Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver

Abstract

Recent investigations have reported many markers associated with human liver stem/progenitor cells, “oval cells,” and identified “niches” in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as “micro-niches.” Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease.

Introduction

The human liver is a highly specialized structure with an exquisitely organized cellular composition that is required to carry out functions essential to life. ¹ While hepatocytes constitute the majority of liver cells, these are dependent upon other cell types, including liver sinusoidal endothelial cells, hepatic stellate cells, and Kuppfer cells, for hepatic homeostasis. ² Persistent cycles of chronic liver injury and repair commonly lead to fibrosis, nodule formation, architectural reorganization, and cirrhosis, along with novel cell clones, including altered liver function, genetic changes and susceptibility to cancer.^3,4 Under these circumstances, hepatic stem/progenitor cells with oval shaped nuclei (designated, “oval” cells) may proliferate in localized areas.^5–14 At the histological level, cirrhotic livers display a diversity of parenchymal cell morphology, including small and large hepatocytes, as well as, additional epithelial cell types in ductular or periductular areas.^15–21 Some of these cells may include adult stem/progenitor cell populations. The canals of Herring in periductular areas have been identified as a source of progenitor cells in adult human liver.^22–28 Stem/progenitor cells in fetal human liver are found within the hepatic acinus even before ductal plate formation or biliary morphogenesis, followed by persistence of such stem/progenitor cells in ductal structures of the adult human liver.^15,25,29,30

To better understand the cellular mechanisms of hepatocyte and bile ductular repopulation of severely diseased human liver, distinct sets markers for stages of differentiation of stem/progenitor cells are needed. In a previous study, we used a novel triple staining approach to identify cell lineages in regions of oval cell proliferation. ³¹ This demonstrated specific “bipolar” structures containing both bile ductular and hepatocytic lineages. We found proliferating oval stem/progenitor cells were positive for neural cell adhesion molecule (NCAM), and these cells progressed along the biliary lineage leading to cytokeratin 19 (CK19)-positive, NCAM-negative mature bile duct cells. Cells progressing to the hepatocyte lineage were also identified by HepPar1 staining. However, in that report, we showed that the step immediately preceding final hepatocytic differentiation was marked by proliferation of “triple negative” cells that lost all the tested markers. This left need to identify a biochemical marker for these cells in the differentiation lineage.

As hepatic differentiation in pluripotent stem cells first recapitulates the fetal stage, we considered whether hepatocyte repopulation in severely cirrhotic end-stage livers would hold parallels with the process of fetal liver development. For instance, during the early stages of commitment and specification of hepatic stem/progenitor cells in mice, expression of the multifunctional transcription factor, FOXA2 (Forkhead box A2), is established as a pioneer and competency factor for the endodermal lineage that is required for later stages of hepatic specification.^32,33 Similarly, FOXA2 is expressed early during fetal liver development in humans and animals^34,35 and serves roles in directing lineages in the mature liver, including those involved in liver cancer. ³⁵ As FOXA2 is not expressed in mature hepatocytes or bile duct cells, we reasoned that FOXA2 might represent an excellent marker of early stage liver stem/progenitor cells, including the triple-negative cells. ³¹ Therefore, we considered whether FOXA2 expression characterized only triple-negative oval stem/progenitor cells defined above, or only lineage committed oval stem/progenitor cells in bile ductular regions or both cell types.

In a recent review, ²⁸ the liver stem cell niche is examined in great detail, and structures are identified in severely diseased livers that greatly resemble those in this report. Two markers of stem progenitor cells that were reviewed and identified in ductular structures in the diseased livers were CK7 and Sox-9. ²⁸ In pluripotent stem cells, additional markers are expressed before hepatic lineage specification and commitment, for example, OCT4, stage-specific embryonic antigens (SSEAs),^36,37 and thus, we incorporated OCT4 expression analysis in our studies. Moreover, we examined whether specific microRNAs (miRNA), which play roles in liver differentiation,^38,39 could be identified within stem/progenitor lineages or in adjacent cells to illuminate further differentiation mechanisms. The combination of markers we used, FOXA2, HepPar1, Albumin, and CK19, and miRNAs, have enabled us to identify distinct hepatocytic and bile ductular stem cell lineages in defined “micro-niches” in severely diseased human livers. Furthermore, we determine that FOXA2 is a marker for the previously identified triple-negative cells. In a final figure, we present a unified flow diagram of illustrating the cell lineage relationships that are supported by our data.

Materials and Methods

Specimen Collection

Diseased liver specimens were collected as part of a liver transplant program. All specimens were collected under institutional review board (IRB) approval from Albert Einstein College of Medicine and were obtained from diseased explanted livers. All three specimens reported in this study were diagnosed as described in Table 1.

Table 1.

Description of Liver Tissue Used in This Study.

Age	Sex	Clinical Diagnosis	Presumed Etiology	Therapeutic Intervention
66 year	M	Chronic hepatitis C infection	Hepatocellular carcinoma	Lobectomy
37 year	F	Portal cirrhosis	Methyldopa-related sub acute liver failure	Transplant
10 months	M	Biliary cirrhosis	Biliary atresia	Transplant

Immunostaining

The liver specimens were fixed in neutral formalin embedded in paraffin and sectioned at 5 µm intervals. Routine hematoxylin and eosin (H&E) stained slides were made for all specimens. Detailed description of double and triple immunostaining is in Supplementary Methods. Briefly, immunostaining was performed with goat anti-human mouse monoclonal antibodies against HNF3b/FOXA2 (1:100, AF2400; R&D Systems, Minneapolis, MN), human CK19 (1:50, SAB4700775; Sigma, St. Louis, MO), human HepPar1 (1:35, M7158; Dako, Glostrup, Denmark; Carpenteria, CA) or human Albumin (1:70, A6684, Sigma). Secondary antibodies and details of color development are as previously reported ³¹ and in “Supplementary Methods.” In regard to color development in this report, HepPar1 was always detected as blue staining and CK19 as red staining. FOXA2 staining was grey after single staining, however, after CK19 and HepPar1 staining in our triple staining protocol, the grey staining darkened to become light to dark brown as shown in the figures in this report. For the double staining, the color of FOXA2 was light to dark brown and Albumin was reddish-orange. To estimate nonspecific binding of monoclonal antibodies, parallel staining was carried out using appropriate isotype controls listed below. Control antibodies and sera used were as follows: Normal Goat IgG control (1:100, AB-108-C; R&D Systems) was used as a control for the anti-HGF3b/FOXA2 antibody. Mouse IgG2a isotype control (1:50, 02-6200; Invitrogen, Carlsbad, CA) was used as a control for the anti-CK19 and anti-Albumin antibodies. Mouse IgG1 isotype control (1:35, 02-6100, Invitrogen) was used as a control for the anti HepPar1 antibody.

Criteria for Categorization of Structures

The general guidelines for categorizing Ductular Reactions and oval/stem progenitor cells are in our previous report. ³¹

In Situ Hybridization

Detection of miRNA transcripts for miR-122, miR-23b cluster miRNAs (miR-23b, miR-27b, and miR-24), and miR-125b was carried out using in situ hybridization using the method previously described. ³⁸

Results

Standard Histological Analyses Identify Regions of High Bile Ductular Proliferation in Decompensated End-stage Livers

Patients reported herein range in age from 66 years to 10 months old. As shown in Table 1, the patients liver disease had a range of etiology, including chronic hepatitis C virus with cirrhosis, bridging fibrosis, ductular proliferation, and hepatocellular carcinoma that typically evolves over decades of low-grade chronic liver injury. A second patient had portal cirrhosis and acute liver failure due to alpha-methyldopa that causes massive destruction of mature hepatocytes calling for a large degree of hepatocyte proliferation to replace lost liver parenchyma. The third patient was a child with severe biliary atresia needing a liver transplant. All the livers had the common feature of heterogeneous portal cirrhosis and periportal fibrosis, bridging septae and parenchymal nodule formation. Regenerative nodules were present in all, along with marked and extensive ductular proliferation and loss of native duct structures in portal areas. Thus, the findings in this study are not limited to a specific etiology of liver disease but are the result of severe chronic liver disease with extensive ductular and stem/progenitor, “oval” cell proliferation that are general characteristics of livers needing transplant.

Our working hypothesis was that in highly decompensated end-stage livers, a survival response occurs via recruitment of stem/progenitor cells to replace lost hepatocytes. In livers with unusually high levels of bile ductular and oval cell proliferation, histological analysis using trichrome, reticulin, CD45, and H&E staining (Fig. 1) confirmed presence of hepatic nodules with reorganized acinar structure. Also, we noted enlarged areas containing disorganized bile ductular elements embedded within fibrotic matrix along with cells of highly variable morphology and inflammatory cells. These histological changes included classic “oval” cells in ductular areas and loosely identified “transitional” hepatocytes with large, rounded hepatocyte-like nuclei and scant cytoplasm, as well as typical mature hepatocytes. We considered this was in support of stem cell proliferation and differentiation in the livers; however, standard histological analysis was insufficient for discerning lineage relationships from within the diversity of cell types observed.

Figure 1.

(A) Hematoxylin and eosin (H&E) staining of cirrhotic liver revealing candidate newly differentiated hepatocytes originating from a cluster of oval cells. Scale bar = 10u. (B) Trichrome stain of cirrhotic liver revealing transitional hepatocytes embedded in a fibrotic matrix. Scale bar = 10u. (C) Reticulin staining of cirrhotic liver revealing disorganized hepatic sinusoids. Scale bar = 100u. (D) CD45 staining of cirrhotic liver revealing the presence of inflammatory cells scattered throughout the liver, some of which are adjacent to oval cells. Scale bars: A and B = 10u; C and D = 100u.

Identification of Hepatocytic Differentiation in “Micro-Niches” in Highly Decompensated Cirrhotic Livers

Tissue staining for FOXA2 (brown) in combination with HepPar1 (blue) selectively identified “micro-niches” in cirrhotic human livers (Fig. 2). In the context of this report, we consider a “micro-niche” to be a group of ~100 to ~1000 cells adjacent to each other that are organized into a discernable structure containing cells with different differentiation states. The micro-niches that we describe are generally bordered by bridging fibrosis in highly cirrhotic diseased livers. The model we suggest is that these micro-niches are the sites of active oval/stem progenitor cell differentiation. For instance, a low magnification view of cirrhotic liver revealed candidate micro-niches bordered by bridging cirrhosis, these micro-niches contain cells at various degrees of hepatocytic differentiation (Fig. 2A). Many micro-niches contained a mixture of brown-stained FOXA2 cells and blue-stained hepatocytes (Fig. 2A, single asterisk). Other micro-niches contained predominantly blue-stained hepatocytes (Fig. 2A, marked by two red asterisks). This suggested that oval stem/progenitor cells progressing along the hepatic lineage were supported by unique and unknown signaling mechanisms within “micro-niches” that promoted stem cell differentiation.

Figure 2.

(A) Section from cirrhotic liver containing two micro-niches with increasing hepatocyte predominance. Forkhead box A2 (FOXA2) = Brown stain. HepPar1 = Blue stain—hepatocyte marker, *Micro-niche with mixture of FOXA2-positive stem/progenitor cells (brown) and hepatocytes (blue). **Micro-niche that is predominantly hepatocytes (blue). This section contained very weak cytokeratin 19 (CK19) staining. (B) Higher magnification of a micro-niche from Fig. 1A, that contains a mixed population of weakly FOXA2-positive proliferating stem/progenitor cells (*, light brown stain) plus highly FOXA2-positive primitive ductular structures (**, dark brown stain) and individual cells within FOXA2-positive ductular structures, the blue stain indicates hepatocytic differentiation (***, blue HepPar1 stain). The newly differentiated hepatocytes initially remain highly FOXA2-positive. (C) FOXA2-positive duct structures (brown) with increasing hepatic differentiation (blue) and distinct cord structures. Mixture of hepatocytes with high- or low-FOXA2 levels is present. (D) Hepatocyte nodule with distinct cord structure arising in region containing hepatocyte birthing zones. Scale bar = 100u.

The frequency of micro-niches, identified as above, varied extensively from case to case. In the case shown in Fig. 2A, micro-niches were extensive, and at least one micro-niche was identified in 18 of 20 low-power fields. The degree of ductular proliferation, hepatocytic, or bile ductular differentiation varied extensively.

An example of a micro-niche that contained a mixture of differentiating cell types, as defined by FOXA2 and HepPar1 staining, is shown in Fig. 2B. Three key histological features of this micro-niche include, first, a large cluster of weakly FOXA2-positive oval cells that were highly disorganized (Fig. 2B; cells marked by single red *). These regions with FOXA2-positive cells did not contain stem cells with pluripotent capacity, as indicated by absence of OCT4 staining, whereas pluripotent human embryonic stem cells expressed OCT4 (data not shown). Second, within this micro-niche, selected oval cells became very highly FOXA2-positive (strong brown nuclear staining) and were simultaneously organized into primitive duct-like structures (Fig. 2B; cells marked by double red asterisks). Third, the next differentiation phase was, apparently, a cell autonomous, single step, differentiation into hepatocytes of individual highly FOXA2-positive cells in ducts (Fig. 2B; cells marked by triple red asterisks). Remarkably, the number of hepatocytes in the primitive duct structures increases until the entire duct structure is composed of newly differentiated hepatocytes (Fig. 2C). Subsequently, these structures progress to bona-fide hepatocyte nodules with a disorganized cord-like structure (Fig. 2D). The cell differentiation mechanisms driving this process are unknown; however, the histological characteristics suggest the possibility of paracrine signaling within ductal structures, possibly driving epigenetic changes that establish a stable hepatocytic phenotype.

Higher magnifications of the above, “newly born,” hepatocytes present in the micro-niche ductular structures described above are shown in Fig. 3A to D. Distinctive features of these hepatocytes included enlarged HepPar1 positive (blue) cytoplasm and round nuclei typical of fully differentiated hepatocytes (Fig. 3A and B). Importantly, hepatic differentiation was observed exclusively within the high-expression FOXA2-positive duct-like structures, and it involved sequential side-by-side cells “popping” into a hepatocytic phenotype until the entire duct structure is hepatocytes (Fig. 2B and 3A–D). In some cases, we visualized the complete progression of hepatic lineage (Fig. 3B). We identified cells with weak HepPar1 staining adjacent to those with strong staining, suggesting newly formed hepatocytes adjacent to mature hepatocytes (Fig. 3C and D). Such newly arising hepatocytes formed cord structures with retention, at least initially, of strong FOXA2 expression.

Figure 3.

(A) Newly differentiated hepatocyte (blue) within a strongly Forkhead box A2 (FOXA2)-positive (brown) primitive ductular element. Scale bar = 10u. (B) Cellular lineage of highly FOXA2-positive stem/progenitor cells (dark brown) leading to hepatocytic differentiation (blue). Scale bar = 10u. (C) Dramatic example of three newly committed hepatocytes (HepPar1, light blue) clearly deriving from strong FOXA2-positive (brown) stem/progenitor cells. Scale bar = 10u. (D) Newly differentiated hepatocytes proceeding from low- to high-level staining for hepatocyte marker HepPar1 (blue). Scale bar: A, B, and C = 10u; D = 100u.

To link FOXA2 expression directly with another hepatocyte differentiation marker, we double stained for FOXA2 (dark brown) and Albumin (red-orange). This again identified weakly FOXA2-positive oval cells in primitive ductular structures (Fig. 4A, single red asterisk) and highly FOXA2-positive oval cells in more complex ductular structures (Fig. 4B, double red asterisk). Higher magnification views of a highly FOXA2-positive oval cell cluster (Fig. 4B, double red asterisk) clearly revealed the enlarged cytoplasm and altered nuclear phenotype (“popping”) of cells within the duct structure becoming Albumin positive hepatocytes (Fig. 4C, triple red asterisk). Fully differentiated hepatocytes that are weakly FOXA2-positive and Albumin-positive were also in proximity to the duct structures, indicating lineage progression as viewed by a lower magnification (Fig. 4D).

Figure 4.

(A) Low magnification of cirrhotic liver section double stained for Forkhead box A2 (FOXA2) (dark brown) and Albumin (orange/red). Weakly (*) and strongly (**) FOXA2-positive stem/progenitor cells and differentiated hepatocytes (***). (B) Higher magnification of section of A. (C) Strongly FOXA2-positive duct structure (**) with Albumin-positive hepatocytes (***) emerging from one end of the structure. (D) Low magnification of double stained section. Albumin = red-orange, FOXA2 = brown/black. Scale bar = 100u.

Micro-Niches Supporting Predominantly Bile Ducts

In the examples above, we described micro-niches where hepatocytic differentiation predominated over biliary differentiation. Here we describe micro-niches in which bile duct formation (marked by red, CK19 expression), and not hepatocyte differentiation, are essentially the sole differentiation type of a large cluster of FOXA2-positive oval cells (Fig. 5A–C). In the micro-niche shown in Fig. 5A, weakly FOXA2-positive oval cells are found immediately adjacent to highly FOXA2-positive duct-like structures (as in Fig. 1 and 2). However, in this micro-niche, virtually all further differentiation was toward the bile ductular lineage as seen by the strong CK19 staining of the duct structures. In this case, the triple staining only identified a single “lone” hepatocyte, marked with blue HepPar1 staining (Fig. 5A). In another example, a micro-niche containing a large cluster of FOXA2-positive oval cells was exclusively associated with CK19 positive ductular elements (Fig. 5B). The final stage of this lineage is fully differentiated, CK19 positive, FOXA2-negative bile ducts (Fig. 5C).

Figure 5.

(A) Forkhead box A2 (FOXA2)-positive ductular elements (brown) differentiating predominantly toward the bile duct cytokeratin 19 (CK19)+ (red) lineage. *Weakly FOXA2-positive stem/progenitor cells. **Strongly positive FOXA2 primitive ductular structures. ***Strongly FOXA2-positive ducts differentiating along the bile ductular lineage as marked by CK19 red stain. ****A “lone” hepatocyte deriving from a highly FOXA2-positive ductular structure. (B) Increasingly higher magnification of area containing FOXA2-positive stem/progenitor cells (*) where surrounding cellular milieu almost exclusively drives bile ductular differentiation (**) with only a few isolated cells becoming hepatocytes (excepting the independent large hepatocyte nodule immediately adjacent to the oval cell micro-niche). (C) Differentiated bile ducts (CK19, red) that become FOXA2 negative (clear nuclei). Scale bar = 100u.

Although the biochemical mechanisms determining cell fate in these highly specialized micro-niches is unknown, the cell–cell contact between cells in the duct structures may play an important role in cell fate determination. In the above case, cell–cell contact was clearly not sufficient, and a single hepatocyte was “born” within the mileu of ductular differentiation, perhaps suggesting that the hepatocyte pathway is the “default” mechanism that must be overcome or inactivated to obtain the ductular phenotype. Such a mechanism could be controlled by activating the TGFβ pathway for ductular differentiation and inactivating it for hepatocytic differentiation. ³⁸

Micro-Niches Supporting Both Hepatocytes and Bile Ducts

Another type of micro-niche included cells with progression to both hepatic and biliary lineages. Increasing magnification of this type of micro-niche revealed a distinct “bipolar” arrangement of cells with differentiation markers. Thus, the micro-niche was characterized by emergence of hepatocytes from one side (left) and of bile duct cells from another side (right; Fig. 6A). In all such cases, weakly FOXA2-positive oval cells were present in regions that generated differentiated structures (Fig. 6B). Also, highly positive FOXA2 ductular structures were present within the cluster of weakly FOXA2-positive oval cells (Fig. 6C), as shown in other micro-niches that yielded either hepatocytes or bile ductular cells.

Figure 6.

(A) Low magnification of a stem/progenitor micro-niche supporting both hepatocytic differentiation to the left (brown = FOXA2, blue = HepPar1) and bile ductular differentiation to the right (red-cytokeratin 19 [CK19]). Note mixtures of cells differentiating in either direction extending out from the center of micro-niche. (B) and (C) Two additional examples of micro-niches with oval cell proliferation and differentiation toward hepatocytic or bile ductular differentiation. Same color scheme with slightly different color intensities. Scale bar: B = 100u; C = 10u.

Differential Expression of Hepatocyte Specific miR-122 in Selected Primitive Ductular Structures

The miRNA, hsa-miR-122 is a molecular marker for fetal and adult hepatocytes.^37–40 Moreover, miR-122 expression may advance hepatic differentiation in stem cells. ⁴¹ Therefore, to further characterize the hepatocytic nature of cells in micro-niches, we used in situ hybridization for miR-122. We observed miR-122 expression in some primitive duct-like structures (Fig. 7A, single asterisk), while other structures in the same micro-niche were negative for miR-122 (Fig. 7A, double asterisk). This served as an internal negative control and was consistent with progression to hepatocytic commitment within selected cells in the FOXA2-positive duct structures.

Figure 7.

(A) In situ hybridization for hsa-miR-122 (black speckle staining) over primitive ductular structures in cirrhotic liver. Signals for miR-122 are localized to cytoplasm, and nuclei are negative. Included are ductular structures with high (*) or low (**) expression of miR-122 revealed by intensity of blue-black color (see “Materials and Methods”). (B) In situ hybridization for miR-122 identifies positive duct structures (*) adjacent to strongly positive hepatocytes (**). Blue-black signals marking miR-122 are cytoplasmic. Nuclei are negative as expected for miR-122. (C) In situ hybridization detecting a mixture of miRs-23b, 27b, and 24 (all from the miR-23b polycistron) over a primitive ductular structure. Scale bar = 100u.

Many of the miR-122 positive ductular structures (Fig. 7B, single asterisk) were immediately adjacent to fully differentiated hepatocytes that expressed miR-122 to a higher level, as judged by the density of hybridization over the cytoplasmic regions of the hepatocytes (Fig. 7B, double asterisk).

miRNAs from the miR-23b gene cluster (that comprises miRNAs 23b, -27b, and -24) are expressed in mouse hepatoblasts and are able to promote hepatocytic differentiation in murine liver stem cells. ³⁸ Therefore, we looked for their expression in the micro-niches. Again we detected specific expression of the miR-23 cluster miRNAs in ductular structures in the micro-niches as expected for stem cells undergoing cell fate commitment (Fig. 7C). The presence of cells without miRNA expression in proximity with miRNA expressing cells again verified the specificity of in situ hybridization signals, and control sections incubated without probes were negative (not shown).

Discussion

Chronic liver disease has multiple etiologies that may lead to severe bridging fibrosis or cirrhosis requiring liver transplantation. In these extreme situations, replacement of lost hepatocytes requires activation of the liver stem/progenitor cell compartment, but key steps in this process remain unknown. We endeavored to understand changes during differentiation of putative liver stem/progenitor cells into hepatocytes and bile duct cells. For this, we used a multi-marker approach comprising detection of FOXA2, HepPar1, Albumin, CK19, and miRNA expression for the identification of differentiation within stem cell lineages.

A foundational finding from this approach was presence of micro-niches in cirrhotic livers that contained clearly identifiable cell lineages. These micro-niches also displayed distinct organization in regard to the polarity of differentiated cell markers and the abundant presence of what we describe as primitive ductular structures also referred to as “reactive ductules” in other reports. ²⁸ The structures we have reported are very similar to structures recently reviewed in a report that focused on the hepatic niche and the presence of Hepatic Stem Cells (HpSCs) in “reactive ductules.” ²⁸ In Fig. 4A of that report, HpSCs that are CK7+ and Sox9+ are identified at the edge of reactive ductules. These reactive ductules are most likely equivalent to the high FOXA2-positive ductules we have identified (Fig. 2, 3, and 5). Because these structures were not co-stained for a hepatocyte marker, such as HepPar1, it is not possible to identify whether some of the reactive ductules contain hepatocytes. Interestingly, there are large numbers of cells that are not identified by any marker in the center of the structures (only blue nuclei identified). According to our data summarized below and in Fig. 8, the negative cells in their structures are most likely the low FOXA2 proliferating oval cells that we identify in our micro-niches (Fig. 8-2). Thus, our use of the FOXA2 marker for oval cells and primitive duct cells, in combination with HepPar1, marking cells on the hepatocyte lineage, and CK19 marking cells on the bile ductular lineage, has enabled us to identify the two distinct cell lineages in stem cell niches.

Figure 8.

Composite illustration of cell lineage pathways in liver “micro-niches” revealed through triple staining described in this report. Images are taken directly from cells in Fig. 1 to 6 that demonstrate the full cell lineages present in micro-niches. Sizes are referred to in the original figures.

The histological characteristics of the micro-niches we report, in combination with the distinct marker patterns that are clearly evident in Fig. 2A–D, support a cellular model of hepatocyte and bile duct replacement. Figure 8 is a composite illustration of the cellular structures and their biochemical markers that define the bi-directional differentiation on micro-niches. The illustration begins with small, weakly FOXA2-positive oval cells that proliferate in isolated micro-niches in cirrhotic liver (Fig. 8-1 and -2). Then, within the oval cell milieu, some oval cells are induced to express high levels of FOXA2, and this is associated with their organization into primitive ducts (Fig. 8-3). These cells are bipotent and can be induced toward the bile ductular or hepatocytic lineages. Differentiation toward the hepatocytic lineages is initially observed when individual cells within the FOXA2-positive ducts become HepPar1 positive (Fig. 8-7). Furthermore, the prevalence of the HepPar1 positive, hepatocyte committed cells apparently spreads due to cell-to-cell contact until the entire ductular structure is HepPar1 positive (Fig. 8-8). Hepatocyte nodules arising from the micro-niches initially exhibit very distinctive duct-like structure, and FOXA2 expression is turned off since cell fate has been established (Fig. 8-9). The second branch of the cell fate differentiation begins when cells in Fig. 8-3 initiate expression of CK19 and progress down the bile duct pathway (Fig. 8-4 and -5). Once in this pathway, CK19 expression increases, and FOXA2 expression is extinguished in fully differentiated bile ducts (Fig. 8-6).

Our data support the notion that key cell fate decisions occur within the context of high FOXA2 expression in duct-like structures present in micro-niches. We note the balance between the two lineages describe above varies in different micro-niches, and we do not, at present, know the signaling mechanisms that regulate the balance in one direction or the other. However, prior work suggests that the TGFβ pathway may play a fundamental role. ³⁸ Thus, selective activation of the TGFβ signaling response in bipotent cells (Fig. 8-3) pushes them toward bile ductular differentiation. ³⁸ In contrast, inactivation of the TGFβ pathway occurs by down-regulation of Smads. miRNAs of the miR-23b polycistron, including miRs-23b, 27b, and 24, have been shown to target Smads, ³⁸ and these miRNAs are expressed in the appropriate cells undergoing cell fate decisions shown in Fig. 7 of this report. Thus, miRNAs cited above will undoubtedly be relevant for cell fate decisions in micro-niches.

The examples we report are from extensively compromised livers with end-stage disease requiring liver transplantation for salvage. However, such processes may occur on a smaller scale in other stages of liver injury during natural attempts at regeneration of hepatocytes and bile duct cells. Thus, different outcomes of cell differentiation in various micro-niches in cirrhotic liver is most likely affected by diverse signaling molecules associated with stromal cells, extracellular matrix components, and so on. Therefore, protocols that increase the number of such stem/progenitor cells that may help regenerate the liver should be of considerable therapeutic interest and value.