1.1 Hepatocytes and progenitor cells both contribute to liver repair

Hepatocytes and biliary epithelial cells (cholangiocytes) in the normal liver are quiescent. In response to liver injury or loss of liver mass, mature, differentiated liver cells proliferate as a first-line defense to restore homeostasis. Despite the low rate of hepatocyte turnover in the healthy liver, serial transplantation studies have demonstrated that differentiated hepatocytes are able to proliferate through many divisions in hosts in which the transplanted hepatocytes have a survival advantage (Overturf et al., 1999). In patients or in animal models of chronic liver disease, however, hepatocytes and cholangiocytes are blocked from proliferating, and a second mechanism of regeneration comes into play as small bipotential progenitor cells with the ability to differentiate into hepatocytes or cholangiocytes become activated (Thorgeirsson, 1996, Alison et al., 1998, Bisgaard et al., 2002, Tan et al., 2002, Fausto, 2004). These facultative stem cells, described in rodent models as oval cells or intermediate hepatobiliary cells, and referred to here as liver progenitor cells, have the appearance of small hepatocytes with scant cytoplasm. Whether or not small numbers of pluripotent stem cells also persist in the adult liver in addition to these bipotential progenitor cells is still unresolved ((Zhang et al., 2008) and reviewed in (Bird et al., 2008, Duncan et al., 2009)).

In addition to their role in liver repair, liver progenitor cells have received significant attention because they may contribute to a subset of hepatocellular cancers exhibiting stem cell characteristics. Progenitor cells are also of great interest to the biomedical community as a potential alternative to organ transplantation. These topics are beyond the scope of this article, and we direct readers to several recent reviews on the role of liver progenitor cells in stem cell cancers (Sell and Leffert, 2008, Mishra et al., 2009) and cell transplantation therapy (Oertel and Shafritz, 2008, Dalgetty et al., 2009).

2.1 The ductular reaction and fibrosis

“The ductular reaction” is the term given to a duct-like proliferation of a population of liver progenitor cells in the setting of fibrosis. The extent of the ductular reaction appears to correlate directly with impaired hepatocyte replication, consistent with the notion that progenitor cells are only recruited when replication of mature liver cells is inhibited. Multiple investigators have shown a compelling direct association between the extent of the ductular reaction and the severity of fibrosis in patients with chronic liver diseases from such diverse etiologies as hepatitis C (Clouston et al., 2005), non-alcoholic fatty liver disease (Richardson et al., 2007), and the pediatric cholangiopathies Alagille syndrome and biliary atresia (Fabris et al., 2007), suggesting that progenitor cell activation plays an important if undefined role in fibrosis.

A detailed marker analysis of ductular reactions in livers from cirrhotic patients demonstrated that these ductular reactions had a bipolar structure, with bidirectional differentiation to either hepatocytic or biliary lineages (Zhou et al., 2007). Other groups have not observed mutually exclusive expression of hepatocytic and biliary markers during progenitor cell proliferation but instead describe “intermediate hepatocytes” that express both sets of markers (Zhang et al., 2008, Fabris et al., 2007). These intermediate hepatocytes appear in contiguity with ductular progenitor cells when liver inflammation and necrosis is moderate or severe, suggesting that differentiation of progenitor cells towards the hepatocytic lineage is directly related to the extent of parenchymal injury (Lowes et al., 1999, Libbrecht et al., 2000, Roskams et al., 2003, Katoonizadeh et al., 2006). Regardless of the details of progenitor cell activation and marker expression, these observations strongly support the concept that hepatic progenitor cells of the ductular reaction contribute directly to the fibrogenic process, most likely through cell-cell cross talk between the progenitor cells and nearby mesenchymal populations and via recruitment of inflammatory cells (Strick-Marchand et al., 2008, Ruddell et al., 2009). The signals that mediate progenitor cell quiescence and activation, however, are still unclear. Nguyen and colleagues made the seminal observation that rat liver progenitor cells (oval cells), unlike hepatocytes, are resistant to the growth inhibitory effects of the growth factor TGF-β, providing at least a partial mechanistic basis for their differential activation in the TGF-β-rich environment of fibrosis and cirrhosis (Nguyen et al., 2007).

3.1 Stem cells as a source of myofibroblasts

Myofibroblasts, defined operationally as fibrogenic cells expressing α-smooth muscle actin (α-SMA), are the primary source of fibrillar collagens and other abnormal extracellular matrix (ECM) proteins in fibrosis (Hinz et al., 2007). Hepatic stellate cells, a heterogeneous population of vitamin A-storing cells normally located in the space of Disse, have long been considered the major source of myofibroblasts in fibrosis, although other resident myofibroblast precursor cells including portal fibroblasts and mesenchymal cells are increasingly recognized as providing equal if not more significant contributions (Guyot et al., 2006, Beaussier et al., 2007, Tuchweber et al., 1996, Li et al., 2007). The embryonic origin of hepatic stellate cells and portal fibroblasts is debated, although studies using transgenic mice suggest that hepatic stellate cells as well as certain perivascular mesenchymal cells originate from the mesoderm of the septum transversum (Loo and Wu, 2008, Asahina et al., 2009, Villeneuve et al., 2009, Kalinichenko et al., 2003, Friedman, 2008). Portal fibroblasts and other mesenchymal cells appear during early development (Loo and Wu, 2008, Villeneuve et al., 2009), during which time stellate cells and portal fibroblasts share at least one specific marker (p75^NTR) (Suzuki et al., 2008). Whether progenitor cells also contribute to the population of stellate cells, portal fibroblasts, and other myofibroblasts in the setting of significant or chronic injury in the adult liver is less clear.

Bone marrow transplants have been touted as a potential therapy for chronic liver disease (Houlihan and Newsome, 2008); thus, understanding the relationship between bone marrow stem cells and matrix producing cells in fibrosis is particularly important. There are now multiple reports suggesting that progenitor cells from the bone marrow can migrate to the injured liver and differentiate into stellate cells and myofibroblasts. In the most quantitative study, Russo et al. carried out male to female bone marrow transplants and found that 70% of fibrogenic myofibroblasts were derived from transplanted marrow cells. Transplantation of cells from mice with a mutant, degradation-resistant collagen I (col 1a1^rr) resulted in increased fibrosis (Russo et al., 2006). A variety of other studies, most involving whole bone marrow transplantation but at least one in which the clonal progeny of a single hematopoietic stem cell were transplanted, yielded similar evidence that marrow-derived progenitor cells could generate fibrogenic myofibroblasts (Miyata et al., 2008, Kisseleva et al., 2006, Baba et al., 2004). A more recent study by Higashiyama et al., however, showed no evidence that any collagen expression in the injured liver was from bone marrow-derived cells (Higashiyama et al., 2009). In an elegant review, Kallis and Forbes summarized the existing studies, suggesting that heterogeneity in the population of bone marrow-derived stem cells explains these divergent results (Kallis and Forbes, 2009). Mesenchymal stem cells, which are resistant to radiation, and hematopoietic stem cells, which are radiosensitive, appear to have different abilities to repopulate the liver, and differences in experimental techniques may select for one versus the other. Interestingly, mesenchymal stem cells may also have indirect effects on fibrosis via the modulation of hepatic stellate cell phenotype (Parekkadan et al., 2007). Ultimately, improved marker analysis for bone marrow stem cells may be required in clinical applications in order to carry out selective transplantation of cells with minimal fibrogenic potential.

4.1 Activated hepatic stellate cells and other myofibroblasts may mediate progenitor cell differentiation

As noted above (section 1), there is evidence in support of at least four stem cell niche locations in the injured liver (Kuwahara et al., 2008). As these are better defined, their relationship to myofibroblasts and myofibroblast precursors should become clearer. There is already significant evidence suggesting that hepatic stellate cells, functioning as support cells in the niche, play an important role in liver progenitor cell expansion and differentiation. Numerous studies have demonstrated a close physical relationship between stellate cells, the extracellular matrix, and oval cells, and have suggested that the two cell types proliferate in tandem (Roskams, 2008, Zhang et al., 2009, Braun et al., 2003, Paku et al., 2001). Co-culture of stellate cells and progenitor cells (either resident liver progenitor cells or circulating mesenchymal stem cells) results in increased proliferation and hepatocytic differentiation of progenitor cells (Nagai et al., 2002, Lin et al., 2008, Deng et al., 2008). Whether this requires cell-cell contact is not clear; nor is it known what the soluble mediators are, although some studies suggest that hepatocyte growth factor and sonic hedgehog play a role (Nagai et al., 2002, Lin et al., 2008, Paku et al., 2001). Progenitor cell expansion is affected by multiple soluble factors, many of which are produced by stellate cells ((Santoni-Rugiu et al., 2005) and see section 2). Notably, in all studies, the hepatic stellate cells used for co-culture experiments have been activated (myofibroblastic); in the one study that addressed this question specifically, activated (rather than quiescent) stellate cells were required for the observed effects on progenitor cells (Deng et al., 2008). The relationship between stellate cells and progenitor cells may be bidirectional: one group found that co-culture with mesenchymal stem cells resulted in stellate cell activation (Parekkadan et al., 2007).

Several studies using the choline-deficient, ethionine-supplemented diet have shown that there is a close physical association between progenitor cells and myofibroblasts (which may or may not be derived from stellate cells) and that the extent of fibrosis correlates with the degree of progenitor cell expansion (Knight et al., 2007b, Knight et al., 2007a). Ruddell and colleagues found that lymphotoxin-β (LTβ)-expressing progenitor cells were adjacent to LTβ receptor expressing myofibroblasts, and have suggested that there is a paracrine loop operational between the two cell types, mediated by LTβ (Ruddell et al., 2009).

The controversial topic of Thy-1 positive cells may provide further insight into the role of mesenchymal cells in the progenitor cell niche. The hematopoietic cell marker Thy-1 was originally identified as a marker of oval cells (Petersen et al., 1998). More recently, several groups have shown that Thy-1 positive cells are actually mesenchymal cells and are an essential component of the progenitor cell niche, required for oval cell differentiation (Hoppo et al., 2004, Koenig et al., 2006, Yovchev et al., 2008, Dudas et al., 2007, Dezso et al., 2007). Although the identity of Thy-1 positive cells remains unresolved, the literature on this topic has highlighted several interesting points: 1) that oval cells, whether or not Thy-1 positive, are in close physical contact with myofibroblasts and other mesenchymal cells in models of oval cell proliferation; 2) that oval cells themselves may express mesenchymal markers, including desmin and type I collagen; and 3) that Thy-1 positive cells are heterogeneous, some although not all appearing myofibroblastic (expressing α-SMA). Unpublished work for our laboratory suggests that the Thy-1 positive cells are negative for elastin (a marker of portal fibroblasts and myofibroblasts), suggesting that they are not portal fibroblasts, while others have shown that they are not hepatic stellate cells (Yovchev et al., 2009). This raises the possibility that there is an important and as yet undefined mesenchymal cell population contributing to the oval cell niche. In our studies of Foxl1 and bipotential progenitor cells in the liver, however, we observed that progenitor cells were encircled by α-SMA negative cells expressing elastin, apparently portal fibroblasts (Fig. 1) (Sackett et al., 2009). The relationship between Foxl1-expressing progenitors and oval cells remains unclear, as does the relationship between the elastin positive cells surrounding Foxl1 progenitors and the possible Thy-1 positive mesenchymal cells surrounding oval cells.

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Fig. 1

Lineage tracing of hepatic progenitor cells

Foxl1-Cre positive progenitor cells (aqua blue) are encircled by portal fibroblasts (royal blue) in the portal areas of DDC injured liver, suggestive of a progenitor cell niche. Brown pigment represents heme breakdown product of DDC.

These studies in aggregate raise interesting questions about cross-talk between hepatic stellate cells, myofibroblasts, other mesenchymal cells, and progenitor cells in wound healing: if myofibroblasts mediate progenitor cell proliferation and differentiation, do progenitor cells in turn mediate stellate cell activation and the differentiation of other myofibroblast populations? Is it an essential feature of wound healing that scar formation and parenchymal reconstitution go hand in hand? Even more interesting is the question of whether activated stellate cells and other myofibroblasts are part of the progenitor cell niche or rather the means whereby progenitor cells exit the niche, and whether quiescent stellate cells may serve the opposite function of retaining progenitor cells in the niche. Future research will undoubtedly be aimed at understanding the heterogeneity of both progenitor cells and the supporting cells of their niches.

R.G.W. is supported by NIH R01-DK058123 and by a grant from the Fred and Suzanne Biesecker Pediatric Liver Center. L.E.G. is supported by the Penn Institute for Regenerative Medicine and NIH Center Grant P-30-DK050306.