Hypoxia-Enhanced Wharton's Jelly Mesenchymal Stem Cell Therapy for Liver Fibrosis: A Comparative Study in a Rat Model

Abstract

Liver fibrosis is a progressive disease that can lead to cirrhosis and liver failure, with limited treatment options. Wharton's jelly-derived MSCs (WJ-MSCs) have immunomodulatory and antifibrotic potential. Hypoxia preconditioning enhances MSC survival and paracrine activity, but its effects in liver fibrosis remain unclear. This study compares hypoxia and normoxia WJ-MSCs in a CCl₄-induced liver fibrosis rat model. Sprague-Dawley rats received chronic CCl₄ to induce fibrosis. At Week 8, normoxia or hypoxia WJ-MSCs were injected via the portal vein. Liver function was assessed using biochemical markers (ALT, AST, T-Bil, albumin), PET/MR imaging, and qPCR for IL-1β and IL-6. Fibrosis regression was evaluated via ultrasound, histology, and collagen quantification. Regeneration was analyzed through Ki67 immunostaining and qPCR for Ki67 and HGF. MSC engraftment was determined by hNA immunohistochemistry. Both normoxia and hypoxia WJ-MSCs improved liver function, with hypoxia WJ-MSCs showing greater AST and T-Bil reductions. PET/MR imaging demonstrated superior metabolic recovery in the hypoxia group, with greater ¹⁸F-FDG uptake reduction. Histological analysis confirmed more significant fibrosis regression and collagen reduction in the hypoxia group. Gene expression analysis showed stronger suppression of TGF-β, α-SMA, and collagen I. Liver regeneration markers Ki67 and HGF were significantly upregulated with a greater HGF increase in the hypoxia group. Additionally, hypoxia WJ-MSCs exhibited higher engraftment and reduced pulmonary entrapment, indicating improved liver homing. Both normoxia and hypoxia WJ-MSCs improved liver fibrosis, but hypoxia preconditioning further enhanced liver function, fibrosis regression, and metabolic recovery, supporting its therapeutic superiority.

Keywords: Wharton's jelly mesenchymal stem cells (WJ‐MSCs); fibrosis regeneration; hypoxia preconditioning; liver fibrosis; stem cell therapy.

FIGURE 1

Ultrasound imaging analysis of liver fibrosis progression in rats. Ultrasound imaging was performed for the rat's liver at Week 0, 4, 8, and 12. These images represented the right lobe of the liver of the different groups. The liver of the fibrosis group at Week 8 and Week 12 showed obvious macronodular cirrhotic change of the parenchyma with a mild to moderate amount of ascites (white arrow) while the liver of the normoxia WJ‐MSCs and hypoxia WJ‐MSCs transplantation groups showed only mild macronodular cirrhotic change without ascites.

FIGURE 2

Serum biochemical analysis in rats over a 12‐week period. Blood samples were collected at Week 0, 2, 4, 6, 8, 10, and 12 for serum biochemical analysis. The values of AST (A), ALT (B), total bilirubin (C), and albumin (D) were measured. *p < 0.05, compare with control group. ^# p < 0.05, compare with fibrosis group. ^& p < 0.05, compare with normoxia WJ‐MSCs group.

FIGURE 3

PET/MR imaging and gene expression reveal metabolic and inflammatory changes in rat liver fibrosis. Abdominal PET/MR imaging of rats at Week 8, coronary section (A). Abdominal PET/MR imaging of rats at Week 12, coronary section (B). Quantitative real‐time PCR of the inflammatory gene expression of the rat's liver, including IL‐1β (C) and IL‐6 (D). *p < 0.05, compared with control group. ^# p < 0.05, compared with fibrosis group. ^& p < 0.05, compared with normoxia WJ‐MSCs group.

FIGURE 4

Liver fibrosis morphology, quantification, and MSC effects on fibrosis‐related gene and protein expression. H&E staining, Masson's trichrome staining, and Sirius Red staining of rat liver at Week 12 (A), scale bar = 100 μm. Quantitative analysis of the liver fibrosis area at Week 12 (B). Collagen content analysis of the liver tissue using a collagen assay at Week 12 (C). Quantitative real‐time PCR of the fibrosis‐related gene expression of the rat's liver, including TGF‐β, α‐SMA, and Collagen 1 at Week 12 (D). *p < 0.05, compare with control group. ^# p < 0.05, compare with fibrosis group. ^& p < 0.05, compare with normoxia WJ‐MSCs group.

FIGURE 5

Analysis of liver regeneration markers in rat liver. Immunofluorescence staining of Ki‐67 at Week 12 (A). Scale bar = 200 μm. Quantification of the percentage of the Ki‐67 positive cells in rat liver at Week 12 (B). Quantitative real‐time PCR of the liver regeneration‐related gene expression of the rat's liver, including Ki‐67 and HGF at Week 12 (C). *p < 0.05, compare with control group. ^# p < 0.05, compare with fibrosis group. ^& p < 0.05, compare with normoxia WJ‐MSCs group.

FIGURE 6

Detection of MSC presence in liver and lung tissues at Week 12. Immunofluorescence staining of human nuclear (yellow arrow) in liver of WJ‐MSCs transplantation group rat at Week 12 (A). Immunofluorescence staining of human nuclear (yellow arrow) in lung of WJ‐MSCs transplantation group rat at Week 12 (B). Scale bar = 100 μm.