Mesenchymal Origin of Hepatic Stellate Cells, Submesothelial Cells, and Perivascular Mesenchymal Cells During Mouse Liver Development

Mice

The 560-base-pair Δ4Msx2-hsplacZ transgenic mouse (Msx2-lacZ) and MesP1-Cre knock-in mouse were described previously.^13,14 Male MesP1-Cre mice were crossed with female R26R mice.¹⁵ The use of animals for this study was approved by the Institutional Animal Care and Use Committee of the University of Southern California.

X-gal Staining and Fluorescence Immunohistochemistry

Whole embryos were fixed with 4% paraformaldehyde and stained with 1% X-gal solution at 37°C for 4 hours.¹³ For detection of lacZ activities in sections, frozen sections (7 µm) were incubated in X-gal solution as above and counter-stained with eosin Y (Sigma, St. Louis, MO).

For immunostaining, cryosections were permeabilized with phosphate-buffered saline (PBS) containing 0.1% Triton X-100. After washing with PBS, the sections were blocked with 0.2% bovine serum albumin and 5% serum that is from the same species as that for secondary antibody for 30 minutes. After blocking, the sections were incubated with diluted primary antibodies for 1 hour. The primary antibodies used in immunostaining are listed in Table 1. For double immunostaining, the sections were washed with PBS and incubated with primary antibodies for 1 hour. After washing with PBS, the sections were incubated with secondary antibodies conjugated with AlexaFluor 488, 555, or 568 (Invitrogen, Carlsbad, CA) for 30 minutes. The sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and fluorescence images were visualized under a microscope (Nikon Eclipse 90i; Nikon, Tokyo, Japan). For assessment of proliferative activity, we prepared cryosections from three different embryos and performed double immunostaining with Ki-67 and activated leukocyte cell adhesion molecule (ALCAM). After staining, a total of 55 photographs were taken, and the Ki-67⁺ cells were counted.

FACS Analysis of LacZ⁺ Liver Cells

E12.5 livers were digested in Dulbecco’s modified Eagle’s medium containing 0.2 U/mL Dispase I (Roche Applied Science) for 20 minutes at 37°C.¹⁶ For detection of lacZ activity in liver cells, we loaded fluorescein di-β-D-galactoside (FDG, Invitrogen) into the liver cells by hypotonic shock as previously described.¹⁷ Dead cells were detected using 5 µM propidium iodide. For FACS analysis, the cells were analyzed using FACS Vantage SE (Becton Dickinson, San Jose, CA), and the data were processed using CellQuest Pro software (Becton Dickinson).

Cell Depletion Using Antibodies and Magnetic Beads

The lacZ⁺ cells sorted by FACS were incubated with antibody-coated magnetic beads as previously reported.¹⁶ Briefly, 5 × 10⁵ beads (Dynabeads Sheep antirat immunoglobulin G; Invitrogen) were coated with each of rat anti-mouse E-cadherin (ECCD-1; Takara Bio, Shiga, Japan), F4/80, Flk1, CD45, TER-119 (eBioscience, San Diego, CA), and CD31 (BD Biosciences) antibodies. After washing, each of the magnetic beads was combined and incubated with the lacZ⁺ cells for 30 minutes on ice. The antibody⁺ cells were depleted with a magnetic particle concentrator (Invitrogen).

Quantitative Polymerase Chain Reaction

Total RNAs were extracted with RNAqueous Micro (Ambion, Austin, TX). Complementary DNAs were synthesized from 30 ng total RNAs using SuperScript III and Oligo(dT)₂₀ primers (Invitrogen). Quantitative polymerase chain reaction (qPCR) was performed with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) in M×3000P QPCR System (Stratagene, La Jolla, CA) as previously described.⁵ The primers are listed in Table 2.

Microarray Analysis

From each 60 ng total RNA prepared from E12.5 liver cells and lacZ⁺Ab⁻ cells, we obtained 26 µg and 18 µg cDNAs using the Ovation RNA amplification system V2 (NuGen, San Carlos, CA). Then, 3.75 µg from each cDNA was fragmented and labeled with Biotin using FL-Ovation cDNA Biotin module V2 (NuGen). The labeled probes were hybridized with GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) and signals were analyzed at the USC/CHLA Genome Core Laboratory.

Isolation and Culture of ALCAM⁺ Cells

E12.5 livers were digested with 0.05% trypsin-ethylene diamine tetraacetic acid for 5 minutes, and the blood cells were depleted using a lineage cell depletion kit (Miltenyi Biotec, Auburn, CA). After blocking with anti-CD16/32 antibodies (eBioscience), the blood cell lineage⁻ cells were incubated with phycoerythrin-labeled rat anti-mouse ALCAM antibodies (eBioscience) for 30 minutes. For FACS analysis, the cells were analyzed using FACS Vantage SE (Becton Dickinson), and the antibody positive and negative fractions were sorted. After FACS, 1 × 10⁴ cells were plated on 24-well plates in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and were subjected to immunostaining and qPCR. Collagen gel culture was performed as described previously.¹⁸ Briefly, ALCAM^high cells were mixed with PureCol (Inamed, Fremont, CA) and loaded in 24-well plates (1 × 10⁴ cells/ well). On day 4 in culture, the cells were treated with 5 µM retinol (Sigma) with 100 µM palmitic acid (Sigma) for 24 hours.¹⁹ For lipid staining, cells were incubated with 0.2% Oil Red O (Sigma) for 5 hours.⁵

LacZ Expression in the Msx2 Transgenic Mouse Liver

TheMsx2-lacZ embryos showed X-gal staining in the developing limbs and head of E12.5 to E14.5 whole embryos (Fig. 1A, B).¹³ The lacZ expression was also found in developing liver, which was not previously reported (Fig. 1C). The developing liver at E13.5 is macroscopically divided into the right and left median lobes (RML, LML), left lateral lobe (LLL), and superior and inferior right lobes (SRL, IRL), and intense X-gal staining was detected, particularly at the ventral surface of the RML, SRL, and LLL (Fig. 1C). The lacZ expression was found in mesenchymal cells in the parenchyma and around the blood vessels (Fig. 1D–G). The “flat cell layers” covering the median lobe right adjacent to heart were also lacZ⁺ (Fig. 1E). This tissue appears to be the transitional structure from septum transversum to diaphragm. Between liver lobes, some mesothelial cells and mesenchymal cells beneath the mesothelial cells also expressed lacZ (Fig. 1F). Because the staining patterns by lacZ immunostaining and X-gal staining were shown to be identical (Fig. 1G, H), subsequent analysis was performed by lacZ immunostaining.

LacZ Expression in Liver Mesenchymal Cells

We determined whether lacZ⁺ liver mesenchymal cells express desmin or α-SMA by immunostaining of E12.5 embryos. Desmin was expressed in the fetal HSCs, perivascular mesenchymal cells, and mesenchymal cells beneath the mesothelium (Fig. 2A). Almost all desmin⁺ cells expressed lacZ (Fig. 2A–D). The lacZ⁺ flat cell layers covering the median lobe weakly expressed desmin (Fig. 2D). Many of the perivascular mesenchymal cells expressed α-SMA (Fig. 2E, G) and coexpressed desmin and lacZ (Fig. 2F, H). Near the vessels, there were rare desmin⁺ α-SMA⁺ HSCs, which accounted for 3.7% of the desmin⁺ HSCs (Fig. 2E, F, arrows). The lacZ⁺ mesenchymal cells beneath the mesothelium was negative for α-SMA (Fig. 2I, J). Expression of Flk1 and CD31, markers for SECs, F4/80, a marker for Kupffer cells, and E-cadherin and albumin, markers for hepatoblasts, were not colocalized with lacZ⁺ staining in E12.5 (Fig. 2K–O) and E13.5 livers (data not shown). These data indicate that desmin⁺ cells including fetal HSCs, perivascular mesenchymal cells, and mesenchymal cells beneath the mesothelium, express Msx2 messenger RNA, and the lacZ expression is a suitable marker for these three populations of fetal liver mesenchymal cells in this model.

Isolation of LacZ⁺ Cells

To identify markers for liver mesenchymal cells, including fetal HSCs, we attempted to isolate lacZ⁺ cells from Msx2-lacZ fetal livers by FACS using FDG. Fig. 3A illustrates the procedure of fetal liver mesenchymal cell isolation. When liver cells prepared from E12.5 lacZ⁺ livers were incubated with FDG, 2% to 3% of the cells showed high FL1 caused by hydrolysis of FDG to fluorescein by β-galactosidase (right panel, Fig. 3B). However, the cells prepared from the LacZ⁻ livers also showed approximately 1% FL1 cells, which appeared to be caused by endogenous β-galactosidase–like activity (left panel, Fig. 3B). LacZ⁺ cells sorted from E12.5 lacZ⁺ livers expressed desmin messenger RNA, although other cell markers, including albumin, CD31, and CD68, were also detected because of inevitable contamination (data not shown). Using antibodies that recognize cell surface markers of hepatoblasts (E-cadherin), SECs (CD31, Flk1), Kupffer cells (F4/80), and blood cells (CD45, TER-119) plus magnetic beads, we depleted these cells to further purify the lacZ⁺ cells (Fig. 3A). From 1.6 × 10⁷ E12.5 liver cells treated with FDG, 8.9 × 10⁴ (0.6%) lacZ⁺ and 6.9 × 10⁶ (43.1%) lacZ⁻ cells were obtained by FACS. Then, the LacZ⁺ cells that were pull-down with beads (LacZ⁺ Ab⁺) and other remaining cells (LacZ⁺ Ab⁻) were subjected to qPCR analysis for the cell type markers. The data were compared against those obtained from isolated liver cells without FACS and magnetic bead separation. As shown in Fig. 3C, the LacZ⁺ Ab⁻ fraction was enriched by the cells expressing desmin, Foxf1, and Lhx2, markers for HSCs, whereas LacZ⁺ Ab⁺ cells expressed other liver cell markers. The LacZ⁺ Ab⁻ cells expressed 5-fold and 32-fold higher lacZ and Msx2, respectively, validating the enrichment efficiency and confirming endogenous expression of Msx2 by this population.

Microarray Analysis

Having isolated the lacZ⁺ Ab⁻ cells, we surveyed their differentially expressed genes by comparing with total isolated liver cells using the cDNA microarray. Among 45,037 genes analyzed, 4793 showed 5-fold or higher expression in the lacZ⁺ Ab⁻ cells than the total liver cells. Table 3 is a partial list of the upregulated genes that include those previously reported to be expressed by HSCs. Confirmation of the microarray results by qPCR analysis was performed for some of these genes (Fig. 3D).

Identification of Submesothelial Cells Beneath the Liver Surface

Of the up-regulated genes confirmed by qPCR (Fig. 3D), we selected CD antigens, including activated leukocyte cell adhesion molecule (ALCAM/CD166), p75NTR/CD271, and platelet-derived growth factor receptor α (PDGFRα/CD140a) and developmental markers including type IV collagen, podoplanin, and WT1 for immunohistochemical analysis in E12.5 livers. ALCAM staining was found along the liver surface (Fig. 4A). Type IV collagen, a marker for basal lamina, is deposited beneath the mesothelial cells at the liver surface (Fig. 4B). Along the dorsal liver surface, the mesenchymal cells were separated from the mesothelial cells by the type IV collagen (Fig. 4B). Immunostaining of ALCAM was found in the mesenchymal cells beneath the mesothelium (Fig. 4B, arrowheads). ALCAM expression was overlapped with type IV collagen and was also detected at the basolateral side of mesothelial cells (Fig. 4B). ALCAM was co-expressed with desmin in the flat cell layers on the surface of the median lobe (Fig. 4C, arrowheads). The ALCAM⁺ mesenchymal cells beneath the mesothelial cells coexpressed desmin (Fig. 4F, arrowheads), but not α-SMA (Fig. 4D). Conversely, fetal HSCs (Fig. 4C,F, arrows) and perivascular mesenchymal cells (Fig. 4E) were negative for ALCAM. From these staining patterns, we defined the desmin⁺ ALCAM⁺ cells associated with type IV collagen of the basal lamina as “submesothelial cells.” Near the liver surface, desmin⁺ ALCAM⁺ cells, not associated with the basal lamina, were also identified in the parenchyma a short distance (3–5 hepatoblast layers) away from the submesothelial cells (Fig. 4F, G, double arrows), suggestive of transitional cells migrating inward from the submesothelial cells.

Podoplanin Is a Marker for Liver Mesothelial Cells

Podoplanin expression was exclusively found in the mesothelial cells positive for pan-cytokeratin or WT1, but not in the submesothelial cells expressing WT1 (Fig. 5A, B). Podoplanin⁺ mesothelial cells were located right above the basal lamina expressing type IV collagen (Fig. 5C) below which desmin⁺ submesothelial cells existed (Fig. 5D). ALCAM immunostaining was evident in the basal lamina side of both podoplanin⁺ mesothelial cells and podoplanin⁻ submesothelial cells (Fig. 5E).

Definitions of HSCs, Submesothelial Cells, Mesothelial Cells, and Perivascular Mesenchymal Cells in the Developing Mouse Liver

p75NTR was expressed in desmin⁺ HSCs, ALCAM⁺ submesothelial cells, and α-SMA⁺ perivascular mesenchymal cells (Fig. 6A–C). PDGFRα expression was detected in the ALCAM⁺ submesothelial cells (Fig. 6D). From these expression patterns and location, we classified three populations of mesenchymal cells in developing liver: HSCs in the liver parenchyma (desmin⁺ p75NTR⁺ αSMA^+/−), submesothelial cells beneath the mesothelium (desmin⁺ p75NTR⁺ ALCAM⁺ PDGFRα⁺), and perivascular mesenchymal cells in the vicinity of the vessels (desmin⁺ p75NTR⁺ α-SMA⁺) (Fig. 6E). The mesothelial cells were identifiable with podoplanin. Immunohistochemistry analysis suggests that cells showing the desmin⁺ ALCAM⁺ submesothelial cell phenotype, but not associated with the basal lamina near the liver surface, are transitional cells between the submesothelial cells and HSCs.

Proliferative Activity of Submesothelial Cells

We determined the proliferative activities of submesothelial cells, mesothelial cells, and transitional cells by double immunostaining of Ki-67 and ALCAM (Fig. 6F). Mesothelial cells were identifiable at the liver surface (Fig. 6G, I, white circles), and 30.0% were Ki-67⁺. Conversely, 41.8% of the ALCAM⁺ submesothelial cells were Ki-67⁺ (Fig. 6H, I, red circles). Transitional cells were identified by ALCAM expression and irregular nuclei apart from the liver surface (Fig. 6H, I, white squares), and 22.8% were Ki-67⁺. These staining data indicate that submesothelial cell are actively dividing in the fetal livers as compared with mesothelial cells and transitional cells.

Differentiation Potential of ALCAM ⁺ Cells In Vitro

To test differentiation potential of submesothelial and mesothelial cells, we isolated the ALCAM⁺ cells by FACS for culture. After depletion of lineage⁺ blood cells, the lineage⁻ population was incubated with anti-ALCAM antibodies. FACS analysis showed three distinct populations: ALCAM^high, ALCAM^low, and ALCAM⁻ in E12.5 liver cells (Fig. 7A). Quantitative PCR revealed that the ALCAM^low population highly expressed albumin (Fig. 7B), implying that hepatoblasts weakly express ALCAM at the level undetectable by immunohistochemistry (Fig. 4A) but detectable by qPCR. Conversely, the ALCAM^high population expressed the genes related to submesothelial cells and mesothelial cells, but not albumin, indicating successful enrichment of these two cell types (Fig. 7B). The ALCAM^high cells highly expressed hepatocyte growth factor (Hgf) and pleiotrophin, but did not express Gfap.

One day after plating, 13.9% of the ALCAM^high cells attached to plastic well (Fig. 7C) and showed a fibroblastic morphology expressing α-SMA, desmin, or both desmin and podoplanin (Fig. 7D). Alpha-Sma messenger RNA showed a 393-fold increase after 1 day culture as compared with the preculture level (Fig. 7E). After 8 days in culture, all cells showed myofibroblastic morphology expressing α-SMA (Fig. 7D). Many of the cells were stained for desmin (86%), and 49% of these cells expressed podoplanin (Fig. 7D). Ten percent of the cells were podoplanin⁺ desmin⁻. Quantitative PCR showed that high expression levels of α-Sma, desmin, podoplanin, and β-actin were maintained in 8-day culture (Fig. 7E). In contrast, p75ntr, Pdgfrα, Hgf, pleiotrophin, Lhx2, and Hlx were down-regulated in the 8-day cells (Fig. 7E). No Gfap expression was found in the cultured cells. These results suggest that ALCAM^high submesothelial cells and mesothelial cells rapidly acquire a myofibroblastic phenotype in vitro. Conversely, only 1.3% of the ALCAM^low population attached to plastic well (Fig. 7C), and these attaching cells showed a fibroblastic morphology expressing α-SMA on day 8 (data not shown). The ALCAM⁻ population did not attach to the well (Fig. 7C).

Next, we tested whether the ALCAM^high population has the ability to store lipids in vitro as HSCs do. The ALCAM^high cells were cultured on a plastic well or in collagen gel for 4 days, and then incubated with retinol and palmitic acid for 24 hours. Oil Red O staining showed that 45% of the cells showed lipid storage in collagen gel (Fig. 7F), but no staining was found in the cells cultured on plastic dish despite the same treatment (Fig. 7F). These results suggest that the ALCAM^high population may acquire a phenotype characteristic of HSCs in three-dimensional collagen culture.

Cell Lineage Analysis of Liver Mesenchymal Cells

Finally, we determined whether the three populations of liver mesenchymal cells described previously are derived from a mesoderm lineage during development by using MesP1-Cre mouse. MesP1 is known to be transiently expressed in the nascent mesoderm during gastrulation, and MesP1-expressing cells contribute to the cranial–cardiac mesoderm in the lateral plate mesoderm.¹⁴ We crossed MesP1-Cre and R26R mice and traced a mesodermal lineage during liver development (Fig. 8A). X-gal staining was found in the liver surface and mesenchymal cells (Fig. 8B). Immunofluorescence staining showed that desmin⁺ HSCs, α-SMA⁺ perivascular mesenchymal cells, and ALCAM⁺ submesothelial cells expressed lacZ (Fig. 8C–G). Some podoplanin⁺ liver (visceral) mesothelial cells coexpressed lacZ (Fig. 8H). Conversely, parietal mesothelial cells at the body wall were negative for lacZ (Fig. 8H). Vascular endothelial cells expressed lacZ (Fig. 8F, I). However, lacZ staining was rarely found in Flk1⁺ SEC (Fig. 8I). LacZ staining was also rare in CD45⁺ (Fig. 8J) or TER-119⁺ blood cells (data not shown). No lacZ expression was detected in hepatoblasts (Fig. 8K) or Kupffer cells (Fig. 8L). These staining patterns demonstrate that HSCs, submesothelial cells, and perivascular mesenchymal cells are derived from the MesP1-expressing mesoderm during development.