV. Shown is a colony of donor hepatoblasts were stained histochemically for DPPIV. Shown is a colony of donor hepatocytes strongly positive for DPPIV. (Photograph provided by Rhonda Simper, Rhode Island Hospital, Providence, RI.)

Fig. 6. Colony of DPPIV-positive hepatocytes in liver of DPPIV-deficient rat. Frozen liver sections prepared 2 mo after transplantation of E12-13 fetal liver cells depleted of TuAgl-positive hepatoblasts were stained histochemically for DPPIV. Shown is a colony of donor hepatoblasts were stained histochemically for DPPIV. Shown is a colony of donor hepatocytes strongly positive for DPPIV. (Photograph provided by Rhonda Simper, Rhode Island Hospital, Providence, RI.)

is complete clonal repopulation of the host liver by donor hepa-tocytes. Overturf et al. (1996, 1999) reported similar findings following transplantation of P-gal-positive mouse liver cells into host mice lacking the enzyme fumarylacetoacetate hydrolase (FAH). The lack of this gene produces symptoms similar to those observed for hereditary tyrosinemia, type 1, a condition that produces extensive liver damage that accelerates and promotes repopulation of the liver by Wt P-gal+, FAH+ donor cells. Another relatively new model system that is currently enjoying wide use is the retrorsine model of Laconi et al. (1998, 2001). In this system, DPPIV-negative host rats are pretreated with retrorsine, a DNA alkylating agent that promotes the selective expansion of donor liver cells by impairing the ability of host hepatocytes to proliferate following PH. After several weeks, donor cells repopulate as much as 80% of the liver. Similar effects can also be obtained by pretreatment with galactosamine (Gupta et al., 2000) or radiation, but the degree of repopulation by donor cells is considerably lower.

An unanswered question in all of these animal models is, Does the introduction of exogenous genes into donor cells or the abnormal host environment needed to promote donor cell expansion have any effect on the differentiation capabilities of donor cells? This may be particularly important for putative progenitor cells, which are likely to be more sensitive to changes in microenvironment than mature hepatocytes or ductal cells. Introduction of Bcl2, e.g., may enhance the ability of transplanted cells to survive genetic changes that would normally induce apoptosis, thereby creating a population of damaged cells that may be at higher risk for neoplasia. Wesley et al. (1999) have reported that DPPIV

suppresses the malignant behavior of melanoma cells by reversing a block in differentiation and by restoring growth factor-dependent cell survival, activities mediated by serine proteases. Although previous investigations by Coburn et al. (1994) led to the conclusion that DPPIV proteolytic activity was not necessary for immune competence, more recent studies suggest that DPPIV enzymatic activity is essential for certain T-cell activation pathways and is involved in the inactivation of chemokines (Iwata and Morimoto, 1999).

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