Introduction

Hepatic stellate cells (HSCs), a mesenchymal cell type in the liver, are characterized by long cytoplasmic processes with Vitamin A containing fat droplets in the cytoplasm. HSCs are pericytes [1] of liver sinusoidal endothelial cells (SECs). Recent evidence suggests a role as a liver-resident antigen-presenting cell, presenting lipid antigens to and stimulating the proliferation of NK/T cells [2]. Their transformed myofibroblastic cells are involved in several liver diseases, liver fibrosis, and cirrhosis [3]. HSCs also have a relationship with hepatic angiomyolipoma as they share a similar gene expression profile [4].

HSCs exhibit both mesenchymal and neuronal cell features by expressing vimentin and desmin, as well as glial fibrillary acidic protein (GFAP), neural cell adhesion molecule (N-CAM), synaptophysin, and nestin, respectively [5,6,7,8,9].

Cell migration is a central process in the development and maintenance of multicellular organisms [10]. Three cytoskeletal proteins, including intermediate filaments (IFs), microfilaments, and microtubules are critical regulators of cell migration and cell shape maintenance [11]. IFs are formed with 10-nm-diameter intracellular filaments and are subcategorized into five types based on similarities in amino acid sequences and protein structure [12].

Nestin, a Type VI intermediate filament, was first identified in the multipotent stem/progenitor cells of the central nervous system (CNS), and has been regarded as a marker of neuronal and non-neuronal stem/progenitor cells [13,14,15]. However, a large number of studies have confirmed nestin expression in several non-neuronal tissues during not only their developmental stages but also in conditions of injury or repair [14], which is widely expressed during development but is very rare in adult organs. Nestin has been detected in several proliferating regions of the CNS and in developing mesenchyme and blood vessels during embryogenesis [16,17,18].

Subsequently, nestin was shown to be upregulated in stem cells of various organs, such as the pancreas [19, 20], testis [21], kidney [22], and liver [23]. Nestin expression generally decreased upon cellular differentiated. However, nestin expression is known to characterize mature cells and begin stem cells in a variety of linages. It can be induced during stem cell activation in response to liver [6], skeletal muscle [24] and the CNS [25] injuries.

In this study, we strived to determine by immunohistochemistry whether HSCs and another (myo)fibroblast derive from nestin-expressing mesenchymal stem cells during rat hepatogenesis. We discovered that a high expression of nestin was detectable in the mesenchymal cells between the liver bud and the septum transversum mesenchyme at ED 10.5. Nestin-expressing cells were also expressed in desmin, a rat HSC marker, during early hepatogenesis. However, desmin-positive HSCs decreased in nestin expression at ED 16.5 and later. At ED 20.5, nestin-positive cells were observed only in mesenchymal cells around the central veins and in the portal tracts. Our results suggest that nestin-expressing mesenchymal cells differentiate into HSCs, perivenular (myo)fibroblasts, and portal (myo)fibroblasts.

Materials and methods

Embryonic and adult normal liver

Pregnant rats of Wistar strain were purchased from SLC (Shizuoka, Japan). All animals were maintained under 12-h light/dark cycles with food and water available as desired.

Liver samples of embryos from adult rats were used at various times of gestation, beginning at ED 10.5. Embryos were removed from the uteri of mothers under ether anesthesia. A portion of the liver was immersed in 20% phosphate-buffered formaldehyde for immunohistochemical analysis, fixed in 4% paraformaldehyde (PFA) for immuno-electron microscopy and whole-mount immunohistochemistry or fixed in 2.5% glutaraldehyde for transmission electron microscopy. For RT-PCR examination, liver samples were immersed in RNA-later (Applied Biosystems, CA, USA) and stored at − 20 °C until use. Samples at all embryonic stages were prepared from at least three different litters.

Animal models of liver injury

Male Wistar rats, 200–250 g body weight, were used in the carbon tetrachloride (CCl4) models. CCl4 acute damaged liver was induced as described [26, 27]. Liver tissues were obtained 24 and 48 h after CCl4 treatment. These samples were immersed in 20% phosphate-buffered formaldehyde for immunohistochemical staining.

Whole-mount immunohistochemistry

Each embryo was fixed in 4% PFA in phosphate-buffered saline (PBS) for 4 h and immersed in 100% methanol for 20 min at − 20 °C. The fixed samples were then bleached with 2% hydrogen peroxide in methanol for 1 h at room temperature to block endogenous peroxidase. The rehydrated specimens were first incubated with 1% normal rabbit serum in 0.1% TritonX-100 in PBS (PBST) consisting of 1% skim milk for 1 h at 4 °C, followed by incubation with anti-nestin antibody in PBST at 4 °C overnight. After washing eight times with PBST each for 30 min at 4 °C, the specimens were incubated with a peroxidase anti-mouse antibody (Simple Stain Nichirei, Tokyo, Japan) overnight at 4 °C. After unbinding the second antibody by washing with PBST, the specimens were incubated in 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, Mo. USA) and additionally hydrogen peroxidase to 0.02% (v/v) for 1 h. The enzymatic reaction was allowed to proceed until the desired color intensity was reached. The specimens were then rinsed three times in PBST. Specimens were photographed with a digital camera mounted on a microscope.

Immunohistochemical detection

Paraffin sections (3 µm thick) were cut and mounted on silane-coated glass slides. The deparaffinized sections were microwaved for 15 min in 10 mM citric acid (pH 6.0) for antigen retrieval. The sections were incubated with 10% normal rabbit serum in 0.01 M PBS for 10 min at room temperature, and then with each monoclonal antibody for anti-nestin (rat401 chemicon Carpinteria, CA, USA), desmin (D33, Dako, Glostrup Denmark; 1:50) with PBS containing 0.1% bovine serum albumin at 4 °C overnight. After washing with PBS, the sections were incubated with biotinylated rabbit anti-mouse immunoglobulin (Dako Glostrup Denmark; 1:200) for 1 h at room temperature, washed again with PBS, and incubated with avidin–biotin peroxidase complex (ABC; Vector Laboratories, Inc., Burlingame, CA, USA) solution. Finally, the sections were immersed in a substrate solution of DAB. As a control, sections were processed without incubation with the primary antibody.

Double immunofluorescence analysis

To identify which cells expressed nestin, a double immunofluorescence method was carried out for several markers: desmin (HSCs and smooth muscle cells). The sections were incubated with 10% normal rabbit serum in 0.01 M PBS for 10 min at room temperature, and then with each monoclonal antibody for anti-nestin with PBS containing 0.1% bovine serum albumin (BSA) at 4 °C overnight. After washing with PBS, the sections were incubated with biotinylated rabbit anti-mouse immunoglobulin for 1 h at room temperature, washed with PBS, and incubated with FITC-labeled streptavidin (Invitrogen Corp., Carlsbad, CA, USA) solution. Immunolabeled sections were then incubated in 10 mM citric acid (pH 6.0) for 30 min at 95 °C for antigen deactival. Subsequently, the specimens were immersed in anti-desmin antibody at 4 °C overnight. After washing with PBS, the sections were treated with secondary antibody (biotin-labeled rabbit anti-mouse immunoglobulin) for 1 h, washed with PBS, and incubated with Texas Red-labeled streptavidin (Invitrogen Corp., Carlsbad, CA, USA). Finally, the slides were covered with vectashield mounting medium with DAPI (Vector Laboratories, UK) to visualize cellular nuclei. As a control, sections were processed without incubation with the primary antibody. Specimens were photographed with an Olympus digital camera (DP70, Olympus Corporation, Tokyo, Japan) mounted on an Olympus fluorescent microscope (BX50).

Electron and immuno-electron microscopy

Embryonic tissues fixed in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 4 h at 4 °C were then post-fixed in 1% osmium tetroxide in PBS for 1 h at 4 °C. This was followed by dehydration and embedding in epoxy resin. To select optimal areas, semi-thin sections were stained with toluidine blue. Ultra-thin sections were stained with uranyl acetate and lead citrate then examined with a JEM 100S electron microscope (JEOL, Tokyo, Japan).

Whole fetuses (ED 12.5) were fixed in 4% PFA solution for 6 h. Following fixation, the specimens were incubated overnight with 10% sucrose in PBS. Frozen sections were cut into 10-µm slices and washed with PBS. The sections were pre-incubated with 10% normal rabbit serum for 30 min, and then incubated for 24 h with anti-nestin antibody at 4 °C. Sections were then incubated with biotinylated rabbit anti-mouse immunoglobulin overnight at 4 °C. Finally, the sections were incubated with ABC solution for 1 h at room temperature. The peroxidase reaction was developed by incubating sections with 0.02% DAB in 0.05 M Tris–HCl (pH 7.6) for 1 h at room temperature and then 0.02% DAB in 0.05 M Tris–HCl (pH 7.6) containing 0.01% H2O2 for 10 min. The tissue sections were post-fixed with 1% OsO4 in a 0.1 M PBS (pH 7.4) for 1 h, dehydrated in a graded ethanol series, and covered with Epon812. Ultra-thin sections were observed with an electron microscope.

RT-PCR

Total RNA was extracted from frozen tissue using TRIzol reagent (Invitrogen, Corp., Carlsbad, CA) according to the manufacturer’s instructions. First-strand cDNAs were synthesized with Oligo (dt) primer and the Superscript III first-strand synthesis system (Invitrogen, Corp., Carlsbad, CA). Each single-stranded cDNA was diluted for subsequent PCR amplification. Standard PCR procedure was performed in 15 microvolumes of PCR buffer. To detect rat nestin, the primers 5′-AACCACAGGAGTGGGAACTG-3′ (rat nestin forward) and 5′-TCTGGCATTGACTGAGCAAC-3′ (rat nestin reverse) were used. As a quantitative control, glyceraldehydrate-3-phosphate dehydrogenase (GAPDH) was amplified with primer 5′-CTCATGACCACAGTCCATGC-3′ and 5′-TTCAGCTCTGGGATGACCTT-3′. PCR conditions were an initial denaturation at 94 °C for 7 min, following by 30 cycles of 94 for 30 s, 55 for 1 min, and 72 for 1 min, and a final extension step of 72 for 10 min. Each PCR product (15 µg/µl) was visualized by ethidium bromide staining on 6% acrylamide gel.

Results

Analysis of nestin protein expression in rat embryos by whole-mount immunohistochemistry

At ED 11.5, whole-mount immunohistochemistry showed the expression of nestin in several regions. Strong staining was detected in the neural tube, the definitive gut endoderm, branchial arches, and liver bud (Fig. 1a). When examined at ED 12.5, the liver, somites, forelimb buds, and branchial arches were stained strongly (Fig. 1b).

Fig. 1
figure 1

At ED 11.5, whole-mount immunohistochemistry revealed strong nestin expression in the neural tube (nt), branchial arches (ba) and liver bud (lb) (a). At ED 12.5, the liver (arrow), somites (s), forelimb buds (fb) and branchial arches (ba) were strongly stained (b)

Full size image

Distribution of nestin-positive cells in developed rat liver

At ED 10.5, the ventral foregut endoderm started to develop and form a liver bud. Nestin was expressed in mesenchymal cells adjacent to the liver bud. Cell types such as neural epithelial cells, neural crest, and mesenchymal cells of somite were also positive for nestin (Fig. 2a, b). At ED 11.5, the nestin expression was detected in mesenchymal cells mixed with migrating hepatoblasts in septum transversum mesenchyme (Fig. 2c). From ED 12.5 to 16.5 strong expressions of nestin were observed in the sinusoidal cells, such as HSCs and SECs around the hepatic sinusoid (Fig. 2d, e). In the rat liver at ED 20.5, numbers of nestin-positive sinusoidal cells were markedly decreased in the liver parenchyma (Fig. 2f). The expression levels of nestin gradually decreased after this stage; however, mesenchymal cells around the central and portal veins continued to express nestin at high levels (Fig. 2g). In the postnatal liver, strong nestin-positive cells were observed around the portal vein and immature bile duct in the portal areas (Fig. 2h). In the adult liver, sinusoidal cells were negative for nestin, whereas the mesenchymal cells around the central and the portal vein and the periductular stromal cells adjacent to biliary epithelium were positive (Fig. 2i). Other cell types, such as hepatocytes and biliary epithelial cells, were not reactive with anti-nestin antibody.

Fig. 2
figure 2

At ED 10.5, nestin was expressed in neural epithelial cells, neural crest, and mesenchymal cells of somite (a). Higher magnification of boxed area in a (b). Nestin-positive cells were observed in mesenchymal cells around and adjacent to the liver bud (lb) in the septum transversum (st) (c). At ED 11.5, the nestin expression was detected in mesenchymal cells mixed with migrating hepatoblasts in the septum transversum. From ED 12.5 to 16.5 nestin expression was observed in the sinusoidal cells, such as hepatic stellate cells and sinusoidal endothelial cells around hepatic sinusoid [d (ED 12.5), e (ED 14.5), f (ED 16.5)]. In the rat liver at ED 20.5, numbers of nestin-positive sinusoidal cells were markedly decreased in the liver parenchyma (g). The expression levels of nestin gradually decreased after this stage; however, mesenchymal cells around the central and portal veins continued to express nestin at high levels [h (postnatal day 3)]. In the adult liver, the mesenchymal cells around the central and the portal vein, and periductular stromal cells adjacent to biliary epithelium were positive (i). Bars: 200 µm (a), 100 µm (e–h), 50 µm (b, c, i)

Full size image

Double immunofluorescence analysis

Double immunostaining for nestin and desmin was performed to examine whether sinusoidal cells were positive for desmin-expressed nestin during liver development. At ED 10.5, nestin-expressing cells were observed around the liver bud, and a small number of these cells expressed in desmin (Fig. 3a–d). At ED 11.5, many, but not all, nestin-positive cells were co-expressed desmin (Fig. 4a–c). At ED 14.5, a large number of desmin-positive cells existed in the liver parenchyma; however, desmin-positive HSCs were largely negative for nestin (Fig. 4d–f).

Fig. 3
figure 3

Transverse section of an ED 10.5 embryo. Double immunostaining for nestin and desmin was performed to examine whether immature hepatic stellate cells (hsc) were positive for desmin-expressed nestin during liver development (a: H&E). Desmin was considered a marker for HSC. Nestin-expressing cells (b, d: green) were observed around the liver bud (lb) in the septum transversum (st). A small number of nestin-positive cells expressed desmin (c, d: red). Nestin and desmin double-positive cells were indicated by white arrows. Bars: 50 µm

Full size image
Fig. 4
figure 4

At ED 11.5, a small number of nestin-positive cells co-expressed desmin (a–c). At ED 14.5, large numbers of nestin-positive cells existed in the liver parenchyma; however, desmin-positive hepatic stellate cells were largely negative for nestin (d–f). Arrows: both nestin- and desmin-positive hepatic stellate cells. Bars: 50 µm

Full size image

Ultrastructural finding of liver development

At an ultrastructural observation at ED 10.5, the liver bud contacted the mesenchymal cells in the septum transversum through cellular protrusions (Fig. 5a, b). At ED 11.5, immature SECs were encircled by immature mesenchymal cells and migrating hepatoblasts. They had narrow lumens without blood cells. Mesenchymal cells in septum transversum mesenchyme were stellate in shape, having many slender processes (Fig. 5c). Likewise, at ED 12.5, cell–cell adhesions between hepatoblasts were loose. Mesenchymal cells existed under the immature SECs. These cells had many cellular processes and contact with other mesenchymal cells (Fig. 5d).

Fig. 5
figure 5

Electron microscope. At ED 10.5, the liver bud (lb) contacted the mesenchymal cells (mc) in the septum transversum through cellular protrusions (arrows in a, b). At ED 11.5, immature sinusoidal endothelial cells (sec) were encircled by mesenchymal cells and migrating hepatoblasts (hb), and had narrow lumens without blood cells. Mesenchymal cells in the septum transversum were stellate in shape, having many slender processes (c). At ED 12.5, immature hepatic stellate cells (hsc) existed under the immature SECs, These cells had many cellular processes and contacts with other mesenchymal cells (d). Immuno-electron microscope using anti-nestin antibody at ED 12.5. Strong positive cells were detected as electron-dense deposits in the cytoplasm of immature HSC under the immature sinusoidal endothelial cells (e). Similarly, it is recognized that nestin-positive cells were both SECs and HSCs in areas with many hematopoietic cells (f). EB: erythroblasts; S: sinusoid; arrow: immature SEC; double arrow: HSC process; bars: 3 µm (a, d–f), 1 µm (b), 10 µm (c)

Full size image

By immuno-electron microscopy and using anti-nestin antibody, strong positive cells were detected as electron-dense deposits on the cytoplasm of HSCs under the SECs at ED 12.5. More mature SECs were negative for nestin (Fig. 5e). Similarly, nestin-positive cells were identified as both SECs and HSCs in areas with many hematopoietic cells in early hepatogenesis (Fig. 5f).

Analysis of nestin mRNA expression in rat embryos and adult liver

Temporal expression profiles of nestin mRNA were examined by RT-PCR analysis. As shown in Fig. 6, the transcripts were detected throughout all embryonic stages from ED 13.5 to 20.5 and the adult liver. Nestin mRNA gradually decreased during liver development.

Fig. 6
figure 6

Nestin mRNA was detected throughout all embryonic stages from ED 13.5 to 20.5 and in the adult liver. The expression of nestin gradually decreased during liver development

Full size image

Distribution of nestin-positive cells in injured liver

Necrotic hepatocytes were observed around the central veins at 24 and 48 h after CCl4 injection (Fig. 7). In this stage, nestin-positive cells were distributed in activated spindle-shaped HSCs (myofibroblast-like cells) around the pericentral areas. In addition, nestin-negative HSCs were existent around the necrotic tissue.

Fig. 7
figure 7

Necrotic hepatocytes were observed around the central veins at 24 and 48 h after CCl4 injection. In this stage, nestin-positive cells were distributed in activated spindle-shaped HSCs around pericentral areas at 24 h (b) and 48 h (d–f) after CCl4 injection (a hepatocyte without CCl4 injection as control). The majority of desmin-positive hepatic stellate cells were nestin positive; however, only nestin-positive cells were also observed in the necrotic areas. Nestin-negative HSCs were existent around the necrotic tissue. Bars: 100 µm (a), 50 µm (b–f) c central vein

Full size image

Discussion

Nestin, an intermediate filament protein, was first described as expressed in central nervous system stem cells [15], and later in several other tissues and cells, including cardiac myogenic cells [16], Leydig cells [28], and adrenocortical cells [29, 30]. Pancreatic stellate cells and vascular endothelial cells in the pancreas [20, 31,32,33,34], kidney [22, 35], and many other cells have been reported to contain nestin. In addition, nestin has been suggested to be expressed in activated HSCs in injured rat livers, not in quiescent HSCs in normal livers [6].

We analyzed the expression of nestin during the development of rat livers by immunohistochemistry from ED 10.5 to ED 20.5 as well as the adult normal and injured liver. The results showed that nestin-positive cells are expressed in the mesenchymal cells adjacent to liver bud at ED 10.5. In this stage, epithelial cells of the neural tube and mesenchymal cells of the neural crest and somite were positive for nestin. At ED 12.5–16.5, nestin-positive cells were detected in sinusoidal cells, both HSCs and SECs, in the liver parenchyma and in mesenchymal cells around the large blood vessel. Starting at ED 16.5, the intensity of nestin positivity in sinusoidal cells gradually decreased. In the adult normal rat liver, sinusoidal cells were negative for nestin, whereas the mesenchymal cells around the central veins, the portal vein, and bile duct epithelial cells were positive.

HSCs, a mesenchymal cell type in the liver, are characterized by long cytoplasmic processes with vitamin A containing fat droplets in the cytoplasm. HSCs are pericytes of liver sinusoidal SECs. Activation of HSCs into myofibroblastic cells is involved in several liver diseases, liver fibrosis, and cirrhosis. HSCs show features of both mesenchymal cells and neuronal cells by expressing vimentin, and desmin, as well as GFAP, N-CAM, synaptophysin, respectively [5,6,7,8,9]. In addition, nestin expression was detected in activated HSCs, myofibroblast-like cells, and in CCl4-induced liver fibrogenesis [6]. Our observation also showed that myofibroblast-like cells derived from HSCs were positive for nestin around the pericentral areas after acute liver injury with CCl4.

On the other hand, the embryonic origin of HSCs in the liver has remained obscure. It has been postulated that the origin of HSCs is related to the mesenchymal lineage [36], the neuro-ectoderm [6, 37], the endoderm [38], or the mesothelial live capsule [39, 40]. However, Cassiman et al. reported that HSCs are not derived from the neural crest in transgenic mice expressing yellow fluorescent protein in all neural crest cells [39]. Furthermore, it was reported that bone marrow cells or hematopoietic stem cells become HSCs in the adult injured liver [41, 42]. Recently, HSCs were derived from the submesothelial cells beneath mesothelial cells in developing mouse liver [43, 44]. However, in rats there was no report that HSCs were derived from submesothelial cells. In our observation, submesothelial cells were positive for desmin, not nestin (data not shown) might be one of the origins of HSCs. In addition, HSCs in the embryonic or adult liver might be of a different origin.

In conclusion, we found that nestin, a Class VI intermediate filament protein, is transiently expressed in HSCs during rat hepatogenesis, but not in an adult normal liver. At ED 10.5, when the rat liver starts, these results may imply that nestin is a significant marker for undifferentiated and activated rat hepatic stellate cells.