In pursuit of the ?holy grail? ? stem cells, hepatic injury, fibrogenesis and repair
Authors: Nick Shackel, Don Rockey
Journal: Hepatology (2004)

An important goal of stem cell research is to treat tissue injury. However, the quest for therapeutic utilization of stem cells must await a better understanding of how these cells participate in normal tissue homeostasis as well as the response to injury. The liver has been a major target for stem cell–based therapy. Indeed, stem cell research has focused on a variety of intrahepatic models of injury. However, few studies have directly examined stem cells in intrahepatic fibrogenesis. The classical paradigm that intrahepatic cell populations are replenished from resident oval cells has now been revised to include extrahepatic stem cells.1 However, the mechanism and extent to which extrahepatic stem cells contribute to the repair process (including regeneration) following injury remains controversial; for example, there is increasing investigation of extrahepatic stem cell involvement in hepatocyte regeneration. The type of injury (or experimental model utilized) seems to play a major role in determining whether stem cells differentiate into hepatocytes (the process of stem cell "plasticity"), fuse with hepatocytes (the process of cell "fusion"), or change to form other cell lineages (the process of "trans-differentiation") (Fig. 1).2, 3 In contrast to the partially characterized stem cell–hepatocyte relationship, the involvement of extrahepatic stem cells in nonhepatocyte liver cell homeostasis and pathobiology is poorly understood. Confusion surrounds whether circulating or resident stem cell populations become or differentiate into nonparenchymal cell populations, such as hepatic stellate cells, endothelial cells, and Kupffer cells. Understanding the stem cell relationship to nonparenchymal hepatic cells may be central to understanding how stem cells influence liver injury. This raises fundamental questions about the origin of these intrahepatic nonparenchymal cell populations. Bone marrow-derived circulating stem cells appear to contribute to hepatic cell subpopulations in liver injury. (A) A schematic outline of the passage of circulating bone marrow stem cells to the liver. The true identity of the circulating stem cell population and the factors responsible for recruitment/chemotaxis are poorly understood. Attachment and migration into the liver parenchyma is not understood (*). Further, differentiation to a committed specific liver cell subpopulation may occur outside the liver parenchyma. (B) Outline of the mechanisms by which a stem cell may give rise to a specific liver cell subpopulation. The terms "plasticity" and "transdifferentiation" are often used interchangeably. However, "plasticity" refers to the ability of stem cells to differentiate or retro/de-differentiate from self-renewing stem cells through to tissue specific cell populations (1). "Transdifferentiation" typically refers to stem plasticity in which stem or progenitor cells differentiate into a cell type characteristic of another tissue or cell lineage (2). Importantly, circulating bone marrow-derived cells may contribute to specific hepatic cell populations by the process of "fusion" (3). Of note, the increased karyotype typical of fusion may be lost in the progeny due to reduction division of chromosome number, thus concealing the fusion event. MMP, matrix metalloproteinase; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; NGFR, nerve growth factor receptor; SDF, stromal cell–derived factor. Hepatic stellate cells are pivotal mediators of the response to liver injury. Given that stellate cell activation, proliferation, and apoptosis are essential determinants of fibrogenesis, studies examining the role that stem cells play in this response may answer key questions about liver disease pathogenesis. In the December issue of HEPATOLOGY, Sakaida et al. demonstrate that extrahepatic bone marrow derived stem cells reduce fibrosis in a murine model of CCl4 injury.4 This study demonstrated a survival benefit after bone marrow stem cell treatment in mice with liver injury. Further, the authors partially characterised the cell population of bone marrow–derived cells (Liv8 antigen negative) that are responsible for this survival benefit. Finally, the study begins to address the potential role of stellate cells in intrahepatic fibrogenesis. A major issue in the current study is that the mechanism of improvement in fibrosis after bone marrow transplantation is unknown. Sakaida and colleagues suggest that bone marrow–derived cells cause a reduction in stellate cell numbers, possibly by apoptosis.4 However, it is plausible that the observed decrease in fibrosis is mediated by intrahepatic cell subpopulations other than bone marrow–derived stellate cells. Additionally, a reduction in stellate cell numbers may result from the effect of a large cell infusion (i.e., with subsequent signalling that leads to induction of stellate cell apoptosis or inhibition of proliferation) rather than hepatic engraftment of bone marrow–derived cells. Therefore, an important control would be to determine if the reduction of fibrosis is seen after infusion of a cell population depleted of stem cells. Although it is reasonable, and even exciting, to hypothesize that bone marrow–derived stellate cells mediate an antifibrogenic effect, it is unclear whether the observed reduction in fibrosis is due specifically to a stem cell effect. The embryonic and intrahepatic origin of hepatic stellate cells is unknown. Available data suggest that circulating bone marrow cells can give rise to hepatic stellate cells.4 Previous morphological analysis in human liver fibrosis demonstrated that 6.8% to 22.2% of activated stellate cells (smooth muscle α actin positive) had cytogenetics consistent with a bone marrow origin.5 Further, recent work has demonstrated the presence of bone marrow–derived stellate cells in C57/BL6 and BALBc mice treated with CCL4.6 Importantly, this study demonstrated that the bone marrow–derived stellate cells express collagen α2(I) messenger RNA and found no evidence of cell fusion upon examining the karyotype of the bone marrow–derived cells.6 Similarly, the current paper by Sakaida and colleagues demonstrates intrahepatic matrix metalloproteinase (MMP)-9 immunoreactivity and green fluorescent protein (GFP) expression under the control of a β-actin promoter on presumed marrow-derived stellate cells in animals administered isolated GFP labeled bone marrow cells.4 Stellate cells express mesenchymal markers such as vimentin, desmin, and neural crest proteins (glial fibrillary acidic protein [GFAP] and nestin).7, 8 Further, it is not known whether resident oval cells give rise to hepatic stellate cells or if these cells have another origin. The possibility that the bone marrow may be a source of stellate cells is suggested from studies in which CD34+ and CK7/8+ stem cells have been described that are CD13+, CD59+, and nerve growth factor receptor (NGFR)+.7 Additionally, circulating stem cell populations expressing tissue-specific markers have been identified, suggesting the presence of either lineage-specific multipotent precursor cells or ongoing extra organ stem cell differentiation.9 These distinctions are important as they have fundamental implications in understanding the mechanism of stem cell involvement in intrahepatic injury. One of the most intriguing aspects of this study was the method used to make the chimeric animals.4 In this study, bone marrow–derived cells were infused without bone marrow ablation.4 Previous studies suggested that haematopoietic reconstitution following ablation is necessary for stem cell engraftment in the liver.10 Additionally, significant engraftment of bone marrow–derived cells during liver injury is thought to require in vivo selection. For example, there may be an engraftment advantage for normal hepatocytes in the fah−/− model of liver injury.11, 12 However, the current study and previous work by the same group support the notion that bone marrow ablation is not required for stem cell engraftment in the liver.13-15 The study by Sakaida begs the following questions: (1) is hepatic stem cell engraftment in liver injury a function of circulating precursor frequency? (2) Is there a functional impairment of stem cells with liver injury? Importantly, these experiments used the same C57BL6 genetic background for both the donor and recipient. However, stem cells used in future therapy may not be as immunologically privileged. If stem cell therapies are to be moved into the clinical arena for treatment of liver disease, then a better understanding of the immune response and tolerance to infused cell populations will be necessary. The identification of bone marrow–derived cells in the liver and the precise identification of the engrafted cell phenotype can be technically demanding and are frequently problematic.16 Therefore, positive marker molecules such as GFP and β-galactosidase are often utilized. Unfortunately, GFP expression is variable on a background of liver autofluorescence, and β-galactosidase activity is frequently weak and difficult to reliably detect. Alternate approaches utilize cytogenetics (sex-mismatched chimeras and karyotype studies) or immunohistochemistry markers (such as Ly 5.1/5.2).16 Technical limitations with each of these methodologies mean that exact co-localization is often difficult and inferred rather than strictly demonstrated.16 Therefore, the lineage of bone marrow–derived cells is frequently misrepresented. Indeed the discrepancy in many studies examining hepatocyte chimerism appears to be due to the erroneous identification of bone marrow–derived liver monocytes as hepatocytes.17 Similar concerns are present for the current study. Ideally, true co-localization requires precise labeling of cells in situ or cell subpopulation isolation, although cell isolation techniques could be confounded by selection bias. Issues surrounding stem cell plasticity and fusion are not investigated in the present study.1, 18-20 Apparently conflicting results suggest that either plasticity or fusion predominates as the mechanism by which the bone marrow–derived cell contribution to intrahepatic cell populations (Fig. 1). What is clear is that there is a significant bone marrow contribution with injury. If plasticity and transdifferentiation are the predominating mechanism, then clonality for the bone marrow–derived cells needs to be demonstrated. Many studies attribute their results to a pluripotent precursor cell population when the infused cells are actually a mixture of multipotent stem cells and circulating oval cells. If clonality is demonstrated this will potentially lead to the ex vivo manipulation of this response. In contrast, if fusion is the underlying mechanism, then the true identity of the bone marrow derived–cell population may not be significant. These distinctions are important in developing potential future therapeutic applications of this technology. The intrahepatic milieu that signals recruitment and/or chemotaxis of bone marrow–derived cells to the liver has been examined to a limited extent. However, as the precursor stem cell population has not been specifically identified the mechanisms of cell attachment and migration are not understood. Further, the signals that determine into which lineage a stem cell differentiates within the liver are also unknown. Another intriguing possibility to be considered is that bone marrow–derived stem cell fate determination happens outside the liver with homing of lineage-specific cells that engraft and proliferate within the liver.9 As in other organs the intrahepatic expression of CXC4 (also known as stromal cell–derived factor [SDF]-1) is important in the homing of bone marrow–derived cells to the liver.9, 21 Further, the hepatic expression of hepatocyte growth factor and MMP-9 are important regulators of stem cell recruitment to the liver.21 Although in the current study stellate cell numbers were reduced there was an increase in the proportion of MMP-9–positive stellate cells. Therefore, the expression of mediators found in the wounding environment (many of which are produced by stellate cells) may be important in stem cell chemotaxis and/or differentiation. The therapeutic potential of bone marrow–derived stem cells has led to increasing research interest focused on hepatic stem cell biology, especially in models of liver injury. However, given the diverse methodologies and models of liver injury utilized, there is now ambiguity and controversy surrounding the role of bone marrow–derived stem cells in each normal homeostasis and injury. Available data suggest that the role of bone marrow–derived stem cells in the normal liver is limited. However, with liver injury the situation is more complex. The study by Sakaida et al.4 is important in that it demonstrates a reduction in fibrosis with stem cell administration and suggests a significant role for bone marrow–derived stem cells in liver injury and fibrogenesis.4 Additionally, this work reminds us that further investigation will be essential in order to understand how stem cells and nonparenchymal cells, including stellate cells, interrelate. While many questions remain, this study provides a further step on the long path towards one of the "holy grails" of hepatic stem cell research—tissue repair.