Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study

Abstract

Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most common chronic liver disorder and can progress to steatohepatitis and fibrosis; although approved pharmacotherapies for metabolic dysfunction–associated steatohepatitis (MASH) with fibrosis remain limited. Autologous chemically induced liver progenitor (CLiP) cells, generated from mature hepatocytes without genetic modification, have shown therapeutic promise in rodents, but their efficacy has not been tested in large animals. Six female Clawn miniature pigs (15–42 kg) were fed a high-fat, high-cholesterol diet to induce MASLD with biopsy-proven fibrosis (Brunt stage ≥1). Animals were assigned to CLiP transplantation (n = 3) or saline control (n = 3). Autologous CLiPs (5 × 10⁷) were generated from laparoscopically resected liver wedges, expanded ex vivo, and infused intraportally. Safety was assessed by monitoring, liver function tests, and lipid profiles. Efficacy was evaluated 1 month later by blinded histology and immunohistochemistry. CLiP transplantation was feasible and well tolerated. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) showed minimal changes in both groups, but total cholesterol and triglycerides decreased in treated pigs and increased in controls. Histologically, two of three CLiP-treated livers regressed from Brunt stage 1 to 0, with resolution of steatosis and reduced stellate cell activation, whereas controls showed no regression. These findings support CLiP therapy as a regenerative option for MASLD.

Keywords

chemically induced liver progenitor liver regeneration cell therapy anti-fibrotic metabolic dysfunction–associated steatotic liver disease porcine model

Introduction

Chronic liver diseases, including metabolic dysfunction–associated steatotic liver disease (MASLD)—referred to as nonalcoholic fatty liver disease (NAFLD) in earlier literature—represent a leading cause of global morbidity and mortality^1–4. MASLD, defined by hepatic steatosis in the context of metabolic dysregulation, affects approximately one-quarter of the adult population and can progress to metabolic dysfunction–associated steatohepatitis (MASH) and liver fibrosis^5–7. Although resmetirom, in combination with diet and exercise, was approved by the US Food and Drug Administration (FDA) in 2024 for adult patients with non-cirrhotic MASH and moderate to advanced fibrosis (F2-F3), drug treatment options remain limited.

Consequently, liver transplantation (LT) remains the only definitive therapy for end-stage disease^6,8. However, LT is constrained by donor organ shortages and procedure-related complications, underscoring the urgent need for alternative therapeutic approaches to prevent or reverse disease progression before liver failure ensues.

Recent advances in regenerative medicine have identified cell-based therapies as promising alternatives to LT for the treatment of chronic liver disorders. One such approach involves the direct infusion of isolated therapeutic cells into the patient’s liver^9,10. Among these, hepatocyte transplantation is the most extensively studied modality, with numerous early-phase trials employing donor-derived primary hepatocytes^9,11,12. However, the inability of mature hepatocytes to proliferate in vitro severely limits their availability and scalability, posing a significant barrier to widespread clinical application^11,13–15. To address the limited availability of primary hepatocytes, alternative cell sources such as pluripotent stem cell (PSC)/induced pluripotent stem cell (iPSC)-derived hepatic cells and organoid-based platforms have been explored^16–19. However, PSC/iPSC-based approaches may face translational barriers, including the need for genetic reprogramming and potential tumorigenicity, which currently limit their clinical applicability^20–22.

An emerging, clinically scalable alternative involves chemically induced liver progenitor cells (CLiPs), which are generated through the direct reprogramming of mature hepatocytes into bipotent progenitor-like cells using small-molecule compounds^23–25. Katsuda et al.²³ first reported that terminally differentiated rodent hepatocytes could be converted into proliferative progenitor cells capable of liver regeneration through treatment with a defined chemical cocktail. CLiPs exhibit robust proliferative capacity in vitro and can differentiate into functional hepatocytes in vivo without the need for exogenous gene integration^23,24. Importantly, under specific conditions, CLiPs can also differentiate into cholangiocyte-like cells, reflecting their bipotential nature²⁵. Given the absence of genetic manipulation in their induction, CLiPs are anticipated to offer a more favorable safety profile compared to iPSC-derived cells^23,24,26,27.

Proof-of-concept studies in rodent models have validated the therapeutic potential of CLiPs^23,26,28,29. In particular, transplantation of CLiPs into mice with diet-induced steatohepatitis has been shown to restore liver function, reduce fibrosis, and attenuate liver injury^26,28. Engrafted CLiPs contributed to hepatic repopulation and were associated with decreased serum liver injury markers and reduced collagen deposition^26,28. However, translating these findings into a large-animal model is a crucial step toward clinical application. Pigs are considered an ideal preclinical model due to their anatomical and physiological similarities to humans³⁰.

In this study, we aimed to assess the safety and efficacy of autologous CLiP transplantation in a miniature pig model of MASLD. We hypothesized that infusion of autologously derived CLiPs into metabolically dysfunctional livers would enhance liver regeneration, reduce steatosis and fibrosis, and improve liver function. This large-animal study serves as a critical preclinical step toward the clinical application of CLiP-based therapies for chronic liver disease.

Materials and methods

Study design and timeline

The study consisted of (1) diet-induced MASLD establishment, (2) post-induction histological assessment by percutaneous liver biopsy, (3) laparoscopic partial hepatectomy to procure autologous hepatocytes for CLiP generation, (4) intraportal infusion of autologous CLiPs, and (5) a 1-month follow-up under continued high-fat/high-cholesterol feeding with serial blood sampling and endpoint necropsy for histological evaluation. The control group underwent the identical diet induction, surgical procedures, and intraportal infusion protocol and received saline vehicle without cells (sham/vehicle control). Thus, the only difference between groups at the time of infusion was the presence or absence of CLiPs in the infused solution. A schematic overview of the timeline, interventions, and sampling points is provided in Supplemental Figure S1.

Animals

Six female Clawn miniature pigs (15–42 kg) were obtained from Kyoto Animal Inspection Center Co. (Kyoto, Japan). Animals were housed and maintained at the Kyoto Animal Inspection Center, and all experimental procedures were performed either at this facility or at the Intervention Technical Center Kobe Laboratory (IVTeC Kobe Lab, Kobe, Japan). All experimental protocols were reviewed and approved by the Animal Ethics Committees of both institutions, under approval numbers IVT23-28 (IVTeC) and ET229038, and ET239058 (Kyoto Animal Inspection Center). To induce steatotic liver disease, all animals were fed a high-fat, high-cholesterol diet. Following this dietary regimen, each underwent percutaneous liver biopsy to evaluate post-induction liver histology. Only animals demonstrating hepatic fibrosis of stage 1 or higher, as defined by Brunt’s histological classification for nonalcoholic steatohepatitis (NASH), were included in the interventional phase of the study. Pigs were sedated with an intramuscular injection of ketamine (10 mg/kg) and xylazine (2 mg/kg). Anesthesia was induced by inhalation of high-concentration isoflurane, followed by tracheal intubation. During surgical procedures, anesthesia was maintained with inhaled isoflurane, and lactated Ringer’s solution was administered intravenously for fluid support.

Hepatocyte isolation and reprogramming of hepatocytes into CLiPs

The protocol for hepatocyte isolation and reprogramming into CLiPs has been described previously^31,32. Briefly, all pigs underwent a laparoscopic partial hepatectomy under general anesthesia, during which approximately 30 g of liver tissue was resected. The excised liver was immediately flushed with Hank’s Balanced Salt Solution, rinsed with ice-cold University of Wisconsin Solution, and transported to Nagasaki University under cold preservation conditions. This workflow was deliberately designed to simulate a clinically feasible autologous CLiP preparation protocol. After approximately 5 h of transport, porcine mature hepatocytes (MHs) were isolated from the resected liver tissue using a modified two-step collagenase perfusion method³².

Isolated porcine MHs were seeded onto collagen-coated dishes (Asahi Techno Glass, Tokyo, Japan) at a density of 2.0 × 10⁴ cells/cm² in Hepato-STIM Culture Medium (Corning, Bedford, MA, USA) to facilitate cell attachment. STIM medium was prepared using the Hepatocyte Culture Media Kit and supplemented with 10 ng/ml epidermal growth factor (EGF), 1× penicillin-streptomycin-glutamine (100×; Gibco, Waltham, MA, USA), and 10% fetal bovine serum (FBS; Gibco). After 1 day of culture, the medium was replaced with a chemically reprogramming medium consisting of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Life Technologies) supplemented with 2.4 g/L NaHCO₃, l-glutamine, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 30 mg/L L-proline, 0.05% bovine serum albumin, 10 ng/ml EGF (all from Sigma-Aldrich Japan, Tokyo, Japan), insulin-transferrin-selenium-Ethanolamine (ITS-X; Life Technologies), 10⁻⁷ M dexamethasone (Fuji Pharma Co. Ltd., Tokyo, Japan), 10 mM nicotinamide (Sigma-Aldrich Japan), 1 mM ascorbic acid-2 phosphate (Wako Pure Chemical), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). The medium also contained three small-molecule inhibitors: 10 μM Y-27632 (AdooQ BioScience, Irvine, CA, USA), 0.5 μM A-83-01 (Wako Pure Chemical), 3 μM CHIR99021 (AdooQ BioScience), and 20 ng/ml recombinant hepatocyte growth factor.

The culture medium was refreshed every 2 or 3 days. Porcine CLiPs reached approximately 90% confluency within 14–16 days after reprogramming of MHs³¹. Cultures were maintained through serial passaging for approximately 2 months, with a final yield of approximately 5 × 10⁷ viable CLiPs per pig for transplantation. Phenotypic and functional characteristics of porcine CLiPs derived from diet-induced MASLD pigs were supported by our prior report³¹. In the present study, EpCAM expression in the CLiP population used for transplantation was confirmed by immunofluorescence (Supplemental Figure S2).

CLiP administration

The six pigs that met the fibrosis threshold were divided into two groups: three assigned to the CLiP transplantation group and three serving as untreated controls. Once autologous CLiPs were prepared, pigs in the CLiP group underwent cell infusion via the portal circulation. Each treated pig received CLiPs generated from its own resected liver specimen (autologous, individual-specific preparation; no pooling between animals). Under general anesthesia, a midline laparotomy was performed, and a catheter was inserted into the splenic vein to access the portal vein. Approximately 5 × 10⁷ CLiPs suspended in saline were slowly infused through the splenic vein. The CLiP dose (5 × 10⁷) was set at approximately 1/1000 of the estimated total hepatocyte number in miniature pigs, based on scaling from our prior rodent studies. Pigs in the control group underwent the same surgical procedure, including midline laparotomy and splenic vein cannulation, but received an infusion of cell-free saline instead. All transplantation and sham infusion procedures were performed under sterile surgical conditions at the IVTeC Kobe facility. Following transplantation, all animals were returned to the Kyoto Animal Inspection Center (Kyoto, Japan) for postoperative care in a dedicated animal facility. Pigs were housed individually and received daily veterinary monitoring throughout a 1-month postoperative period. During this time, all animals remained on the high-fat, high-cholesterol diet to maintain metabolic stress and to allow for potential physiological changes associated with CLiP engraftment.

Histological examination

Liver tissue samples were collected at three key time points during the study: post-induction, pre-transplantation, and endpoint. At post-induction—following the high-fat, high-cholesterol diet induction and prior to any intervention—a percutaneous needle liver biopsy was performed on each animal to assess pretreatment histology and fibrosis stage. Pre-transplantation samples were obtained during laparoscopic partial liver resection to represent the liver status before cell infusion. At the study endpoint (1 month after transplantation), all animals were euthanized under deep anesthesia, and complete necropsies were performed. Extensive liver tissue was collected at that time to evaluate both macroscopic and histological changes, including steatosis, inflammation, and fibrosis stage. All liver specimens were fixed in 10% neutral-buffered formalin, paraffin-embedded, and sectioned for histological analysis. Staining protocols included hematoxylin and eosin (H&E) for assessment of general hepatic architecture, reticulin silver staining to visualize the reticulin fiber network and determine the extent of fibrosis, and immunohistochemical staining for desmin to evaluate hepatic stellate cell activation. All histopathological assessments were conducted in a blinded manner to ensure unbiased evaluation. Semi-quantitative scoring was used to evaluate steatosis, lobular inflammation, and hepatocellular ballooning according to the NASH CRN/NAFLD Activity Score framework (steatosis 0–3, lobular inflammation 0–3, ballooning 0–2). Fibrosis was staged using the NASH CRN/Brunt fibrosis stage (0–4)³³. Hepatic stellate cell activation was assessed on desmin immunohistochemistry using an ordinal activation score (0–3; 0, none; 1, mild; 2, moderate; 3, marked). In addition to blinded semiquantitative histopathological scoring, image-based quantitative analysis was performed for steatosis and desmin immunostaining. Lipid droplet area ratio (%) was quantified on H&E-stained sections as the area of optically empty vacuolar spaces consistent with lipid droplets divided by the analyzed parenchymal region of interest (ROI) area. Desmin-positive area fraction (%) was quantified on desmin-immunostained sections as the desmin-positive area divided by the analyzed ROI area (%). For each animal and time point, five non-overlapping microscopic fields at fixed magnification were analyzed using predefined ROIs, and field-level values were averaged to generate one value per animal/time point. These quantitative values are shown in Table 1 and are presented as mean ± SD across analyzed fields.

Table 1.

Liver pathology scores before and after treatment in individual pigs.

Pig ID	Group	NAFLD activity score			Fibrosis stage (Pre → Post)	Stellate activation (Pre → Post)	Lipid droplet area ratio (%) (Pre → Post)	Desmin-positive area fraction (%) (Pre → Post)
Pig ID	Group	Steatosis (Pre → Post)	Lobular inflammation (Pre → Post)	Ballooning (Pre → Post)	Fibrosis stage (Pre → Post)	Stellate activation (Pre → Post)	Lipid droplet area ratio (%) (Pre → Post)	Desmin-positive area fraction (%) (Pre → Post)
Pig 1	CLiP	3 → 1	1 → 0	1 → 0	1 → 0	2 → 0	12.3 ± 1.0 → 2.8 ± 0.2	2.9 ± 1.2 → 0.2 ± 0.4
Pig 2	CLiP	1 → 0	1 → 0	0 → 0	1 → 0	1 → 0	8.6 ± 1.3 → 1.9 ± 0.3	3.6 ± 2.9 → 0.6 ± 0.9
Pig 3	CLiP	0 → 0	1 → 1	0 → 0	0 → 0	1 → 1	5.7 ± 0.3 → 6.2 ± 0.2	4.1 ± 1.4 → 3.8 ± 0.9
Pig 4	Control	1 → 2	0 → 1	0 → 0	1 → 1	1 → 1	5.2 ± 0.6 → 7.1 ± 0.5	3.1 ± 2.7 → 1.6 ± 0.7
Pig 5	Control	1 → 1	1 → 1	1 → 1	1 → 1	2 → 2	6.6 ± 0.5 → 6.8 ± 0.8	2.2 ± 1.4 → 3.2 ± 1.1
Pig 6	Control	0 → 0	0 → 0	0 → 0	1 → 1	1 → 1	4.5 ± 0.6 → 4.3 ± 0.3	1.3 ± 1.6 → 3.8 ± 3.4

Steatosis, lobular inflammation, and hepatocyte ballooning were graded according to the NAFLD Activity Score system (steatosis and inflammation, 0–3; ballooning, 0–2). Fibrosis stage (0 = none; 4 = cirrhosis) was evaluated on sections stained with a reticulin-based silver method using the NASH CRN/Brunt fibrosis stage. Hepatic stellate cell activation was assessed by desmin immunohistochemistry using a semiquantitative score (0 = none; 3 = extensive activation). In addition, steatosis and desmin immunostaining were quantified by image analysis as lipid droplet area ratio (%) on H&E-stained sections and desmin-positive area fraction (%) on desmin-immunostained sections, respectively. Pigs 1–3 received CLiP transplantation, and Pigs 4–6 served as controls. Two of three CLiP-treated pigs exhibited improvements in steatosis grade, fibrosis stage, and stellate cell activation score after treatment (the third pig had minimal baseline steatosis/fibrosis and remained unchanged), whereas the control pigs showed no improvement in these parameters.

Values for lipid droplet area ratio and desmin-positive area fraction are presented as mean ± SD of five non-overlapping microscopic fields analyzed per animal at each time point (Pre and Post).

Monitoring of biochemical and physiological parameters

Serum biochemistry and body weight were monitored throughout the study as indicators of safety and therapeutic response. Blood samples were collected starting at the initiation of the high-fat, high-cholesterol diet (Week 0), and subsequently at Weeks 4, 8, 12, 16, and 20 (pre-transplantation) to establish baseline levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (T-CHO), and triglycerides (TG). Additional samples were collected on the day of CLiP or saline infusion (Day 0, immediately prior to administration) and on post-infusion Days 1, 3, 7, and 28. For contextual interpretation, published reference intervals in miniature pigs are as follows: AST 14.3–363.45 IU/L, ALT 11.3–49.85 IU/L, total cholesterol 59.45–270.3 mg/dL, and triglycerides 3–141.45 mg/dL³⁴. Body weight was recorded biweekly during the pre-transplantation phase and at the same post-infusion time points as blood collection.

Statistical analysis

Given the pilot nature of this study and the small sample size (n = 3 per group), outcomes are primarily presented descriptively with individual animal-level data and summary statistics (mean ± SD). Exploratory nonparametric analyses were performed to describe potential time-dependent changes and group differences in serum biochemistry. For within-group repeated measures across time (days 0, 1, 3, 7, and 28 after transplantation), the Friedman test was used. Between-group comparisons at each time point were performed using two-sided Mann–Whitney U tests (exact method). No adjustment was made for multiple comparisons; therefore, P values should be interpreted cautiously as hypothesis-generating rather than confirmatory.

Results

Safety and feasibility of CLiP-based autologous cell therapy

In both the CLiP-transplanted and control groups, all interventions—including laparoscopic partial hepatectomy for CLiP preparation, intraportal cell (or saline) infusion via splenic vein cannulation, and perioperative management—were completed without complications. No clinically significant fluctuations in vital signs were observed during or after these procedures, and all animals remained in stable condition throughout the postoperative period. CLiP induction was successful in all three treated pigs (100% success rate), and intraportal transplantation was completed as planned in every case. Each infusion, performed via splenic vein cannulation, was completed within 10 min and was technically comparable to saline administration in the control group. At necropsy, no evidence of portal vein thrombosis or major organ injury was observed, further supporting the favorable safety profile of autologous CLiP transplantation in this preclinical model.

Liver histology outcomes

At pre-transplantation, all pigs exhibited macrovesicular steatosis (grade ≥1 by NAFLD Activity Score) except for one CLiP-treated pig with minimal fat accumulation (grade 0) (Figure 1a and b). One month after transplantation, hepatic steatosis was notably reduced in the CLiP group (Figure 1c). Two of the three treated pigs demonstrated histological improvement: one pig showed a reduction in steatosis from grade 3 to grade 1, and another from grade 1 to grade 0. The third pig remained at grade 0, having had no steatosis at pre-transplantation. In contrast, none of the control pigs exhibited improvement over the same period; in fact, one control animal demonstrated progression from grade 1 to grade 2 as the high-fat diet continued (Figure 1d).

Figure 1.

Representative histological changes after CLiP transplantation (H&E stain). Panels (a–d) show liver sections from one representative pig per group and time point. (a) CLiP-treated pig at pre-transplantation: severe macrovesicular steatosis with numerous large fat vacuoles and lobular inflammatory foci. (b) Control pig at pre-transplantation: similar macrovesicular steatosis and inflammatory cell infiltration. (c) CLiP-treated pig 1 month post-transplantation: marked reduction in fat vacuoles with near-complete resolution of lobular inflammation, restoring normal hepatocyte architecture. (d) Control pig 1 month post-saline infusion: persistent macrovesicular steatosis and inflammatory foci without histological improvement.

A similar pattern was observed for lobular inflammation. In two of the three CLiP-treated pigs, inflammatory foci completely resolved, with the inflammation score decreasing from 1 to 0 (Figure 1c). The third treated pig maintained a stable score of 1, indicating persistent but mild inflammation. By contrast, control animals showed no such resolution: two maintained the same inflammation score as at pre-transplantation (Figure 1d), while the third progressed from a score of 1 to 2, indicating increased lobular inflammatory activity.

Hepatocellular ballooning remained minimal (score 0–1) across all animals and did not change appreciably in either group during the study period. All pigs had only mild or no ballooning at pre-transplantation, and this parameter remained stable, likely reflecting the relatively short duration of diet-induced injury, which was insufficient to elicit more extensive ballooning degeneration.

At pre-transplantation, most animals exhibited mild perisinusoidal or periportal fibrosis (stage 1 on a Brunt stage). One month after transplantation, fibrosis showed regression in the CLiP-treated group. The two treated pigs that initially had stage 1 fibrosis showed complete resolution to stage 0, while the third pig—fibrosis-free at pre-transplantation—remained at stage 0. In stark contrast, none of the control pigs exhibited any improvement; all three remained at stage 1, with no reduction in collagen deposition observed. However, no progression to a higher fibrosis stage was seen in the control group over this short timeframe. These findings suggest that CLiP transplantation facilitated histological resolution of both steatosis and fibrotic changes, effects not observed in untreated controls.

Reticulin silver staining (Gomori’s method for type III collagen) corroborated these findings and revealed a reduction in the fine reticulin fiber network in CLiP-treated livers compared to controls (Figure 2a-d). Collagen fibers were diminished in centrilobular and periportal regions of CLiP-treated pigs, whereas control livers retained the dense fibrous lattice observed at pre-transplantation.

Figure 2.

Fibrosis changes in CLiP-treated and control livers (reticulin silver stain). Panels (a–d) show liver sections from one representative pig per group and time point. Reticulin-based silver staining highlights type III collagen fibers as black strands. (a) CLiP-treated pig at pre-transplantation: mild perisinusoidal fibrosis with thin reticulin-positive fibers around central veins (stage 1). (b) Control pig at pre-transplantation: similar mild fibrotic framework with delicate reticulin-positive fibers (stage 1). (c) CLiP-treated pig 1 month post-transplantation: near-complete loss of reticulin-positive fibers, consistent with regression to stage 0 fibrosis. (d) Control pig 1 month post-saline infusion: persistent reticulin-positive fibers with no appreciable change from pre-transplantation (stage 1).

Desmin immunohistochemistry, used as a marker of hepatic stellate cell activation, showed a substantial decrease in desmin-positive cells in the CLiP-treated group. This reduction was consistent with decreased stellate cell activation following transplantation. Quantitative scoring (0–3 scale) based on desmin staining revealed that two treated pigs had a decline in activation score to 0 (from pre-transplantation scores of 2 and 1), while the third pig maintained a low score of 1. In contrast, no decrease in stellate cell activation was observed in the control group, with all animals maintaining scores of 1–2. These staining results are consistent with the possibility that CLiP therapy may be associated with reduced hepatic fibrosis and attenuated the underlying fibrogenic activity mediated by hepatic stellate cells (Figure 3).

Figure 3.

Hepatic stellate cell activation in CLiP-treated and control livers (desmin immunohistochemistry). Panels (a–d) show representative liver sections from one pig per group and time point. Desmin-positive hepatic stellate cells are visualized as brown-stained cells in the sinusoidal regions (arrow). (a) CLiP-treated pig at pre-transplantation: numerous desmin-positive stellate cells distributed in the liver parenchyma (activation score 1–2). (b) Control pig at pre-transplantation: a similar abundance of desmin-positive stellate cells (activation score 1–2). (c) CLiP-treated pig 1 month post-transplantation: marked reduction in desmin-positive stellate cells (score 0–1). (d) Control pig 1 month post-saline infusion: stellate cell activation persists with abundant desmin-positive stellate cells (score 1–2).

A summary of individual histopathological findings before and after treatment, including blinded semiquantitative scores and image-based quantitative metrics (lipid droplet area ratio and desmin-positive area fraction), is provided in Table 1. Representative images for each animal (both groups) are provided in Supplementary Figure S3.

Taken together, these results suggest the therapeutic effect of CLiP transplantation in ameliorating MASLD-associated histopathology in this porcine model. However, because transplanted cells were autologous and not prospectively labeled, engraftment and survival could not be directly evaluated in this study; therefore, the observed changes should be interpreted as exploratory associations.

Biochemical and functional outcomes

Following induction with a high-fat, high-cholesterol diet, AST and ALT showed no consistent worsening after intraportal infusion and generally remained stable over the 1-month follow-up (Figure 4a and b). At day 28, T-CHO was lower in the CLiP-treated pigs (462.3 ± 89.6 mg/dL) than in controls (699.7 ± 127.4 mg/dL) (Figure 4c). TG was lower in the CLiP-treated pigs (13.0 ± 9.0 mg/dL) than in controls (60.0 ± 57.2 mg/dL) (Figure 4d).

Figure 4.

Time course of biochemical parameters. Blue circles (solid line) represent CLiP-treated pigs (n = 3) and red squares (dashed line) represent controls (n = 3). Data points (mean ± SD) are shown for Days 0, 1, 3, 7, and 28 post-transplant. (a) ALT (U/L). (b) AST (U/L). (c) Total cholesterol (T-CHO, mg/dL). (d) Triglycerides (TG, mg/dL). Serum ALT and AST levels remained within normal ranges and showed minimal change in both groups throughout the study period. T-CHO decreased in CLiP-treated pigs but increased in controls. TG also declined in the treated group, while rising in controls.

Exploratory nonparametric analyses did not identify significant between-group differences for AST or ALT at any time point (all P ≥ 0.65). For T-CHO and TG, between-group differences also did not reach statistical significance at individual time points, but showed trends at day 28 (T-CHO P = 0.10; TG P = 0.10). Within-group repeated-measures testing across time (Friedman test) did not show significant temporal changes for AST, ALT, or T-CHO in either group (all P ≥ 0.19); TG in the CLiP group showed a trend toward temporal change (P = 0.064). Group-level summary values are shown in Table 2, and raw individual-level serum biochemistry values at each time point are provided in Supplementary Table S1.

Table 2.

Summary of biochemical measurements.

Analyte	Group	Day 0	Day 1	Day 3	Day 7	Day 28	Δ(28-0)	Friedman p	Wilcoxon P (0 vs 28)	Δ P (MW)
AST	CLiP	34.0 ± 7.0	47.3 ± 18.9	33.3 ± 6.1	29.7 ± 6.5	32.7 ± 9.8	−1.3 ± 9.7	0.339	1.000	0.700
AST	Saline	51.0 ± 52.8	42.7 ± 15.5	38.0 ± 11.5	35.3 ± 9.0	33.3 ± 1.2	−17.7 ± 52.3	0.900	1.000	0.700
ALT	CLiP	14.7 ± 1.5	16.7 ± 2.1	13.3 ± 0.6	13.0 ± 2.0	13.0 ± 2.6	−1.7 ± 3.1	0.282	0.750	0.700
ALT	Saline	17.0 ± 3.5	15.7 ± 2.5	15.0 ± 3.6	14.7 ± 2.5	14.0 ± 1.0	−3.0 ± 3.6	0.767	0.180	0.700
T-CHO	CLiP	554.7 ± 158.8	393.7 ± 90.2	453.7 ± 62.2	455.3 ± 82.5	462.3 ± 89.6	−92.3 ± 178.1	0.549	0.500	0.200
T-CHO	Saline	447.7 ± 175.0	408.3 ± 78.8	431.7 ± 139.3	565.7 ± 102.7	699.7 ± 127.4	252.0 ± 299.9	0.189	0.500	0.200
TG	CLiP	30.0 ± 27.6	25.7 ± 11.8	67.3 ± 55.5	68.0 ± 53.8	13.0 ± 9.0	−17.0 ± 23.6	0.064	0.500	0.100
TG	Saline	21.0 ± 10.1	46.3 ± 34.5	29.3 ± 19.6	38.7 ± 9.5	60.0 ± 57.2	39.0 ± 48.1	0.483	0.250	0.100

Note. Values are shown as mean ± SD (n = 3 per group). Tests are exploratory; no multiple-comparison adjustment was applied.

Discussion

In this preclinical pilot study, autologous CLiP transplantation was feasible and well tolerated, and it was associated with exploratory histological and biochemical improvement trends in a large-animal diet-induced MASLD/MASH model.

Recent clinical translation efforts have evaluated hepatocyte-derived progenitor-like cell products in patients with Hepatitis B virus-related cirrhosis³⁵. In contrast, MASLD/MASH is driven by persistent metabolic dysfunction, lipotoxicity, and chronic inflammation, creating a disease milieu that can continue to injure the liver even after an intervention. Thus, demonstrating feasibility and exploratory signals consistent with improvement in a metabolically stressed liver represents a non-trivial translational step. To our knowledge, the present study provides a proof-of-concept evaluation of autologous CLiPs in a large-animal diet-induced MASLD model. Our intent is not to claim MASLD-specific mechanisms, but to establish that a clinically scalable autologous workflow (tissue procurement, ex vivo expansion, and portal delivery) can be implemented safely and may be associated with histological and metabolic improvement trends even under continued dietary challenge.

Mechanistically, CLiPs can differentiate into functional hepatocytes in vivo²³, which may increase the liver’s capacity to uptake and metabolize lipids, thereby reducing steatosis and circulating lipid levels. In addition, CLiPs likely exert paracrine effects, secreting regenerative or anti-fibrotic cytokines that suppress hepatic stellate cell activation and dampen inflammation. Previous studies in rodent MASH models have shown that transplanted CLiPs upregulate matrix-degrading enzymes (e.g. MMP-2 and MMP-9) and downregulate pro-fibrogenic pathways, supporting an anti-fibrotic mechanism²⁶. Consistent with this, our CLiP-treated pigs demonstrated reduced stellate cell activation and prevention of progression. Furthermore, crosstalk between CLiPs and hepatic immune cells—such as Kupffer cells or monocyte-derived macrophages—may help shift the intrahepatic environment toward an anti-inflammatory and pro-regenerative state^26,28,29. Clarifying these mechanisms will be essential for optimizing therapy and identifying biomarkers of response.

These local histological improvements were paralleled by systemic metabolic benefits. Serum cholesterol and triglyceride levels were numerically lower in the CLiP group than in controls, although these differences should be interpreted cautiously given the pilot sample size. This is consistent with improved hepatic lipid handling and reduced lipid export from the liver. Given that dyslipidemia is a core component of MASLD and an independent cardiovascular risk factor, such improvements could provide both hepatic and extrahepatic clinical benefits.

If translated into clinical practice, autologous CLiP transplantation could represent a viable therapeutic strategy for patients with MASLD/MASH. In practice, this would involve isolating hepatocytes from a portion of the patient’s liver (via biopsy or resected wedge), chemically reprogramming them into CLiPs ex vivo, expanding the cells, and re-infusing them into the patient’s liver, typically via the portal vein. Because the cells are autologous, the risk of immune rejection would be minimal, and long-term immunosuppression may be avoided. Recent work has shown the feasibility of generating human CLiPs from chronically damaged liver tissue using small-molecule cocktails, indicating that autologous CLiPs can be derived even from diseased livers³². Notably, the fact that CLiP therapy was associated with fibrosis regression in this model despite continued metabolic stress suggests a robust anti-fibrotic effect. This distinguishes CLiPs from dietary interventions and even from many pharmacological agents evaluated in MASH, which rarely demonstrate histologic fibrosis stage improvement over short treatment period^8,36–38. This approach could be particularly useful for patients with advanced fibrosis who have not yet progressed to decompensated cirrhosis, offering the possibility of regenerative intervention prior to the need for LT. Moreover, CLiPs could potentially be applied to other chronic liver diseases (e.g. alcoholic steatohepatitis, viral hepatitis) provided the underlying cause is adequately controlled.

Several challenges must be addressed before clinical translation. These include scaling up CLiP production to meet the demands of human liver volumes, ensuring genomic and epigenetic stability during expansion, and determining optimal timing and dosing of therapy. Repeated CLiP infusions or combination with other modalities (e.g. anti-inflammatory agents) may enhance therapeutic outcomes. Standardization of cell preparation protocols and long-term safety evaluation will also be essential.

This study has several limitations. First, the sample size was small (n = 3 per group), reflecting the logistical and ethical constraints of large-animal experiments; accordingly, inter-individual variability was evident, with one treated pig showing only minimal improvement. Second, because the transplanted cells were autologous and not prospectively labeled or genetically marked, we could not directly assess CLiP engraftment, survival, or engraftment efficiency in vivo. This limits mechanistic interpretation and precludes definitive causal attribution of the observed changes to CLiPs. Future studies will incorporate prospective tracking strategies (e.g. labeling) and study designs enabling unambiguous detection to quantify engraftment and persistence. Third, while the sham/vehicle control was appropriate for evaluating feasibility and procedure-related effects (i.e. the incremental impact of adding CLiPs to an otherwise identical intraportal infusion procedure), the absence of additional cell-type controls prevents us from distinguishing CLiP-specific effects from non-specific effects of cell infusion and ex vivo handling. Fourth, the 1-month follow-up allowed assessment of early histological and biochemical changes but remains relatively short, and we did not evaluate long-term durability of response or long-term engraftment. Long-term studies, including the potential need for repeated dosing (e.g. monthly infusions), are warranted. Finally, our diet-induced model resulted in mild-to-moderate fibrosis, limiting evaluation in more advanced disease. Models that extend the induction period or combine a high-fat diet with additional injurious stimuli may better recapitulate bridging fibrosis or early cirrhosis and allow evaluation of CLiPs in more advanced stages of liver injury, including whether any benefit depends on disease severity and ongoing metabolic stress.

Conclusions

Autologous CLiP transplantation is a promising regenerative strategy for MASLD. In a miniature pig model, intraportal delivery of autologous CLiPs was feasible and well tolerated and was associated with improvement trends in liver histology and serum biochemistry compared with vehicle controls. These findings suggest that CLiPs may support hepatic repair and functional recovery even under sustained metabolic stress. While further studies are needed to optimize dosing and timing and to evaluate durability, our results provide a preclinical foundation for translating CLiP-based regenerative therapy into clinical application. Ultimately, CLiPs may offer a novel avenue for reversing chronic liver injury in MASLD and potentially other liver diseases—an advance that could transform the treatment paradigm and reduce the burden of end-stage liver disease.

Supplemental Material

sj-docx-6-cll-10.1177_09636897261446732 – Supplemental material for Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study

Supplemental material, sj-docx-6-cll-10.1177_09636897261446732 for Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study by Takanobu Hara, Yasuhiro Maruya, Daisuke Miyamoto, Masayuki Fukumoto, Hajime Imamura, Hajime Matsushima, Akihiko Soyama, Tomohiko Adachi, Takahiro Ochiya and Susumu Eguchi in Cell Transplantation

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Supplemental material, sj-tiff-1-cll-10.1177_09636897261446732 for Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study by Takanobu Hara, Yasuhiro Maruya, Daisuke Miyamoto, Masayuki Fukumoto, Hajime Imamura, Hajime Matsushima, Akihiko Soyama, Tomohiko Adachi, Takahiro Ochiya and Susumu Eguchi in Cell Transplantation

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Supplemental material, sj-tiff-2-cll-10.1177_09636897261446732 for Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study by Takanobu Hara, Yasuhiro Maruya, Daisuke Miyamoto, Masayuki Fukumoto, Hajime Imamura, Hajime Matsushima, Akihiko Soyama, Tomohiko Adachi, Takahiro Ochiya and Susumu Eguchi in Cell Transplantation

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Supplemental material, sj-tiff-3-cll-10.1177_09636897261446732 for Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study by Takanobu Hara, Yasuhiro Maruya, Daisuke Miyamoto, Masayuki Fukumoto, Hajime Imamura, Hajime Matsushima, Akihiko Soyama, Tomohiko Adachi, Takahiro Ochiya and Susumu Eguchi in Cell Transplantation

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Supplemental material, sj-tiff-4-cll-10.1177_09636897261446732 for Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study by Takanobu Hara, Yasuhiro Maruya, Daisuke Miyamoto, Masayuki Fukumoto, Hajime Imamura, Hajime Matsushima, Akihiko Soyama, Tomohiko Adachi, Takahiro Ochiya and Susumu Eguchi in Cell Transplantation

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Supplemental material, sj-tiff-5-cll-10.1177_09636897261446732 for Autologous chemically induced liver progenitor cell transplantation ameliorates steatosis and fibrosis in a preclinical miniature pig model of metabolic dysfunction–associated fatty liver disease: A pilot study by Takanobu Hara, Yasuhiro Maruya, Daisuke Miyamoto, Masayuki Fukumoto, Hajime Imamura, Hajime Matsushima, Akihiko Soyama, Tomohiko Adachi, Takahiro Ochiya and Susumu Eguchi in Cell Transplantation

Footnotes

ORCID iDs

Takanobu Hara

Daisuke Miyamoto

Hajime Matsushima

Akihiko Soyama

Susumu Eguchi

Ethical considerations

All experimental protocols were reviewed and approved by the Animal Ethics Committees of both institutions, under approval numbers IVT23-28 (IVTeC; approved 20 May 2023) and ET229038 (Kyoto Animal Inspection Center; approved 8 September 2022) and ET239058 (Kyoto Animal Inspection Center; approved 29 March 2024).

Author contributions

TH and YM drafted the manuscript. TH, YM, DM, and MF performed animal experiment and collected data. HI, HM, AS, and TA critically reviewed and revised the manuscript. TO and SE conceived and designed the study, supervised the project, and led the research. All authors have approved the submitted version of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number JP24bk0104152. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The raw individual-level serum biochemistry data are provided in the Supplementary Table S1. Representative histological images from all animals are provided in the Supplementary Figures S3. Additional materials are available from the corresponding author upon reasonable request.

Statement of human and animal rights

This article does not contain any studies with human or animal subjects.

Statement of informed consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental material

Supplemental material for this article is available online.

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