3D-ESC-derived MSCs with Enhanced Immunomodulatory Capacity Repair APAP-Induced Liver Injury
Authors: Liu Tingdang , Hayierhan Ayidana , Dai Ximing , Cheng Ke , Zhou Qi , Pan Yueyuan , Lin Jie , Liu Qin , Xie Qingdong , Zhou Xiaoling , Sun Pingnan 
Published: June 2023

Abstract
Background
Mesenchymal stem cells (MSCs) possess the ability to self-replicate, self-proliferate, and differentiate into various cell types, and hence have been extensively studied in the field of regenerative medicine. Despite the promising clinical applications of MSCs, their limited quantity and in vitro expansion potential from human tissues remain major concerns. Alternatively, MSCs can be derived from human embryonic stem cells (hESCs) that share similar phenotypic features, making hESC-MSCs a potential candidate for cell therapy. Our study aimed to investigate the efficacy of 3D-ESC-MSCs, obtained through a 3D differentiation system, as an immunoregulatory agent for treating liver damage caused by acetaminophen (APAP).
Methods
We differentiated human ESCs into MSCs using a 3D culture method involving a horizontal shaker. We characterized MSCs by detecting surface-specific markers through flow cytometry and qPCR, and validated their differentiation potential using in vitro lipid, bone, and cartilage differentiation assays. MSC proliferation and safety were tested using MTT, cell survival at 4°C, and nude mice tumorigenicity assays. The immune regulatory potential of 3D-ESC-MSCs was studied by transfecting polyI:C into these cells. We further investigated the effects of 3D-ESC-MSCs on APAP-induced liver injury by preconditioning hepatocyte cell line L-O2 with 3D-ESC-MSC conditioned medium and evaluating their cell viability through MTT assay. Additionally, we assessed the number of surviving cells following co-culturing with L-O2 cells stimulated with APAP. Finally, we administered 3D-ESC-MSCs to mice, via tail vein injection, with APAP-induced acute liver injury, and analyzed the repair effects by detecting ALT and AST levels in mouse serum, creating liver pathological sections, and HE staining.
Results
3D-ESC-MSCs were positive for CD73, CD90, and CD105 surface markers, and negative for hematopoietic markers CD45 and HLA-DR in. The cells expressed low levels of pluripotent genes OCT4 and NANOG. Compare to umbilical cord mesenchymal stem cells (UCMSCs), 3D-ESC-MSCs displayed excellent proliferation and low-temperature resistance, and lower concentrations of polyI:C were required to induce immune regulatory genes IDO1, IF71, IRF7, and ISG15. They also exhibited higher expression levels of immunomodulatory。In vitro experiments demonstrated that the conditioned medium of 3D-ESC-MSCs increased L-O2 cell activity under low concentrations of APAP, and the survival of L-O2 cells co-cultured with 3D-ESC-MSCs was higher compared to L-02 cells cultured alone under the same conditions. Animal experiments revealed that the ALT and AST levels in APAP-treated mice injected with 3D-ESC-MSCs were reduced, and the necrotic area of the liver in the 3D-ESC-MSC group was reduced. The therapeutic effect was similar to that of the UCMSC group.
Conclusions
3D-ESC-MSCs, differentiated from ESCs, exhibit stronger immunomodulatory effect and can be utilized to repair acute liver injury caused by APAP. This study highlights the clinical potential of 3D-ESC-MSCs in treating human diseases.