From hair to liver: emerging application of hair follicle mesenchymal stem cell transplantation reverses liver cirrhosis by blocking the TGF-β/Smad signaling pathway to inhibit pathological HSC activation

Experimental animals

Wild-type healthy C57BL/6J mice (male, 20–22 g) were purchased from the Animal Center of the Second Affiliated Hospital of Harbin Medical University (Harbin, China). The mice were housed in the animal center for 1 week at 25 °C to adapt to the environment before the experiment. The mice were allowed to obtain feed and tap water normally. Our experimental protocol was performed according to medical ethics principles, and the mouse experiments were approved by the ethics committee of the Experimental Center of the Second Affiliated Hospital of Harbin Medical University (No. SYDW2019-240).

Humane endpoints were defined when mice showed persistent self-harm behavior and nonhealing wounds, and we euthanized the mice before the end of the experiment. Pentobarbital sodium was used to alleviate pain or fear during euthanasia, after which the mice were transferred to a new cage and placed in a quiet and undisturbed place. No animals survived at the end of the experiment.

Isolation, culture, characterization and staining of HF-MSCs

The extraction of HF-MSCs was as reported previously (Sun et al., 2021). Six- to seven-day-old mice were used for the extraction of primary HF-MSCs. The mouse vibrissa pads were dissected and cut vertically into several small tissues. The tissues were then incubated with 0.1% type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 4 h. The subcutaneous fat and connective tissue around the vibrissa were peeled off and then the hair follicles were separated from the connective tissue sheath with micro tweezers under a binocular microscope. The hair was cut above the bulge area. One or two hair follicles were inoculated in the middle of each well of a 24-well plate with complete medium, containing 15% fetal bovine serum (FBS) (ScienCell, San Diego, California, USA), 100 U/mL penicillin–streptomycin (Beyotime, Shanghai, China), and DMEM/F12 (Gibco, Gaithersburg, MD, USA), at 37 °C with 5% CO₂. The cells were passaged when they reached 70–80% confluence, and the medium was changed the day before passaging to maintain cell viability. The operations described above involving cell culture were strictly performed under aseptic conditions.

We identified HF-MSCs by detecting the expression of the surface marker (CD29, CD90, CD31 and CD34) and their potential to differentiate into adipocytes and osteocytes. Third-passage HF-MSCs with good growth conditions were stained with the low-toxicity cell membrane dye PKH67 (Sigma–Aldrich) to locate HF-MSCs in the damaged liver after transplantation. Then, the stained HF-MSCs and their distribution in multiple organs were captured under an upright fluorescence microscope (BX51; Olympus, Japan).

Animal model of cirrhosis and experimental design

The results of the preliminary experiment showed a significant difference when the sample size of mice was six. Thirty-five experimental mice were used in this study. carbon tetrachloride (CCl₄) was intraperitoneally injected at a concentration of 10% to establish a mouse LC model with a dose of 1 mL/kg and a frequency of two times per week. Cirrhosis was evaluated by serology and histology after the last injection in the 12th week. Five mice that died during modeling were excluded from the experiment, and the surviving mice were used for subsequent experiments and analyses. Then, the CCl₄-induced model mice were divided into the following two groups at random (based on a random order generator): (1) the HF-MSC group (n = 6), which was injected with 1 × 10⁶ HF-MSCs dissolved in PBS, and (2) the model group (n = 6), which was injected with an equivalent volume of saline. Mice in the control group (n = 6) were injected with 0.9% saline. Different groups of mice were assigned to different cages. Then the mice were administered 250 µl of the same injection volume by tail vein injection. Three weeks after cell transplantation, the liver tissues and serum obtained from the left ventricle were collected for subsequent analysis (Fig. 1A).

Hepatic stellate cell line and TGF-β1-induced HSC activation

The immortalized murine hepatic stellate cell line JS1 (Otwo Biotech, ShenZhen, China) was used to explore the effect of HF-MSCs on HSCs in vivo. JS1 cells were activated by cocultured with TGF-β1 (Peprotech, NJ, USA) at a concentration of five ng/mL for 48 h (Ma et al., 2018). JS1 cells were cultured in complete medium consisting of high-glucose DMEM (Gibco). Then, the activated JS1 cells were used for the Transwell assay.

Transwell assay

Transwell assays were performed to coculture JS1 cells and HF-MSCs in a double-cell coculture system using Transwell chambers with a 0.4 µm semipermeable membrane. HF-MSCs (2 × 10⁵ cells/well) were inoculated in the upper chamber of 6-well culture plates, and the same number of activated JS1 cells was mixed and inoculated in the lower chamber of the same well. HF-MSCs and IS1 cells were cocultured with high-glucose DMEM. JS1 cells alone were used as a control. After coculture for 48 h with saturated humidity and 5% CO₂, cells in the lower chamber were harvested for a Western blot assay.

Western blot analysis

Liver tissues from all groups that had been stored in liquid nitrogen were ground into a powder in a mortar and placed in an EP tube. Protease inhibitors and phosphatase inhibitors were added to lysis buffer to prevent the decomposition and dephosphorylation of the target proteins, and the entire lysis process was performed on ice for 30 min. The supernatant was transferred to an EP tube after centrifugation. After measuring the total protein concentration, the same amount of protein from each group was separated on 8% or 10% SDS–PAGE gels according to their molecular weights. Then, the proteins in each lane were transferred onto polyvinylidene difluoride membranes (EMD Millipore Corporation, Billerica, MA, USA) using a membrane transfer apparatus (Bio-Rad, Hercules, CA, USA). Blocking buffer (Bio-Rad) was used to block nonspecific binding. After an incubation with primary antibodies overnight, the membranes containing the proteins were then incubated with secondary antibodies for 1 h at room temperature. The following primary antibodies were used: anti-β-actin (ab8226, 1:1000; Abcam, Cambridge, UK), anti-TGF-β1 (#3711, 1:1000; Cell Signaling Technology, Beverly, MA, USA), anti-p-Smad2/3 (#8828, 1:1000, Cell Signaling Technology), anti-Smad2/3 (AF6367, 1:1500; Affinity, Changzhou, China) and anti-α-SMA (14395–1-AP, 1:2000; Proteintech, Wuhan, China). Images of the specific protein bands were captured using an Omega-Lum G imaging system after an incubation with enhanced chemiluminescence solution and analyzed using ImageJ software.

Labeling HF-MSCs with DiR and in vivo imaging

DiR is a lipophilic fluorescent dye that can be used to stain cell membranes. DiR (D12731; Invitrogen) labeled HF-MSCs were used to track HF-MSCs in vivo. Adjust the third-generation HF-MSCs concentration to 1 × 10⁶/L, and add 5 uL of three mmol/L DIR storage solution to one mL of cell suspension. The DiR solution and cells were incubated for 30 min. Then the stained cells were transplanted via tail vein. After 48 h, the biodistribution of DiR-labeled HF-MSCs were performed by in vivo imaging System (Night OWL II LB983).

Immunofluorescence staining

The frozen liver, kidney, lung and spleen tissues were sectioned to a thickness of 5–6 µm. Goat serum (abs933, Absin, Shanghai, China) was used to block the sections. The slices were incubated at 4 °C overnight with the following antibodies: anti-cytokeratin 18 (CK18) (ab181597, Abcam) and anti-albumin (ALB) (ab207327; Abcam). Sections were then incubated with a goat anti-rabbit secondary antibody (SA00013–4; Proteintech) for 1 h at room temperature on the second day. Next, after 3 rinses with TBST, the sections were incubated with 4,6-diamidino-2-phenylindole (DAPI; Beyotime) for 4 min. Then, the stained sections were mounted with antifade mounting medium.

Pathological analysis

The dried paraffin-embedded liver tissue sections were completely immersed in xylene and different concentrations of ethanol and then rinsed several times with PBS. Slices were stained with an aqueous hematoxylin solution and differentiated with an acid solution in alcohol. After an incubation with ammonia, the slices were stained with eosin and dehydrated and sealed with neutral gum.

For Masson’s trichrome staining, the slices were stained with Weigert iron hematoxylin solution for 7 min. The sections were differentiated with an acidic ethanol differentiation solution for 6 s and then returned to blue with Masson’s blue solution for 4 min. After rinses with distilled water, the sections were stained with Ponceau red magenta solution for 5 min. The slices were washed with phosphomolybdic acid solution and then immersed in aniline blue staining solution for 2 min. After sealing with neutral gum, they were observed under a microscope.

Serological analysis

Blood was collected from the left ventricle into a coagulation-accelerating vacuum tube when the mice were sacrificed. During blood collection, contamination with mouse hair was avoided, and the blood was directly collected into the bottom of the tubes to avoid hemolysis. The supernatant was collected after the blood coagulated in an EP tube. ELISA kits were used to evaluate serum ALB (ab207620; Abcam), alkaline phosphatase (ALP) (SEB472Mu; Cloud-Clone Corp, Wuhan, China), alanine aminotransferase (ALT) (CSB-E16539 m; Cusabio, Wuhan, China) and aspartate aminotransferase (AST) levels (ab263882; Abcam).

Statistical analyses

At least three independent replicates were performed of each experiment. All data are presented as the means ± standard deviations (SD) and were analyzed using GraphPad Prism 8.0 software (GraphPad Prism Software, San Diego, CA, USA). Student’s t test was used for comparisons between two groups, and one-way ANOVA with Tukey’s test were used to determine significant differences between multiple groups. A nonparametric test was used for data that did not exhibit a normal distribution. Differences were considered statistically significant when P values were less than 0.05.

Characteristics of HF-MSCs

The primary HF-MSCs showed adherent growth and a paving stone-like appearance on the fifth day of cell culture (Fig. 1B). Primary cells grew rapidly after adherence and were subcultured after 10 days. Cells of the second generation showed a typical spindle-shaped morphology (Fig. 1C). The multilineage differentiation potential of HF-MSCs was determined by performing osteogenesis and adipogenesis experiments (Figs. 1D–1E). The extracted cells expressed CD29, CD90, while the expression of CD31 and CD34 was negative (Figs. 1F–1I). Cells labeled with the PKH67 green fluorescent dye are shown in Fig. 1J. This result indicates that we successfully extracted HF-MSCs.

Evaluation of the cirrhosis model

After 12 weeks of CCl₄ administration, healthy mice and model mice were used to identify whether the model was successfully established. HE staining showed that the structure of hepatocytes in the normal group was clear without necrosis or inflammatory cell infiltration, and collagen fibers were rarely observed in the perisinusoidal space. In the model group, the arrangement of hepatocytes was altered, and the hepatocytes showed fatty degeneration and necrosis. Collagen fibers around the central vein in the liver tissue proliferated and formed pseudolobules (Figs. 2A–2B).

Serum assays were performed to assess hepatic function. The ALT, AST and ALP levels in the model group were increased to varying degrees compared with those in the normal group (all P values <0.05, Figs. 2C–2E). These features were consistent with the typical histopathological and serological changes of LC induced by CCl₄.

HF-MSCs alleviated histological injury and improved liver function in mice

The mice were sacrificed to collect liver tissue and serum after 3 weeks of cell therapy. The degree of LC was evaluated by performing HE and Masson’s trichrome staining. HE staining of hepatic paraffin sections from the model group showed degeneration and necrosis of hepatocytes and a large area of pseudolobular tissue structure, and Masson’s trichrome staining showed thick and deeply stained deposited collagen fibers extending outward from the portal area. However, these pathological changes related to LC were significantly attenuated by HF-MSC treatment (Figs. 3A–3B). Measurements of liver function indicated remarkable increases in the levels of ALT (175.49 ± 7.29 U/L) and AST (238.42 ± 19.44 U/L), while the serum level of ALB (18.22 ± 0.78 g/L) exhibited a decreasing trend in LC mice. However, these changes were reversed in the treatment group (Figs. 3C–3E). In summary, HF-MSCs promoted the repair of liver injury.

Labeled HF-MSCs migrated to the injured liver and expressed hepatocyte-specific markers

Three weeks after cell transplantation, PKH67-labeled HF-MSCs resided in the injured liver tissue and showed green fluorescence (Figs. 4A–4E). The hepatocyte-specific markers CK18 and ALB were detected in liver sections from cirrhotic animals (Figs. 4B and 4F). More importantly, these labeled cells were exactly colocalized with the hepatocyte surface markers ALB and CK18 (Figs. 4D and 4H), indicating that HF-MSCs in the damaged liver expressed hepatocyte surface markers. Notably, PKH67-stained HF-MSCs were rarely observed in the intestine, kidney, lung and spleen tissues (Figs. 4I–4L). Pearson’s correlation coefficient and the overlap coefficient reflecting the degree of colocalization are shown in Fig. 4M. To detect the distribution of DiR-labeled HF-MSCs in vivo, the in vivo imaging was performed. It showed that after transplanting the DiR-labeled HF-MSCs for 48 h, the HF-MSCs were mostly gathered in the lung and the liver (Fig. 4N).

HF-MSCs suppressed the pathological activation of HSCs and inhibited the TGF-β/Smad signaling pathway in vitro

The cell bodies of quiescent JS1 cells were oval or irregular (Fig. 5A). After HF-MSCs were cocultured with JS1 cells for 48 h, the activated JS1 cells displayed enlarged cell bodies, protruding slender pseudopods, and a star-shaped appearance (Fig. 5B). Compared with the JS1 group, the expression of α-SMA, an indicator of JS1 cell activation, was decreased in the cell treatment group (P < 0.05). Furthermore, levels of TGF-β1 and p-Smad3 in the JS1 group were noticeably increased, which were inhibited by cell treatment in the HF-MSC group (P < 0.05) (Figs. 5C–5F). Based on the results described above, we determined that HF-MSCs inhibited pathological activation of JS1 cells and that activation of the TGF-β signaling pathway, as determined by Smad3 phosphorylation, was also suppressed in the HF-MSC group.

HF-MSCs inhibited pathological HSC activation and Smad3 phosphorylation in the LC model

We next studied the role of HF-MSC treatment in regulating HSC activation. The expression of α-SMA, a marker of HSC activation, showed an increasing trend in the model group, which was reversed by HF-MSC treatment (P < 0.05) (Fig. 6). Furthermore, levels of the TGF-β and p-Smad3 proteins in the liver tissue were measured. Immunoblotting showed increased levels of TGF-β1 and p-Smad3 in the model group, but these changes were suppressed to a certain degree and approached the normal level in the HF-MSC treatment group (P < 0.05).