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- Materialsand Methods
During development, the hepatic bud arises from the foregut endoderm [1, –3]. Liver organogenesis takes place by invasion of the endoderm into the mesenchyme of the septum transversum, resulting in the formation of the ductal plates (also called limiting plates), a band of cells around the portal triads. Hepatic stem cells in fetal and neonatal livers have been found recently to be located in the ductal plates  (E. Schmelzer et al., manuscript submitted for publication). These give rise to hepatoblasts, the dominant cell type of fetal and neonatal livers. Hepatoblasts, in turn, give rise to the hepatocytic and biliary lineages, the hepatocytes and cholangiocytes [4, 5] (E. Schmelzer, L. Zhang, A. Melhem, N.G. Moss, E. Wauthier, R.F. McClelland, A. Bruce, H. Yao, W.S. Turner, N. Cheng, M.E. Furth, and L.M. Reid, manuscript submitted for publication). In the adult liver, stem cells have been found associated with the Canals of Hering .
The ratio of hepatic stem cells to hepatoblasts changes depending on developmental stage, with the hepatoblasts being dominant in fetal and neonatal livers and with few if any mature parenchymal cells . In addition, the fetal liver is the primary site of hematopoiesis, consisting of up to 60% erythrocytes at certain developmental stages . By contrast, in pediatric and adult human livers, the hepatic stem cells are the dominant pluripotent progenitor (0.3%–0.7%); the hepatoblasts are few (<0.1%), and the majority (>98%) of the parenchymal cells are diploid and polyploid hepatocytes and biliary epithelia (Schmelzer et al., manuscript submitted for publication). These findings are summarized in a recent review .
Although the identity of hepatic stem cells and hepatoblasts in human livers has been described recently, our knowledge is incomplete on the gene and protein expression profiles of these pluripotent progenitors and whether the profiles change during development. We have shown that epithelial cell adhesion molecule (EpCAM), a 34–40-kDa transmembrane glycoprotein  first described by Spurr et al.  and cloned by Strnad et al.  and Szala et al.  is expressed by hepatic stem cells, hepatoblasts, and committed progenitors but not by mature hepatocytes  (Schmelzer et al., manuscript submitted for publication; Bruce et al., manuscript submitted for publication). Thus, sorting for EpCAM results in only progenitor cells but in distinct ratios of hepatic stem cells to hepatoblasts, depending upon whether the tissue is fetal, neonatal, or adult. In these studies, we analyzed the gene expression profiles in hepatic stem cells and hepatoblasts isolated from fetal, neonatal, pediatric, and adult livers and compared these profiles to those of mature hepatocytes.
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- Materialsand Methods
The gene expression profiles in terms of both RNAs and proteins of two pluripotent human hepatic progenitors, hepatic stem cells and hepatoblasts, have been compared with those of mature human hepatocytes and shown to reveal three distinct patterns: a phenotype for stem cells, a phenotype for mature hepatocytes, and one for hepatoblasts that is a mix of the other two.
The ability to isolate and purify specific subpopulations of human liver cells such as the human hepatic stem cells has been made possible only recently by using immunoselection for a defined antigen such as EpCAM, a transmembrane glycoprotein described by de Boer  and found now to be expressed by hepatic progenitors but not hepatocytes  (E. Schmelzer, L. Zhang, A. Melhem, N.G. Moss, E. Wauthier, R.E. McClelland, A. Bruce, H. Yao, W.S. Turner, N. Cheng, M.E. Furth, and L.M. Reid, manuscript submitted for publication; A. Bruce, L. Zhang, J.W. Ludlow, E. Schmelzer, A. Melhem, M. Kulik, L.M. Reid, and M.E. Furth, manuscript submitted for publication). Subdivision of the hepatic progenitor subpopulations into hepatic stem cells versus hepatoblasts can also be done by culture selection conditions that give rise to clonigenically expanding stem cell colonies (E. Schmelzer, L. Zhang, A. Melhem, N.G. Moss, E. Wauthier, R.E. McClelland, A. Bruce, H. Yao, W.S. Turner, N. Cheng, M.E. Furth, and L.M. Reid, manuscript submitted for publication). The only comparable findings by others are those of Tanimizu et al. [15, 16], who have shown isolation of hepatoblasts from murine and rodent livers by Dlk/Pref-1, a membrane protein with six extracellular epidermal growth factor-like repeats [17, 18].
By immunohistochemical analyses, we have been able to assign EpCAM expression in nonpathologic human livers to hepatic stem cells and hepatoblasts in all developmental stages (i.e., fetal, neonatal, pediatric, and adult livers). Transplantation into the livers of mice of EpCAM+-sorted cells gave rise to human liver tissue expressing human-specific liver proteins (E. Schmelzer, L. Zhang, A. Melhem, N.G. Moss, E. Wauthier, R.E. McClelland, A. Bruce, H. Yao, W.S. Turner, N. Cheng, M.E. Furth, and L.M. Reid, manuscript submitted for publication; A. Bruce, L. Zhang, J.W. Ludlow, E. Schmelzer, A. Melhem, M. Kulik, L.M. Reid, and M.E. Furth, manuscript submitted for publication). Sorting for EpCAM+ cells from pediatric and adult livers resulted in a population mainly comprised of hepatic stem cells, whereas those from fetal and neonatal livers resulted in predominantly hepatoblasts.
We show here that hepatic stem cells from livers at all developmental stages show highly similar expression profiles, defined below as the stem cell phenotype. Phenotypic characterization of gene expression at mRNA and protein levels in pluripotent human hepatic progenitors versus mature hepatocytes indicated two patterns, a stem cell phenotype and a liver-specific phenotype; a gradient from minimal levels was observed in hepatoblasts to maximal levels found in hepatocytes. The stem cell phenotype comprised expression of EpCAM, NCAM, CK19, c-kit, CLDN-3, and weak levels of albumin but no expression of AFP or adult liver-specific proteins such as transferrin, connexins, PEPCK, DPP4, or P450s. The expression of both hepatic (albumin) and biliary (CK19) markers reflects the bipotential nature of the hepatic stem cell toward both lineages of the liver, the hepatic and biliary, which are characterized by albumin and CK19 expression, respectively. In studies published elsewhere, the hepatic stem cells also express components of the Hedgehog signaling pathway, including Indian and Sonic Hedgehog and its receptor, Patched .
The hepatocyte phenotype comprised expression of classic liver-specific genes such as albumin, connexins, transferrin, PEPCK, DPP4, and P450s and loss of expression of the genes typifying stem cells (including EpCAM, NCAM, CK19, c-kit, and CLDN-3). Hepatoblasts proved to be cells in an intermediate position with lowered levels of the stem cell genes and with activation but low levels of adult liver-specific genes and with the unique, defining feature of AFP.
A unique and novel hepatic stem cell surface marker is CLDN-3. Together with occludins, claudins form tight junction proteins  that act as a barrier for the passage of ions and molecules through the paracellular pathway and to the movement of proteins and lipids between the apical and the basolateral domains of the plasma membrane. Claudins comprise a family of more than 20 members (reviewed in ref. ). Morita et al.  demonstrated that CLDN-3 is expressed in mouse lung, liver, kidney, and testis and that the mouse gut shows lineage dependent expression of CLDN-3; liver sections show staining for CLDN-3 in bile canaliculi regions . After partial hepatectomy in rats, CLDN-3 is evident periportally and in sites where regenerative responses are initiated . Combining our results with these previous findings, CLDN-3 is a candidate surface molecule expressed by hepatic stem cells and providing a marker in addition to NCAM, permitting selection uniquely for stem cells.
In summary, this study suggests that the pluripotent hepatic progenitors are evident in both fetal and postnatal livers and remain stable in their phenotypes throughout life, particularly the hepatic stem cells, which persist in relatively constant numbers in livers at all donor ages. The primary changes with age are in the numbers of hepatoblasts that are the dominant cell type in fetal and neonatal livers and then decline in numbers to comprise <0.1% of the parenchyma in normal adult livers. Hepatoblast numbers appear to wax and wane with need for regenerative responses, and the only conditions under which they are observed in high numbers other than in fetal development are in diseased states such as cirrhosis . Their responses suggest parallels with transit-amplifying cells in other tissues.