1. Introduction

End stage liver diseases are serious medical conditions which require liver transplantation as an only life-saving treatment [1]. However, due to the shortage of donations, liver transplantation is not applicable for every patient with end stage liver diseases [1, 2]. In most countries, the hepatitis virus related liver diseases are leading cause of liver cirrhosis [2–4] Even introduction of directly acting antivirals (DAAs) for hepatitis C virus related liver diseases, certain proportion of patients develop liver failure even after viral eradication [3–5]. Also, hepatitis B related liver cirrhosis is mostly managed with nucleot(s)id treatment, whereas some patients with advanced liver fibrosis reportedly deteriorates after intervention [6, 7]. Furthermore, there are increased number of patients with decompensated cirrhosis from several liver diseases such as alcoholic liver cirrhosis or nonalcoholic fatty liver diseases [8, 9]. Thus, it is essential to develop life-saving medical therapy other than liver transplantation [10].

Autologous bone marrow and stem cell transplantation has been clinically applied for liver generation, especially in Japan [11]. However, it is not widely used in a general clinical setting. In the field of regenerative medicine, induced pluripotent stem (iPS) cells prepared by genetic modification have been considered to be promising therapeutic options, and thus, several clinical trials using iPS cells has been conducted [12]. In the past, clinical trials with mesenchymal stem cell (MSC) have been applied for liver disease and improvements of hepatic reserves and hepatic fibrogenesis have been reported [13, 14]. However, recent clinical trials could not find the beneficial effects of MSC transplantation [15, 16]. Although other reports and studies suggested the potential beneficial effects of cellular transplantation as clinical applications, they are not conclusive [17–19]. Thus, development of alternative methods, especially using large animal model was warranted [20].

One of the candidates for cell source is multilineage-differentiating stress enduring (Muse) cells [21, 22]. Muse cells exist in one out of 3000 bone marrow cells and are also found in skin and fat [23]. In addition, Muse cells differentiate into cells of tridermic system (ectoderm, mesoderm, and endoderm). Muse cells are characterized by positivity of both the MSC marker, CD105, and the pluripotent stem cell marker, stage-specific embryonic antigen-3 (SSEA-3) [24]. Since Muse cells could be isolated using marker SSEA-3, and they have enough pluripotency without the transgene techniques [25]. In an in vivo study using Muse cells, unlike embryonic stem (ES) cells and iPS cells, there is no reported formation of teratoma; thus, the risk of developing tumor formation is considered low [26].

In a Muse cell transplantation experiment with a murine liver fibrogenesis model, recognition and accumulation to the damaged organ was observed [15]. As for hepatic regeneration, human Muse cells transplanted into mice model with 70% liver resection, Muse cells differentiated into not only hepatocytes but also bile duct epithelial cells, sinusoidal endothelial cells, and Kupffer cells [27]. However, the efficacy of Muse cell transplantation in large animals has not been investigated before clinical application to human end-stage liver diseases. Liver of mini pigs (Göttingen mini pig) has similar external appearance, volumes, and hematological/biochemical levels to human livers. Thus, we prepared a liver fibrosis model using carbon tetrachloride (CCl₄) to mini pigs to induce hepatic fibrosis model and examined the therapeutic effect of Muse cells transplantation assessed by several indicators.

2. Materials and Methods

3. Result

3.1. Induction of Liver Injury and Liver Fibrosis in Mini Pig Models

The initial dose of 0.15 mg/kg of CCl₄ killed four of the eight animals (50% lethality after 1 day) and the initial dose of 0.12 mg/kg killed one of the eight animals (12.5% lethality after 1 day; Table 1). After an initial dose of 0.12 mg/kg, most of the animals survived the subsequent weekly doses of 0.15 mg/kg. Therefore, as a model protocol for liver fibrosis, CCl₄ was administered at 0.12 mg/kg for the first time, followed by 0.15 mg/kg/week for 12 weeks. The body weight and blood and biochemical findings before (at the beginning of the experiment) and after 12 weeks of administration of CCl₄ were similar to those observed in humans (Table 2). Liver biopsy specimen after 12 weeks of repeated intraperitoneal administration of CCl₄ showed inflammatory cell infiltration and fibrosis formation in the liver parenchyma, similar to that of human cirrhosis (Figure 1).

Table 1. Initial dose of carbon tetrachloride and its lethality in mini pigs after 1 day of CCl₄ administration.

CCl4 dosage (mg/kg)	Total number of individuals	Number of deaths	Number of survivors	Lethality (%)
0.12	8	1	7	12.5
0.15	8	4	4	50.0

Table 2. Laboratory tests before and 12 weeks after CCl₄ administration.

Checklist	Before carbon tetrachloride administration (mean ± standard error) (n = 19)	12 weeks after carbon tetrachloride administration (mean ± standard error) (n = 19)
Weight (kg)	24.5 ± 4.9	23.1 ± 4.4
T.Bil (mg/dL)	0.11 ± 0.04	0.14 ± 0.06
AST (U/L)	23.0 ± 11.5	57.1 ± 30.6
ALT (U/L)	29.0 ± 4.7	82.8 ± 37.1
γ-GTP (U/L)	43.8 ± 6.5	67.7 ± 19.5
ALP (U/L)	199.2 ± 47.9	189.8 ± 54.7
TP (g/dL)	6.73 ± 0.36	6.77 ± 0.48
Alb (g/dL)	4.71 ± 0.23	4.25 ± 0.48
NH3 (μg/dL)	105.8 ± 17.6	95.1 ± 27.8
WBC (/μL)	6721 ± 1,031	6879 ± 1,382
Hb (g/dL)	11.4 ± 1.0	10.5 ± 2.4
Plt (×10/L)3μ	431.1 ± 99.6	613.5 ± 135.2
PT (%)	68.4 ± 8.3	73.3 ± 9.8

• Abbreviations: γ-GTP, gamma-glutamyl transpeptidase; Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCl₄, carbon tetrachloride; Hb, hemoglobin; NH₃, ammonium; Plt, platelet counts; PT, prothrombin time; T.Bil, total bilirubin; TP, total protein; WBC, white blood cell.

3.3. Evaluation of Pig Muse Cells and MSCs After Transplantation

In the liver tissues of the Muse and MSC groups, 1.6 ± 0.2 and 1.2 ± 0.2 GFP (green) and Alb (red) co-positive cells (yellow) were observed in average of one 400x field of view, respectively, confirming the engraftment and differentiation into hepatocytes in both groups (Figure 2).

3.4. Evaluation of Hepatic Reserve and Fibrosis After Cell Transplantation

There were no significant differences among the three groups about the backgrounds at the time of cell transplantation. Regarding the laboratory parameters (T. Bil, AST, ALT, Alb, ammonia (NH₃)), white blood cell count, hemoglobin, and PT%, there were significant differences among the three groups (Table 3). At 4 weeks after transplantation, there was a significant improvement in serum Alb levels in the Muse cell group among the three groups (4.35 ± 0.31 g/dl before transplantation and 4.66 ± 0.19 g/dl at 4 weeks, p < 0.05, Figure 3a). Similarly, NH₃ levels in the MSC group showed significant decrease compared to before transplantation (106.5 ± 29.4 μg/dl before transplantation and 74.6 ± 14.6 μg/dl at 4 weeks, p < 0.05, Figure 3b). Regarding AST values, AST levels in the MSC group showed significant improvement (65.6 ± 38.9 IU/L before transplantation and 23.5 ± 7.2 IU/L at 4 weeks, p < 0.05, Figure 3c). ALT values significantly improved in each group 4 weeks after transplantation (26.6 ± 6.2 U/l in the Vehicle group, 27.3 ± 2.5 U/l in the MSC group, and 29.0 ± 4.6 U/l in the Muse group, p < 0.05, Figure 3d).

Table 3. Laboratory tests of vehicle, MSC, and Muse cell groups at the time of cellular transplantation.

Items	Vehicle (n = 7) (mean ± standard error)	MSC (n = 6) (mean ± standard error)	Muse (n = 6) (mean ± standard error)	p Value
Age (month)	14.1 ± 1.1	15.6 ± 1.3	15.3 ± 2.0	N.S.
T.Bil (mg/dL)	0.14 ± 0.05	0.16 ± 0.10	0.13 ± 0.05	N.S.
AST (U/L)	45.0 ± 22.4	65.6 ± 38.9	61.5 ± 35.7	N.S.
ALT (U/L)	78.5 ± 35.3	62.5 ± 33.3	87.8 ± 31.6	N.S.
Alb (g/dL)	4.30 ± 0.67	4.25 ± 0.24	4.35 ± 0.31	N.S.
NH3 (μg/dL)	83.2 ± 14.4	106.5 ± 29.4	102.5 ± 39.7	N.S.
WBC (/μL)	7084 ± 1158	6373 ± 1777	6778 ± 1438	N.S.
Hb (g/dL)	10.1 ± 3.8	10.4 ± 0.73	11.8 ± 1.69	N.S.
PT (%)	74.4 ± 14.8	68.6 ± 5.75	75.0 ± 6.89	N.S.

PCNA immunostaining showed a significant increase in positive cells in the Muse and MSC groups compared to the Vehicle group 4 weeks after transplantation (0.0029 ± 0.0002 in the Vehicle group vs 0.0091 ± 0.0040 in the MSC group, p < 0.05:0.0029 ± 0.0002 in the Vehicle group vs Muse group 0.0080 ± 0.0036, p < 0.05, Figure 4). Evaluation of liver fibrosis by Elastica Masson staining showed a significant reduction in the Muse group compared to the Vehicle group, with significant fibrosis improvement (Vehicle group 0.073 ± 0.005 vs MSC group 0.067 ± 0.025, p < 0.05, Figure 5). α-SMA immunostaining, significantly decreased α-SMA positive area in the Muse group compared to the Vehicle and MSC groups (0.0041 ± 0.0002 in the Vehicle group vs 0.0048 ± 0.0015 in the MSC group, 0.0041 ± 0.0002 in the Vehicle group vs 0.0025 ± 0.0004 in the Muse group, p < 0.05, Figure 6).

4. Discussion

In this study, we successfully established the mini pig model of hepatic fibrosis by repeated intraperitoneal administration of CCl4. Also, the transplantation of Muse cells into the mini-pig was safe as like MSC transplantation. Furthermore, cellular transplantation in hepatic fibrosis model resulted in an increase in hepatic reserve capacity and cell proliferation activity, as well as improvement of hepatic fibrosis, demonstrating the usefulness of Muse cell transplantation for the first time in large animal models.

Recently, ES cells, iPS cells, and MSCs have been candidates for transplantation cell sources as a liver regeneration therapy, and basic research has been conducted on each of them [28–30]. However, the risk of tumorigenesis is a concern with utilization of ES cells and iPS cells. Muse cells are considered to be pluripotent-like stem cells with low risk of tumorigenesis after transplantation because, unlike ES and iPS cells, they do not require fertilized eggs or exogenous gene transfer. In the area of liver diseases, improvement of hepatic fibrosis has been reported by transplanting Muse cells into a mouse model of hepatic fibrosis [27]. However, these beneficial effects were not evaluated in large animals, thus basic research using large animals is needed for future clinical application to humans.

In this study, we first established a hepatic fibrosis model in mini pigs for the first time. To do this, we fine-tuned the dosage of CCl₄ since the ordinal dosage used in rodents were fatal for female mini pigs since half of those pigs died of hepatic failure within 48 h at this dosage. This may be due to the high sensitivity of mini pigs to carbon tetrachloride which has not been reported elsewhere. The authors reduced this fatality by reducing the initial dose of CCl₄ from 0.15 to 0.12 mg/kg, and increased the maintenance dose as 0.15 mg/kg to maintain hepatic fibrosis assuring the safety of surviving animals. In addition, even CCl₄ administration was discontinued at cellular transplantation, we confirmed that hepatic fibrosis was maintained in the liver tissue harvested after 12 weeks of discontinuation (Figures 5 and 6). Furthermore, the macroscopic appearance, hepatic pathology, and laboratory tests were similar to those of humans (Table 2), suggesting that our mini pig model is an appropriate large animal model for future clinical application to humans.

Four weeks after cellular transplantation of allogeneic Muse cells into a mini-pig liver fibrosis model, GFP and Alb co-expressing cells were observed in the liver tissue, confirming the differentiation of Muse cells into recipient’s hepatocytes (Figure 3). Compared to the reported mouse model (Muse cell engraftment rate: ~6%) [27], the number of GFP-Alb co-positive cells in this study was 1.6 ± 0.2 cells/view (400x) in the Muse group and 1.2 ± 0.2 cells/view (400x) in the MSC group, indicating that fewer cells were engrafted in our model. The reasons for this may include the possibility that the transplanted Muse cells were eliminated by the immune response because the previous mouse model used an immunodeficient model, whereas our study used a normal mini pig model, thus, most of transplanted cells in our model could be eliminated during differentiation into hepatocyte. This issue will be addressed by autologous cellular transplantation model in future studies.

PCNA staining showed a significant increase of PCNA-positive cells in the Muse and MSC group compared to the Vehicle group at 4 weeks after cell transplantation (Figure 4), indicating that cell proliferation activity was enhanced in both cellular transplantation groups. Blood biochemical tests showed a significant increase in Alb levels, and a decreasing tendency in NH₃ levels in the Muse group at 4 weeks after cell transplantation. These results implicated that Muse cell transplantation can improve liver function and regeneration in the liver.

In our study, significant improvement of hepatic fibrosis was observed in the Muse and MSC transplanted groups compared to the vehicle group at 12 weeks after transplantation (Figures 5 and 6). The mechanism involved in this improvement may be derived from possible humoral factors secreted from Muse cells. In a previous study in a mouse model, it was confirmed that MMP-9 was significantly increased in Muse cell transplantation compared to MSCs [27], thus, the similar mechanism may be involved in the present study. Our recent model using surgical hepatic resection model similarly demonstrated the involvement of MMP-9 which is highly expressed in Muse cells [27]. Moreover, in that study, we found mini pig Muse cells could spontaneously differentiate into hepatocytes [28]. In addition, Wakao et al. [31] demonstrated differentiation of human Muse cells into hepatocytes in vitro. Conversely, MSCs are shown to also exhibit phagocytic activity; however, their differentiation potential remains limited, and although they differentiate into cells of the same mesodermal lineage (etc., chondrocytes and adipocytes), they do not differentiate beyond the germline [31]. In this previous report, they showed that Muse cells phagocytose dead differentiated cells (in this case, dead hepatocytes) and recycle the natural pardon factors that were functioning in the hepatocytes, resulting in efficient differentiation in a short period of time [31]. In the brain, it has also been shown in vivo that Muse cells phagocytose the dead neurons and differentiate into neurons [31]. Apart from this mechanism, hepatic stellate cells are known to play the central role in hepatic fibrosis and apoptosis and inactivation of stellate cells as well as antifibrotic chemokines such as CX3CR1 [32, 33] could be evaluated in mini pigs model in future different studies. Moreover, more recent sophisticated techniques such as applying single cell analysis will help our understanding of the precise mechanisms of in vivo cellular mechanisms [34].

Until now, liver fibrosis models have mainly been created in small animals such as mice and rats, although it is unrealistic to apply the dosage of small animals to human applications. Thus, establishing large animal models seems to be feasible to develop novel cell transplantation therapy before human clinical trials. Recently, mini pig hepatectomy model has been reported to apply Muse cell transplantation of surgical hepatic failure model [27]. With this context, we believe that the results of our study strongly support the usefulness of Muse cell transplantation in human diseases such as end-stage liver cirrhosis.

One limitation with this study is that the number of studies is still small. The number of studies was limited due to the expensive cost of mini pigs resulting from limited number of pigs available in Japan for medical experiments. Another limitation is the large individual differences among the mini pigs. In this study, although laboratory studies at the time of liver injury showed no significant differences among the groups, there was a large variation in regard of body weight, which may affect the individual response to the cellular transplantation. We believe this issue could be addressed in future studies. Last, low availability of the molecular and immunohistochemical agents made it difficult to apply modern molecular or immunological approach to analyze possible mechanisms observed in our study. This is one of the fundamental and intrinsic disadvantages of using mini pigs.

In conclusion, although further investigation with more large numbers of animals is needed, current study demonstrates that Muse cell transplantation seems to be safe and beneficial to ameliorate hepatic fibrosis in large animal models. Future studies seem to be feasible to confirm our observations to bridge possible human clinical trials.

Ethics Statement

Approval of animal studies: Yamagata University Animal Experiment Committee Approval No. 28082.

Conflicts of Interest

All of the authors do not have potential conflict of interests regarding this study other than external grant source described funding source.

Funding

This study is supported in part by Japan Agency for Medical Research and Development (AMED) (Grants 15545342 [Mari Dezawa, MIchiaki Unno and Yoshiyuki Ueno]), by Japan Society for the Promotion of Science (JSPS) (Grants 23K1500200 [Taketo Nishina], 21K07998 [Hiroaki Haga], 23K15063 [Keita Maki], 23K0740900 [Tomohiro Katsumi], and 22K07954 [Kyoko Tomita Hoshikawa]), and by Ministry of Education, Culture, Sports, Science and Technology (Grants 19H03632 [Yoshiyuki Ueno] and 23K24313 [Yoshiyuki Ueno]).