In vivo localization of EpCAM+ hHpSCs
Sections of fetal and adult livers were stained for EpCAM and for liver-specific markers (albumin, AFP, and CK19; ). We found that ductal plates, bands of tissue encircling each of the portal triads in fetal and neonatal livers, have small cells (7–10 μm) with a paucity of cytoplasm, and stained intensely both cytoplasmically and at the surface for CK19 and EpCAM, and weakly for albumin, but are negative for AFP. In nondiseased, postnatal (pediatric and adult) livers, cells staining positively for EpCAM by immunohistochemistry appear exclusively in the Canals of Hering in the vicinity of the portal triads of acini. Theise et al. (18
) reported that cells lining these ductules express cytokeratin (CK) 19, which is present in biliary epithelia, but not in hepatocytes. Our data confirm their study and demonstrate that the CK19+ cells within the Canals of Hering express EpCAM, and that subpopulations of them also express albumin (; yellow color is caused by overlap of CK19 and albumin expression). The coexpression of CK19 and albumin is consistent with hHpSCs and corroborates the hypothesis of others that the Canals of Hering comprise a niche for hHpSCs (19
). Shown are data from ex vivo studies of EpCAM+ cells supportive of the interpretation that they include hHpSCs.
Figure 1. Immunohistochemical studies on human fetal livers. Confocal microscopic images on 5-μm liver sections. The antigenic profiles are given in the table (bottom left). In human fetal livers, sections were stained for: EpCAM (green) and CK19 (red) (more ...)
Most parenchymal cells of fetal and neonatal livers consisted of hepatoblasts, slightly larger cells (10–12 μm in diameter) that stained positively for albumin, AFP, and CK19. In hepatoblasts, the distribution of CK19 is more particulate and less intense than that in ductal plate cells. EpCAM expression in AFP+ cells, both in fetal and postnatal livers, occurred at the membrane only. In pediatric and adult livers, hepatoblasts were found as individual cells or small clusters of cells tethered to the ends of the Canals of Hering (). The hepatoblasts in nondiseased, postnatal livers constitute <0.01% of cells and express AFP weakly.
Flow cytometry of EpCAM+ cells
Using flow cytometry, we observed EpCAM+ cells in human liver cell suspensions of all donor ages (). Suspensions from fetal livers from which hemopoietic cells had been purged contained, on average, 12% EpCAM+ cells. However, the percentage could be as high as 20% depending on the gestational age of the fetus. Most of the EpCAM− cells in fetal livers were of nonhepatic lineage and were predominantly hemopoietic. The vast majority (>90%) of the EpCAM+ cells in fetal livers coexpress AFP, albumin, and CK19 (). They could be subdivided into two subpopulations: (a) hepatoblasts showing expression of ICAM1, AFP, albumin, CK19, CD133/1, P450A7, and CD44H (hyaluronan receptor), and (b) hHpSCs, constituting ~5% of EpCAM+ cells from fetal livers, had an overlapping, but distinct, profile; they were positive for albumin (weak), CK19, CD44H, CD133/1+, neural cell adhesion molecule–positive (NCAM+), and claudin 3, but negative for AFP and P450A7.
Figure 2. Flow cytometric characterization of EpCAM+ cells. (A) The percentage of EpCAM+ cells in livers of varying donor ages. The numbers for fetal livers have been previously reported (28), but are presented here for comparison to findings in (more ...)
Cell suspensions from adult human livers averaged 1.3% EpCAM+ cells, and those from neonatal and pediatric donors two- to threefold higher. Preparations having EpCAM+ cell populations at levels >1.5% were typically obtained from livers subjected to ischemia (cold or warm) before organ procurement and/or during transportation. This suggests that the EpCAM+ liver cells are more resistant to ischemia than mature liver cells. The postnatal EpCAM+ liver cells also coexpress CK19 and albumin (unpublished data), but have no detectable AFP+ cells (by flow cytometry), except in rare cases of overt hepatic disease (e.g., cirrhosis; unpublished data). Neonatal livers, including some from premature births, showed rapidly decreasing levels of AFP+ cells as a function of age, falling below detection level by a few months after birth. Based on considerations detailed in our study and from our previously published work (17
), we identify the EpCAM+ cells from pediatric and adult livers as almost exclusively hHpSCs, not hepatoblasts.
Culture selection on plastic and in serum-free Kubota's medium (KM) isolates hHpSCs, but not hepatoblasts
Suspensions of liver cells plated in KM, which is a serum-free medium optimized for ex vivo expansion of hepatic progenitors (8
), either on tissue culture plastic or on embryonic stromal cell feeders, yielded parenchymal cell colonies with two distinct morphologies. Type I colonies consisted of cells forming a cordlike morphology interspersed with clear channels and expressing EpCAM, albumin, CK19, ICAM, and AFP, but not NCAM (Figs. S1–S4, available at http://www.jem.org/cgi/content/full/jem.20061603/DC1
). Type 2 colonies consisted of densely packed, morphologically uniform cells, strongly expressing EpCAM, NCAM, CD44H, and claudin 3; weakly expressing or negative for albumin; and negative for AFP and ICAM-1 ( and Fig. S3). We interpret the type 1 colonies as corresponding to hepatoblasts and the type 2 colonies as corresponding to hHpSCs ().
Figure 3. hHpSCs in culture. A–D show a stem cell colony forming at 2 (A), 4 (B), 7 (C), 10 (D), and 14 d (14) in culture on plastic and in KM. Phase contrast coupled with image of cells with staining for NCAM (E and F), CK19 (G and H), EpCAM (I and J), (more ...)
Antigenic profiles of hHpSC and hepatoblasts
In cultures on plastic, by 5–7 d (mean 5.2 ± 1.6 d; maximum number of days), the hepatoblast colonies disappeared. However, if cultured on STO feeders, hepatoblasts survived for up to 2 mo, continuing to show coexpression of albumin, AFP, and CK19 (Figs. S1 and S2). The hepatoblast colonies typically contained fewer than ~100 cells.
In contrast to hepatoblasts, hHpSC colonies on plastic continued to expand. A time-lapse sequence of a growing hHpSC colony (, and Video 1, available at http://www.jem.org/cgi/content/full/jem.20061603/DC1
), in which the expansion of hHpScs seeded at very low density is shown on day 1, 3, and 8. The hHpSCs can be subcultured after mechanical disaggregation and continue to multiply extensively. Their doubling time on plastic is ~36 h. That doubling time decreased to <24 h if they are plated on specific extracellular matrix substrata (unpublished data). By 2–3 wk, hHpSC colonies typically contained many thousands of cells.
The hHpSC colonies were assessed for expression of lineage markers by immunofluorescent staining. The expression pattern closely resembled that of ductal plate cells in vivo. They were positive for CK19, NCAM, EpCAM, and CD44H (). In addition, they were positive for albumin (weak), E-cadherin, N-cadherin, CK8 and 18, CD133/1, integrin β-1 (CD29), claudin 3, and telomerase (unpublished data). They were negative for AFP, any form of cytochrome P450, hemopoietic markers (CD34, CD45, CD38, CD14, CD90, and glycophorin A), endothelial cell markers (vascular endothelial growth factor receptor [VEGFr], von Willebrand factor, and platelet/endothelial cell adhesion molecule or CD31), and mesenchymal markers, such as those for hepatic stellate cells (CD146, desmin, and α-smooth muscle actin). The expression of NCAM by the hHpSCs is important because previous studies have shown that this marker is present on the ductal plate in fetal livers and evident on liver cell populations proliferating under various disease states (20
Immunoselection using EpCAM isolates hepatoblasts from fetal and neonatal livers; immunoselection using EpCAM or NCAM isolates hHpSCs from livers of all donor ages
To enrich for hepatic progenitors from liver cell suspensions, we explored several fractionation strategies, including separation by buoyant density on Ficoll gradients (Table S1, available at http://www.jem.org/cgi/content/full/jem.20061603/DC1
) and by immunoselection. The most satisfactory results were obtained using magnetic immunoselection. Although FACS was able to yield highly purified cellular subpopulations, the shear forces and the use of buffers (PBS) that are not optimal resulted in low yields of viable cells. We used magnetic microspheres conjugated with monoclonal antibody to EpCAM (Miltenyi Biotec) to immunoselect EpCAM+ cells from liver cell suspensions and obtained robust, highly viable sorted cells that survived and expanded well when cultured ( andTable S2). From postnatal livers, up to 10 billion viable cells were processed in a single pass using the CliniMACS apparatus (Miltenyi Biotec). This yielded >100 million EpCAM+ cells. Purity of the enriched EpCAM+ cells was typically 75–90%, and recovery usually exceeded 90%. Representative fractionations of a fetal liver and of a postnatal liver are depicted in . A cell suspension from the liver of a 2-yr-old donor was found to contain 0.7% EpCAM+ cells. The immunoselected population contained 81% EpCAM+ cells, whereas the flowthrough fraction was almost entirely depleted of EpCAM+ cells. The majority of hepatic EpCAM+ cells were of 8–10 μm in diameter, as judged by Coulter Counter analysis, in contrast to 18–20 μm for diploid hepatocytes, which is the predominant population in the initial liver cell suspension. A small peak of presumptive tetraploid cells also is evident, measuring ~25 μm in diameter. Light scatter (“side scatter”) profiles indicate that the EpCAM+ liver cells are considerably smaller and less granular than the bulk of the parenchymal cell population.
Figure 4. Magnetic immunoselection. (A–F) Flow cytometry on human fetal liver cells stained for EpCAM (D; A is the isotype control for D used for setting the gate shown in pink) indicated 20.7% of the cell suspension was positive for EpCAM. The cells were (more ...)
Magnetic immunoselection for NCAM+ cells from fetal livers enriched for cells capable of forming only hHpSC colonies (). The majority of EpCAM+ cells from fetal liver coexpressed NCAM, whereas only ~40% of those from adult liver were also NCAM+. Therefore, sorting for NCAM+ cells proved useful for isolation of hHpSCs from fetal livers, but less so from adult livers. It is unknown at this time whether NCAM and EpCAM coexpression is a definitive property of hHpSCs. An alternative hypothesis, which is currently being tested, is that NCAM is present on angioblasts or other mesenchymal companion cells that are tightly bound to the hHpSCs such that immunoselection for it results in coselection of the two cell types (see later in this paper for more on this theme). Sorts for KDR (VEGFr) resulted mostly in angioblasts (). However, these sorts also yielded an increase in hHpSC colonies caused by, we assume, coselection of hHpSCs and angioblasts.
Proteins and genes expressed by EpCAM+ cells
Immunoselected EpCAM+ cells from fetal and postnatal livers were examined by flow cytometry for expression of lineage markers characteristic of various cell types that reside in the liver ( and and Table S1). As judged by double-label flow cytometry, ~95% of the immunoselected EpCAM+ cells expressed CK19, and comparable percentages expressed albumin and CD133. Evaluations of many preparations indicated that >90% of the EpCAM+ cells are positive for CD133, which was detected with monoclonal antibodies to two distinct epitopes (CD133-1 with monoclonal antibody AC133; CD133-2 with monoclonal antibody AC141). Virtually all CD133-1+ cells in adult liver cell suspensions were found in the EpCAM+-selected fraction, and mature hepatocytes were clearly negative. However, it appeared that ~40% of liver cells with light scatter profiles consistent with mature hepatocytes were positive for CD133-2. Examination by immunofluorescent microscopy showed that staining for CD133-1 clearly outlines cell membranes, whereas that for CD133-2 shows a more diffuse pattern (unpublished data). It is likely that the staining of many more liver cells by CD133-2 results from a known cross-reactivity with CK18 (24
) that is expressed by hepatocytes and can reportedly be found on the cell surface (25
). Based on the more specific CD133-1 antibody, we conclude that EpCAM and CD133 (prominin) are coexpressed by the vast majority of hHpSCs.
NCAM (CD56), which was previously shown to be expressed by glia, muscle cells, and neurons (26
), was found on the majority of hHpSCs derived from fetal and neonatal livers, but only ~40% of the EpCAM+ cells from adult livers. In our prior studies, NCAM mRNA was enriched strongly in EpCAM+ cells from both fetal and postnatal livers, but expression at the protein level was variable (17
). NCAM staining was most evident at the borders of the hHpSC colonies ().
Less than 1% of the enriched EpCAM+ cells stained for the hemopoietic marker CD45 (leukocyte common antigen), which is found on Kupffer cells (tissue macrophages) and lymphocytes in the liver. The EpCAM+ cells were negative for expression of other hemopoietic markers assayed (CD34, CD14, CD38, CD4, CD90, and glycophorin A), for endothelial cell markers (CD34, VEGFr or KDR, von Willebrand factor, and CD31), and for mesenchymal markers, especially those associated with hepatic stellate cells (CD146, also called Mel-CAM, desmin, and α-smooth muscle actin; unpublished data). Finally, we found AFP expression at the RNA and protein levels in EpCAM+ cells from fetal and neonatal livers, but not from pediatric or adult livers. As noted, small numbers of cells weakly positive for AFP, as judged by immunohistochemistry, were observed to be tethered to the ends of the Canals of Hering in sections from pediatric and adult livers (). In our experience, these cells are too few and express AFP too weakly to permit recognition as a defined subpopulation by flow cytometry.
Assessment by RT-PCR of RNA expression in the EpCAM+ liver cells () gave results consistent with the flow cytometry data. Further details of these findings are reported elsewhere (17
). In brief, EpCAM+ selection from fetal livers more than doubled the expression levels of albumin, AFP, and CK19. Immunoselection for EpCAM+ cells from postnatal livers strongly enriched for transcripts encoding EpCAM, CK19, CD133, and CD117 (c-Kit); these transcripts were barely detectable in the EpCAM− cells. AFP transcripts were not detectable in EpCAM+ or EpCAM− cells from postnatal livers.
Although immunoselected cells are enriched for relative expression of CD117 mRNA, we have not observed the corresponding protein by immunostaining of freshly isolated cells from fetal or postnatal livers, or on cultured cells from postnatal livers. However, we have occasionally observed low levels of CD117 staining on cells at the periphery of hHpSC colonies from fetal livers that are located in regions where hHpSCs overlap with mesenchymal companion cells.
Cytochrome P450 3A4 (CYP3A4), which is a protein expressed by mature hepatocytes, was not found at all in parenchymal cells, either EpCAM+ or EpCAM−, from fetal livers in terms of both mRNA and protein level of it. The level of mRNA for cytochrome P450 in EpCAM+ cells was 20-fold lower relative to EpCAM-negative cells from postnatal livers. The small amount of CYP3A4 RNA in the EpCAM+ cell fraction from postnatal livers could be accounted for by residual hepatocyte contaminants. In contrast, the hepatoblasts, but not the hHpSCs, were found to express P4503A7, which is a protein found in fetal livers (unpublished data). EpCAM+ cells from postnatal livers also showed eightfold lower relative expression of albumin RNA than the flow-through (EpCAM-negative) population. Again, some transcripts can be attributed to incomplete removal of hepatocytes. However, the detection of albumin protein in EpCAM+ cells by flow cytometry, together with the transcript data, demonstrates that these progenitor cells express the albumin gene, albeit at a significantly lower level than differentiated hepatocytes.
Assays for telomerase activity indicate significant levels in freshly isolated EpCAM+ cells from livers of all donor ages and in cultures of colonies of both hHpSCs and hepatoblasts. Full characterization of telomerase activity and its regulation in various fractions of human liver cells from fetal and postnatal donors is presented elsewhere (unpublished data), as are studies on the effects of purified matrix substrata on telomerase activity in cultures of hHpSCs (unpublished data).
Ex vivo clonogenic expansion: evidence for self renewal
Colony formation by committed hepatic progenitors or diploid adult parenchyma involves a limited number of divisions (typically 5–7 divisions) over a relatively short period of time (2–3 wk) (8
). In contrast, self-renewal involves clonogenic expansion that can go on for >100 population doublings with phenotypic stability, which are properties associated with stem cells. We previously found that rat hepatoblasts multiply far more extensively in KM with STO feeder cells than on tissue culture plastic (8
). However, STO feeders and KM were not permissive for clonogenic expansion of human hepatoblasts. Under these conditions, the hepatoblasts survived for a few months, but demonstrated limited growth. In contrast, hHpSCs from livers of all donor ages could undergo clonogenic expansion for >6 mo (>150 population doublings) in culture on tissue culture plastic and in KM with only the native feeders (the companion cells) (). The cells maintained phenotypic stability as assessed by morphology and by antigenic and biochemical profiles ( and ). hHpSC colonies starting from 1–3 cells (Videos 1–3, available at http://www.jem.org/cgi/content/full/jem.20061603/DC1
) grew to cover 4.9 ± 0.3 mm2
in area and contained an average of 1,400 ± 520 cells (3 independent counts of total cells from 50 dispersed colonies). Thus, the cells in this representative experiment had gone through 10–11 population doublings in 2 wk, corresponding to an average doubling time of 31–34 h (Table S2).
Mesenchymal companion cells provide critical paracrine signaling for hHpSCs
The tightly packed colonies of hHpSCs have a prominent ridge at the perimeter (, , and ) at which we have identified mesenchymal companion cells (). As the colonies grow, the companion cells penetrate the colonies and are found throughout them. Time-lapse movies (Videos 1–3) reveal a boundary zone between the companion cells and the hHpSCS in which the companion cells fluctuate back and forth, touching the edge of the hHpSC colony, or traversing it and moving below the colony. When removed from a culture dish, the attachment to the plastic surface is evident only at the edge of the colonies, not in the center. This suggests that attachment to the plastic is mediated either by mesenchymal cells or by cooperative interactions between the hHpSCs and the mesenchymal cells.
Figure 6. hHpSCs shifted to STO feeders erupt hepatoblasts. Passage of hHpSCs from plastic to STO feeders results in cordlike eruptions that morphologically and antigenically are identical to hepatoblasts. (A) An hHpSC colony shortly after passaging. (B–E) (more ...)
Figure 5. Companion cells to the hHpSC colonies comprise hepatic stellate cells and angioblasts. hHpSCs are associated with mesenchymal companion cells with distinct antigenic profiles (Videos 1–3). Two types of companion cells are evident: angioblasts (more ...)
Phenotypic analyses of the companion cells indicates at least two distinct populations: angioblasts (VEGFr+ or KDR, CD133/1+, CD117+, Von Willebrand factor, CD31weak); and hepatic stellate cells (desmin+, α-smooth muscle actin+, CD146+) (). A comparison of their morphological and antigenic phenotypes is given in . Cells rigorously purified away from the companion cells (by repeated immunoselection for EpCAM+ cells) did not survive on culture plastic, but only on STO feeders (unpublished data). Immunoselection of CD117+ cells yielded angioblasts, but neither hepatic stellate cells nor hHpSCs (unpublished data). Immunoselection for other markers found on the companion cells (VEGFr) resulted mostly in selection of the companion cells alone, though we did find coselection for hHpSCs to occur at low and variable frequency (). We still cannot rule out that the consistent enrichment of hHpSCs from fetal liver by immunoselection for NCAM could actually result from coselection of the stem cells via tight association with NCAM+ companion cells.
Proof of pluripotency of hHpSCs
Passaging (transfer) of colonies of hHpSCs (whether derived from fetal or adult livers) from culture plastic onto feeder layers of STO cells resulted, within hours, in eruption of hepatoblasts from the periphery of hHpSC colonies ( and ; ). After the transfer, the morphology and antigenic profile of the cells within the hHpSC colonies proper did not change in most of the cells, although there were occasional cells with distinct gene expression within the colony (). Instead, the colonies of hHpSCs gave rise to cordlike eruptions from their edges, yielding cells with morphology, antigenic, and biochemical profiles identical to that of hepatoblasts. The cells in these erupted areas strongly expressed AFP, ICAM-1, and albumin, and were positive for cytochrome P450-A7 (not depicted), but were negative for NCAM (). In addition, committed biliary progenitors were sometimes observed erupting from a colony of hHpSCs, as shown by staining for CK19, but not for albumin ().
Figure 7. Shift in antigenic profile from hHpSCs to hepatoblasts when on STO feeders. (A and B) The border between the hHpSC colony and hepatoblast outgrowths is marked by arrowheads. (C and D) The antigenic profile of the cords of cells erupting from the parent (more ...)
In cultures of cells from postnatal livers, in colonies stained by double-label immunofluorescence for CK19 and albumin, we have observed distinct sectors positive for one or the other marker, but not both (Fig. S4). This was found most frequently in colonies of hepatoblast morphology. We interpret such sectors as deriving from unipotent cells, corresponding to committed progenitors for biliary and hepatocytic lineages, respectively. The sectoring could occur if at division a bipotent cell gives rise to a daughter cell that was restricted to the biliary or to the hepatocytic lineage. Occasionally, small colonies showed expression of only one of the lineage markers (CK19 or albumin); these colonies are assumed to have arisen from committed progenitors for the corresponding cell type.
EpCAM+ cells and colonies of hHpSCs give rise to human liver tissue in vivo
Transplantation of freshly isolated EpCAM+ cells or of hHpSC colonies, from either fetal or postnatal livers, into livers of NOD/SCID mice resulted in engraftment and the formation of human liver tissue (). Islands of cells staining positive for human albumin, CK19, and AFP were found within 2 d of transplantation (), and they persisted within the livers for weeks (). The extent of engraftment and expansion of human liver cells in vivo was enhanced by treatment of the mice with carbon tetrachloride (CCL4), which is a poison for the pericentral zone of the liver acinus and is often used to create a cellular vacuum in transplanted hosts (). Human-specific DNA sequences were found in the liver of animals that received the human cell transplants, but not in other tissues or in control animals that did not receive human cells (unpublished data). Before transplantation, as expected, the cells were shown to express EpCAM and CK19 strongly and albumin weakly, but were negative for AFP at both the RNA and the protein levels. The liver sections from mice transplanted with hHpSCs contained cells strongly expressing human-specific forms of albumin, CK19, and AFP, but were negative for EpCAM. However, human cytochrome P450 3A was not detectable. Therefore, it appears that after transplantation and expansion in recipient livers, the human cells lost expression of a marker (EpCAM) found only on stem and progenitor cells, and, acquired some, but not yet all, of the functions specific to mature hepatocytes. We think it logical that transferrin, but not P450 3A, is expressed in transplanted cells given that in mature liver, transferrin is expressed by zone 2 parenchymal cells, whereas P450 3A by zone 3 parenchymal cells within the acinus. Thus, P450 3A is a late gene produced by cells at the end of the liver's maturational lineage.
Figure 8. Transplantation of EpCAM+ cells (or colonies of stem cells in culture) results in engrafted liver tissue in NOD/scid mice. NOD/scid mice were transplanted with 106 cells of either freshly isolated and immunoselected EpCAM+ cells or colonies (more ...)
As an independent test of engraftment, we assessed expression of the human transferrin gene, encoding a protein characteristic of mature hepatocytes. We found by quantitative RT-PCR analysis with human-specific primers that the livers of mice sampled 1 wk after injection of the EpCAM+ cells derived from postnatal human livers contained significant levels (2,100 ± 1,140 strands/100 ng) of human transferrin RNA. Such sequences were undetectable in RNA from livers of control mice (<100 strands/100 ng RNA). Although before transplantation >80% of cells in the test cell population expressed EpCAM and CK19, cells in recipient animals were positive for human albumin and were negative for both of the progenitor cell markers. Collectively, the data suggest that within 7 d in vivo, the engrafted hHpSCs gave rise to mature human liver cells.