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Human amniotic epithelial stem cells promote cholangiocyte regeneration via Notch signaling pathway in bile duct injury

Year: 2025

Type: article

Abstract: To the Editor: Bile duct injury (BDI) greatly impacts prognosis after liver transplantation (LT).[1] Cell therapies, including cholangiocyte organoids and human placental mesenchymal stem cells, have been reported for the regeneration and repair of different types of BDIs in preclinical animal models. Human amniotic epithelial stem cells (hAESCs) are a type of stem cell derived from the amniotic membrane of the placenta that possess embryonic stem cell-like proliferation and differentiation capabilities, can successfully modulate the regional immune environment and improve the situation in rat models of intrauterine adhesions and autoimmunity.[2] However, whether the transplantation of hAESCs can reduce BDIs in vivo has not been investigated. The present study was designed to investigate the therapeutic potential of hAESCs for intrahepatic BDIs and explore the underlying mechanisms involved. Detailed descriptions of the data and statistical methods are included in the Supplementary Materials, https://links.lww.com/CM9/C382. First, the morphology and immunophenotype of hAESCs were observed. hAESCs showed a typical appearance of epithelial cells, appearing as cobble-shaped cells under a light microscope [Supplementary Figure 1A, B, https://links.lww.com/CM9/C382]. Flow cytometry revealed that hAESCs were positive for the epithelial marker CD324 and negative for the hematopoietic lineage marker CD34, the endothelial marker CD146, and the immunogenicity marker human leukocyte antigen-DR isotype (HLA-DR), which demonstrate their low immunogenicity [Supplementary Figure 1C, https://links.lww.com/CM9/C382]. The main mechanism of BDI is direct injury to cholangiocytes caused by various risk factors, and the pathological manifestations are atrophy, stenosis, or even the absence of the small bile duct, accompanied by cholestasis and hyperplasia of fiber tissues in the junction area.[3] In this study, a mouse intrahepatic BDI model was established via intraperitoneal and direct bile duct injections of 4,4′-methylenedianiline (MDA) according to a previous study[4] [Supplementary Figure 2A, https://links.lww.com/CM9/C382]. BDI significantly affected the body weight of the mice, resulting in a jaundice-like appearance, including yellow skin and urine [Supplementary Figure 2B, C, https://links.lww.com/CM9/C382]. Biochemical examination confirmed a greater level of serum total bilirubin (TBIL) in BDI mice than in sham mice [Supplementary Figure 2D, https://links.lww.com/CM9/C382]. Furthermore, the serum levels of alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), and alkaline phosphatase (ALP) were also significantly greater in the BDI group than in the sham group, indicating impaired liver function [Supplementary Figure 2E, https://links.lww.com/CM9/C382]. Compared with those of the sham group, the livers of the BDI group were obviously pale in color with scattered nodules grossly [Supplementary Figure 2F, https://links.lww.com/CM9/C382]. As shown by hematoxylin and eosin (H&E) staining, intrahepatic structures were disrupted in BDI mice, and their bile ducts showed hyperplasia, as evidenced by increased layers of cholangiocytes [Supplementary Figure 2G, https://links.lww.com/CM9/C382]. Moreover, obvious deposition of collagen fiber components was observed around the intrahepatic bile duct in the BDI group via Masson and Sirius red staining [Supplementary Figure 2H, I, https://links.lww.com/CM9/C382]. Next, we attempted to rescue mouse BDI by intraductal delivery of hAESCs via gallbladder puncture catheterization [Figure 1A]. To evaluate the survival of transplanted hAESCs, mouse liver samples were immunohistochemically stained with human nucleoli, and immunoreactive cells were observed in the hAESC-treated group, indicating effective colonization [Figure 1B]. The serum levels of ALT, AST, ALP, and TBIL were decreased in the BDI + hAESCs group, indicating improved liver function and reduced BDI after the transplantation of hAESCs [Figure 1C]. Moreover, the intrahepatic bile ducts were more continuous and regular in the BDI + hAESCs group, as shown by H&E staining, which confirmed the improved bile duct structures [Figure 1D]. The collagen fiber components around the intrahepatic bile duct observed by Masson and Sirius red staining were lower in the BDI + hAESCs group than in the BDI + phosphate buffer solution (PBS) group [Figure 1E, F]. Moreover, immunofluorescence (IF) staining of keratin-19 (KRT19)/alpha-smooth muscle actin (α-SMA) revealed that intrahepatic biliary occlusion, stenosis, and increased peripheral fibrosis were more severe in the BDI + PBS group and were relieved after hAESC transplantation [Figure 1G]. The total gene expression levels of the epithelial marker Epcam, key cholangiocyte markers (Krt19 and Sox9), the proliferation marker Ki67, and the stemness-associated marker Esrrb1 were the highest in the BDI + hAESCs group [Supplementary Figure 3A, https://links.lww.com/CM9/C382]. By detecting the protein levels of the apoptosis-related factors B-cell lymphoma-2 (BCL-2), BCL-2 associated X protein (BAX) and cleaved caspase3, we found that hAESC therapy had a better antiapoptotic effect [Supplementary Figure 3B, https://links.lww.com/CM9/C382]. IF staining of KRT19 and Ki-67 was performed, and colocalization was observed in the BDI + hAESCs group, demonstrating that intrahepatic cholangiocyte proliferation was increased, which promoted bile duct regeneration and relieved BDI [Figure 1H]. These results revealed that hAESC transplantation could significantly reduce collagen fiber deposition and promote cholangiocyte proliferation in a mouse BDI model.Figure 1: Transplantation of hAESCs ameliorates intrahepatic BDI. (A) Schematic diagram of hAESC treatment in a mouse intrahepatic BDI model. (B) Representative IHC image of human nucleoli in mouse liver tissues, showing the presence of hAESCs. Scale bar: 100 μm. (C) Mouse serum levels of ALT, AST, ALP, and TBIL in the sham, BDI + PBS, and BDI + hAESCs groups, showing alleviated hyperbilirubinemia and improved liver function. (D) H&E staining of mouse liver tissues, showing improved intrahepatic biliary structures after hAESC therapy. The black arrows indicate the presence of the intrahepatic bile duct. Left column scale bar: 50 μm. (E) Masson staining of mouse liver tissues demonstrating decreased fibrosis in BDI mice treated with hAESCs. The black arrows indicate the presence of the intrahepatic bile duct. Scale bar: 50 μm. (F) Sirius red staining of mouse liver tissues showing reduced collagen deposition after hAESC therapy. The black arrows indicate the presence of the intrahepatic bile duct. Scale bar: 50 μm. (G) IF staining for KRT19, α-SMA, and DAPI, indicating improved biliary structures and alleviated fibrosis. Scale bar: 50 μm. (H) IF staining for KRT19, Ki-67, and DAPI; the white arrows indicate cholangiocyte proliferation and regeneration in the BDI + hAESCs group. Scale bar: 50 μm. (I) GSEA identified gene sets enriched in the BDI + hAESCs group. (J) IF staining for KRT19, NOTCH3, and DAPI; the white arrows indicate colocalization of KRT19 and NOTCH3 in hAESCs from mice. Scale bar: 50 μm. The data are presented as the mean ± SEM. * P <0.05, † P <0.01. ALP: Alkaline phosphatase; ALT: Alanine aminotransaminase; AST: Aspartate aminotransaminase; BDI: Bile duct injury; DAPI: 4′,6-diamidino-2-phenylindole; hAESCs: Human-derived cells; H&E: Hematoxylin and eosin; GSEA: Global student entrepreneur awards; IF: Immunofluorescence; IHC: Immunohistochemistry; MDA: 4,4′-methylenedianiline; NES: Normalized enrichment score; PBS: Phosphate buffer solution; SEM: Standard error of mean; TBIL: Total bilirubin.To clarify the specific mechanism of hAESC therapy, we performed bulk RNA sequencing on six mouse liver samples (BDI + PBS, n = 3; BDI + hAESCs, n = 3). Among the 176 differentially expressed genes (DEGs), Gene Ontology (GO) functional enrichment analysis of molecular functions and biological processes showed that chromatin modification and regulation activities were the most enriched molecular function-related pathways [Supplementary Figure 4A, https://links.lww.com/CM9/C382], and cell cycle and immune-related biological processes were also significantly altered after hAESC therapy [Supplementary Figure 4B, https://links.lww.com/CM9/C382]. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed enrichment of genes related to the immune system and signal transduction, indicating the immunoregulatory role of hAESC therapy in the liver [Supplementary Figure 4C and 4D, https://links.lww.com/CM9/C382]. Notably, global student entrepreneur awards (GSEA) revealed that the Notch signaling pathway was expressed at a significantly greater level in the BDI + hAESCs group than in the BDI + PBS group, whereas Wnt signaling was not different [Figure 1I]. Notch signaling has been reported to be involved in biliary regeneration, which plays a critical role in cell–cell communication and gene regulatory mechanisms of multiple cell differentiation processes during embryonic and adult life, whereas Wnt signaling is responsible for the regeneration of hepatocytes.[5] These results demonstrated that hAESC therapy may exert multiple effects on intrahepatic BDI and that Notch signaling pathway activation may be responsible for the repair and regeneration of damaged cholangiocytes. As the Notch signaling pathway is known to play a critical role in the development and regeneration of the biliary duct, we next examined the expression and distribution of Notch pathway-related genes in BDI mice treated following intrahepatic hAESC transplantation. First, 44 Notch pathway-related genes were significantly upregulated in the BDI + hAESCs group [Supplementary Figure 5A, https://links.lww.com/CM9/C382]. Among these genes, protein–protein interaction (PPI) analysis revealed that Jagged1–Notch1/2/3/4 interacted at high levels, indicating activation of the Notch signaling pathway after hAESC transplantation [Supplementary Figure 5B, https://links.lww.com/CM9/C382]. To further confirm the intrahepatic activation of the Notch signaling pathway after hAESC therapy, we performed quantitative polymerase chain reaction to identify the genetic expression of these Notch-related genes, including the ligands Dll4 and Jagged1; the receptors Notch1 and Notch2; and the downstream genes Hes1, Heyl, and Enos [Supplementary Figure 5C, https://links.lww.com/CM9/C382]. These genes were all significantly upregulated in the treatment group compared with the control group, as expected, demonstrating activated transcription of the Notch signaling pathway. Intracellular Notch receptors only enter the nucleus after being cleaved, and we next examined the protein expression of NOTCH1, cleaved NOTCH1 (C-NOTCH1), and the downstream protein hairy and enhancer of split homolog-1 (HES1) in mouse liver samples. As shown in Supplementary Figure 5D, https://links.lww.com/CM9/C382, the protein levels of both NOTCH1 and cleaved NOTCH1 were the highest in the BDI + hAESCs group among the three groups. Moreover, the downstream protein HES1 was also upregulated, indicating successful activation of the Notch signaling pathway. In the BDI + PBS group, NOTCH expression was scattered throughout the liver [Figure 1J]. NOTCH expression was detected both on the membranes and inside the nuclei of cholangiocytes in the BDI + hAESCs group. These results indicate that the intrahepatic Notch signaling pathway was effectively activated in cholangiocytes after hAESC therapy and successfully promoted the transcription of downstream pathways, which further improved bile duct regeneration and ameliorated BDI. In the present study, we constructed a mouse intrahepatic BDI model to imitate posttransplant cholangiopathy, as previously reported.[4] With direct infusion of primary hAESCs into the biliary tree, the injured bile duct and impaired liver function were significantly improved. Nevertheless, the animal models used in the present study are limited and require different mouse cholangiopathy models or even large animal models and discarded human liver grafts to further investigate the therapeutic effects of hAESCs and their potential for clinical translation. Acknowledgments The authors would like to acknowledge Xun Qiu for assistance in cell preparation. We also thank Nan Zhou and Chun Guo from the Core Facilities, Zhejiang University School of Medicine for their technical support. Funding This research was funded by Hangzhou Medical College “Leading Innovation Team” Clinical Research Project on Organ Transplantation (CXLJ202401), the National Key Research and Development Program of China (No. 2021YFA1100500), the Zhejiang Provincial Natural Science Foundation (No. LY22H160048), and the Key Research & Development Program of Zhejiang Province (No. 2022C03108). Conflicts of interest None.

Source: Chinese Medical Journal

DOI: 10.1097/cm9.0000000000003557