Journal of Hepatology
Volume 45, Issue 1 , Pages 1-4, July 2006

Different types of liver progenitor cells and their niches

  • Tania Roskams

      Affiliations

    • Corresponding Author InformationTel.: +32 16 33 65 96; fax: +32 16 33 65 48.

Head Liver Research Unit, Department of Morphology and Molecular Pathology, University of Leuven, Minderbroederstraat, 12 B-3000 Leuven, Belgium

published online 12 May 2006.

Article Outline

 

A stem cell is an undifferentiated cell capable throughout life of renewing itself as well as of generating one or more types of differentiated cells [1], [2], [3]. Every adult cell has a ‘mother’ (who may have parted from her years before), and a line of ancestors – stem cells – of increasing differentiation potential. The ultimate stem cells are those of the early embryo, which are totipotential. Farther down along the line, in the fetus and also in the adult, we find multipotential (pluripotential) stem cells. Those closer to final differentiation are called progenitor, committed or transit cells.

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1. The Liver: two types of ‘stem cells’ and a range of ‘progenitor cells’ 

Hepatocytes in normal adult liver hardly ever proliferate and have a life span of over a year. After partial hepatectomy however, proliferation of the main epithelial compartments (hepatocytes and cholangiocytes), followed by proliferation of the mesenchymal cells (hepatic stellate cells and endothelial cells), quickly restores the liver [4], [5]. Serial transplantation experiments have shown that hepatocytes have a near infinite capacity to proliferate [6], [7], a stem cell-like property.

When the mature epithelial cell compartments of the liver, hepatocytes and/or cholangiocytes are damaged or inhibited in their replication, a reserve cell compartment is activated [8]. This compartment, in humans called the progenitor cell compartment and in rodents the oval cell compartment, resides in the smallest and most peripheral branches of the biliary tree, the ductules and canals of Hering [9]. The canal of Hering, a channel partly lined by hepatocytes and partly by cholangiocytes, represents the anatomic and physiological link between the intralobular canalicular system of hepatocytes and the biliary tree. It resides along an array of sites that project star-like from the portal tracts. Cells of morphology and immunophenotype intermediate between hepatocytes and cholangiocytes (“intermediate cells”, see below) are not recognized in normal tissue. The progenitor cells are labeled by biliary type cytokeratins (CK) CK7 and 19, oval cell markers OV6 and OV1, neuroendocrine markers chromogranin-A, neural cell adhesion molecule and parathyroid hormone-related peptide, connexin 43, etc … A subpopulation of ductular/progenitor cells express markers of haematopoietic cells (CD34, C-kit, flt-3, Thy-1), which raised the question whether progenitor cells would be directly derived from bone marrow stem cells. Most studies however are concordant with a progenitor cell niche in the ductules/canals of Hering, at the interface between the parenchyma and the portal tract mesenchyme. This is also the location where during embryonic development, bipotential hepatoblasts form the primitive ductal plate, which has the same phenotype as progenitor cells in adult life: ductal plate cells express biliary markers and (immature) hepatocytic markers like alphafoetoprotein and in addition haematopoietic markers like CD34.

Wilson and Leduc were the first to describe activation of a ‘reserve cell compartment’ in mice after severe dietary injury [10]. Subsequently, several models of so-called oval cell reaction in rodents have been described. Mostly, these models employed potential carcinogenic agents to inhibit the proliferation of mature hepatocytes after a regenerative stimulus. E.g. acetaminofluorene intoxication of rats, after partial hepatectomy or after administration of a necrogenic dose of carbon tetrachloride [11] or ethionine intoxication in mice. Also models of fatty liver disease like ObOb-mice or PARP1(−/−) mice are characterized by inhibition of the replication of mature hepatocytes, caused by oxidative stress and show a striking oval cell response [12], [13].

Progenitor cell activation or ductular reaction is also seen in the majority of chronic human liver diseases. The degree of progenitor cell activation increases with the severity of the disease [12], [14], [15]. In moderate and severe degrees of inflammation, intermediate hepatocytes occur, having a phenotype intermediate between progenitor cells/ductular cells and mature hepatocytes. The number of these intermediate hepatocytes gradually increases with higher degrees of inflammation, and also with higher degrees of necrosis in necrotising hepatitis and with more advanced stages of (non)alcoholic steatohepatitis [12], [14], [15]. This highly suggests a higher degree of differentiation of progenitor cells into hepatocytes when there is more hepatocyte damage.

A general trigger for progenitor cell activation is certainly a lack of ability of the mature cell compartments to proliferate. In parallel to what we know from rodent models, also in human liver diseases there is inhibition of replication of mature hepatocytes. Recently it has been shown that hepatocytes are senescent due to telomere shortening, in the cirrhotic stage of a wide variety of chronic human liver diseases. Intriguingly, mesenchymal cells like endothelial cells and hepatic stellate cells do not show this replicative senescence [16], [17]. Probably this hepatocyte replicative senescence is in part the result of ongoing proliferation during 20–30 years of chronic liver disease. Chronic inflammation, presence of growth factors, DNA-damaging agents like reactive oxygen species and nitrogen species also play a role. So, similar to rodent models, replicative senescence of hepatocytes triggers progenitor cell activation, also in man [8], [18]. In cirrhotic livers, especially in (non)alcoholic steatohepatitis, whole cirrhotic nodules can be composed of intermediate hepatocytes, which ultrastructurally look strikingly ‘normal’, without Mallory body formation and without fatty change. This suggests that these intermediate hepatocyte-nodules originate from progenitor cells [12]. In parallel, Falkowski et al. showed in a three dimensional reconstruction study that sequestered hepatocyte ‘buds’ in cirrhosis are always in continuity with reactive ductules, highly suggesting a progenitor cell origin [19]. Progenitor cells seem to be able to survive when hepatocytes are lost due to toxic damage or viral infections. Adenosine triphosphate binding cassette (ABC) transporters play both a secretory and a protective role and a strong up-regulation of apical MDR1 and basolateral MRP1, MRP3 and BCRP in human hepatocytes and in progenitor cell-related bile ductules was observed [20], [21]. We hypothesized that this change in expression offers protection against the accumulation of toxic bile constituents and may render these cells resistant to oxidative stress.

In view of their bipotential differentiation potential, progenitor cells would be very interesting cells to use for cell therapy or in bioartifical livers. One of the problems to study human progenitor cells is the lack of specific markers, so that cell isolation at the current time is not possible. Some attempts have been made using ckit or Neural cell adhesion molecule as markers of subpopulations of progenitor cells [22], [23]. Until now no differentiation of these isolated cells into mature hepatocytes has been accomplished.

In the article in this issue of the Journal Kon et al. [24] further characterize so-called ‘small hepatocytes’, a subpopulation of rat hepatocytes that have a high growth potential in culture [25], [26], [27]. These cells are two times smaller than normal mature hepatocytes, express both hepatocytic and biliary characteristics and clonally proliferate to form colonies that survive more than 5 months in defined medium [25], [28]. These isolated small hepatocytes can differentiate into mature hepatocytes by interaction with nonparenchymal cells [29] or as a result of treatment with Engelbreth–Holm–Swarm gel [30]. Therefore, these cells can be considered as ‘committed progenitor cells’ and resemble the previously described ‘intermediate hepatocytes’. Interestingly, these cells can also proliferate after cryopreservation [31]. Kon et al. looked for markers differentiating small hepatocytes (being committed stem cells) and mature hepatocytes, using microarrays. CD44 was one of the markers which was further examined. Cell sorting of CD44+ cells from a Galacosamine rat model and differentiation of these cells was studied in vitro. If similar cells existed in human liver, this could be an interesting approach to isolate ‘committed’ progenitors, already on their way to hepatocyte differentiation. Especially for the treatment of acute or subacute liver injury because ‘committed’ cells probably need less time to differentiate than less differentiated progenitor cells. In situ immunohistochemistry in the Galactosamine model showed that biliary cells and local progenitor cells (located in the smallest branches of the biliary tree) are marked with CD44 and that at day 4 periportal small hepatocytes are reactive for CD44. This finding is very well compatible with periportal small hepatocytes originating at least in part from local progenitor cells, like is seen in other rat models like CDAAF or Solt Farber model. The maximum progenitor (oval) cell reaction in Galactosamine intoxication is seen at 48h [32]. At 4 days one would expect that already the majority of progenitor cells have differentiated into hepatocytes, which fits with the presence of CD44s-reactive small hepatocytes in the periportal area. When cells mature, it is logical that they lose certain phenotypical characteristics (in this case CK19) and gain others (e.g. CD44v6), like is seen by Kon et al. However, as the authors state, there is also the theoretical possibility that small hepatocytes are a subpopulation of less differentiated/dedifferentiated hepatocytes.

There have been a number of papers illustrating the role of bone marrow stem cells in producing hepatocytes, both in animal models and in humans. Their precise role is not clear, since in several models, it has been shown that cell fusion of bone marrow stem cells with damaged hepatocytes took place [33].

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2. The society of liver cells and the surrounding stroma; the liver ‘stem cell niche’ 

The liver consists of epithelial cell types (hepatocytes, cholangiocytes and progenitor cells), mesenchymal cell types (Kupffer cells, endothelial cells, hepatic stellate cells) and stroma. Stroma is present in the portal tracts and in the Disse’s space.

The importance of this ‘society’ of cells and the stroma is illustrated by the fact that hepatocytes, which have a huge growth potential in vivo, when isolated and put in culture, are very hard to keep alive and differentiated. The ‘small hepatocytes’ used in the paper by Kon et al. have a high growth potential and can form colonies. and differentiate into mature hepatocytes in co-culture with nonparenchymal cells, illustrating the importance of the latter cells. There is a continuous interplay between the different cell types in the liver and the surrounding stroma (Fig. 1). Stellate cells produce growth factors for hepatocytes and progenitor cells, like hepatocyte growth factor. All hitherto growth factors act on all epithelial cell compartments. Recently, a specific growth factor for ‘oval cells’, TWEAK (transforming growth factor like weak inhibitor of apoptosis), was described, which does NOT act on hepatocytes [34]. This is an important step in our understanding of the regulation of oval cell reaction versus hepatocyte proliferation. This growth factor is produced by sinusoidal lining cells. Sinusoidal lining cells like hepatic stellate cells proliferate in close anatomical relationship with progenitor cells; in addition, stellate cells produce growth factors for which progenitor cells have the receptors, suggesting a true interaction between these cell compartments [8]. We also know that there are different subtypes of hepatic stellate cells/myofibroblasts [35]. In general, defining the so-called ‘stem cell niche’ for adult human liver progenitor cells will be an important goal for the near future. The article of Kon et al. in this issue of the Journal provides an interesting model to study the influence of matrix components and of mesenchymal cells on ‘small hepatocytes’ a committed progenitor cell type. Small hepatocytes express CD44, standard form. Preliminary data of the authors suggest that hyaluronic acid (HA), a ligand of CD44s is important for the proliferation of small hepatocytes, but keeps them in their less differentiated state: small hepatocytes plated on HA-coated dishes proliferate and form colonies, retain their CD44s expression and do not mature into mature hepatocytes.

  • View full-size image.
  • Fig. 1. 

    Scheme of the ‘Liver Society of Cells’: When hepatocytes are injured, Kupffer cells secrete interleukin-6 and transforming growth factor-α, which activate hepatic stellate cells. Hepatic stellate cells (HSC) on their turn produce growth factors for hepatocytes and progenitor cells (e.g. hepatocyte growth factor). Recently a specific growth factor which only acts on oval cells/progenitor cells (TWEAK) has been described. HSC also produce matrix components and they produce transforming growth factor-β, which inhibits the growth of hepatocytes. Growth factors like hepatocyte growth factor and epidermal growth factor also come from the circulation. The matrix harbors growth factors for hepatocytes and progenitor cells, bound to e.g. proteoglycans, which can be quickly released when needed. Release of hepatocyte growth factor by the matrix is on its turn inhibited by tissue inhibitor of metalloproteinase. Hepatocytes and progenitor cells produce growth factors for endothelial cells like vascular endothelial growth factor in order to maintain their vascularization.

Important future work will be to study CD44s expression in human liver. If this expression can also be used for cell isolation and cryopreservation, this would be a great step forward.

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PII: S0168-8278(06)00242-X

doi:10.1016/j.jhep.2006.05.002

Journal of Hepatology
Volume 45, Issue 1 , Pages 1-4, July 2006

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