Lisheng Wang

The therapeutic efficacy of mesenchymal stem cells (MSCs) can be improved by enhancing their adaptation to the inflammatory microenvironment. Autophagy mai

Background: Lung adenocarcinoma (LUAD) is highly relapsed and responds poorly to chimeric antigen receptor (CAR)‐T therapy due to antigenic heterogeneity and the immunosuppressive microenvironment. Trophoblast cell‐surface antigen 2 (TROP2), overexpressed in LUAD and linked to poor prognosis, is a promising target. Radiofrequency ablation (RFA) can cause direct tumor necrosis, trigger immunogenic cell death, and enhance immune infiltration through antigen secretion; however, it often fails to eradicate residual tumors. Therefore, combining RFA with CAR‐T‐cell therapy may offer a new and synergistic therapy against LUAD. Objective: This study examined the cytotoxicity of TROP2‐directed CAR‐T cells against LUAD in vitro and observed their therapeutic efficacy and safety in combination with RFA in vivo. Methods: Third‐generation TROP2‐CAR‐T cells were developed and characterized for transduction efficiency, CD4/CD8 ratio, and proliferative capacity. Their antigen‐specific cytotoxicity against TROP2 ⁺ target cells was observed using a luciferase‐based assay and cytokine analysis. An A549‐TROP2‐Luc xenograft model was developed to examine the therapeutic effects of RFA, TROP2‐CAR‐T monotherapy, and their combination. Tumor suppression, Ki67/TUNEL assays, and immunohistochemical (IHC) analyses were performed to evaluate efficacy and safety. Results: TROP2‐CAR‐T cells showed efficient transduction and potent, antigen‐dependent cytotoxicity with significant cytokine secretion in vitro. In vivo, both RFA and TROP2‐CAR‐T therapy suppressed tumor growth, and their combination showed a synergistic antitumor effect without hepatic or renal toxicity. Conclusion: The combination of RFA and TROP2‐CAR‐T therapy demonstrates enhanced antitumor efficacy and improved safety profile in LUAD, highlighting a promising combinatorial immunotherapeutic approach.

Introduction/Objective: This study aimed to examine the effect of a human umbilical cord mesenchymal stem cell-derived exosome (hUC-MSC-Exo) liquid band-aid on wound healing in mice. Methods: hUC-MSC-Exos were prepared from the supernatant via ion exchange chromatography. The composition ratio of the chitosan liquid band-aid was optimized to form a film and encapsulate hUC-MSC-Exo. The biological effects of chitosan exosome liquid band-aid on human umbilical vein endothelial cells (HUVECs) were observed, and its anti-bacterial properties were tested. BALB/c mice with back skin injury were randomly divided into chitosan exosome liquid band-aid group (CS-Exo), chitosan liquid band-aid group (CS), and normal saline control group (Con), and wound healing was evaluated post-treatment. Skin tissue samples posttreatment were collected for H&E staining. Results: The hUC-MSC-Exo was prepared and characterized. The optimum conditions for film formation were 1% chitosan solution and 15% poloxamer 407/poloxamer 188 (pH 5.0 ~ 7.0). The chitosan exosome liquid band-aid promoted HUVEC proliferation and migration and markedly inhibited Escherichia coli and Staphylococcus aureus growth in vitro. In vivo, the wound healing rate in the CS-Exo group was higher than that in the Con and CS groups. Fourteen days post-treatment, the wounds completely healed, and hair grew normally, which was consistent with H&E results. Mouse weights in each group did not change significantly after administration, indicating that the chitosan exosome liquid band-aid had no obvious toxic side effects. Conclusion: Local chitosan exosome liquid band-aid application can promote wound healing in mice, and the mechanism could be related to hUC-MSC-Exo-induced vascular endothelial cell proliferation and migration.

Radiation-induced lung injury (RILI) is associated with alveolar epithelial cell death and secondary fibrosis in injured lung. Mesenchymal stem cell (MSC)-derived exosomes have regenerative effect against lung injury and the potential to intervene of RILI. However, their intervention efficacy is limited because they lack lung targeting characters and do not carry sufficient specific effectors. SARS-CoV-2 spike glycoprotein (SARS-CoV-2-S-RBD) binds angiotensin-converting enzyme 2 (ACE2) receptor and mediates interaction with host cells. MiR-486-5p is a multifunctional miRNA with angiogenic and antifibrotic potential and acts as an effector in MSC-derived exosomes. Ferroptosis is a form of cell death associated with radiation injury, its roles and mechanisms in RILI remain unclear. In this study, we developed an engineered MSC-derived exosomes with SARS-CoV-2-S-RBD- and miR-486-5p- modification and investigated their intervention effects on RIPF and action mechanisms via suppression of epithelial cell ferroptosis. Adenovirus-mediated gene modification led to miR-486-5p overexpression in human umbilical cord MSC exosomes (p < 0.05), thereby constructing miR-486-5p engineered MSC exosomes (miR-486-MSC-Exo). MiR-486-MSC-Exo promoted the proliferation and migration of irradiated mouse lung epithelial (MLE-12) cells in vitro and inhibited RILI in vivo (all p < 0.05). MiR-486-MSC-Exo suppressed ferroptosis in MLE-12 cells, and an in vitro assay revealed that the expression of fibrosis-related genes is up-regulated following ferroptosis (both p < 0.05). MiR-486-MSC-Exo reversed the up-regulated expression of fibrosis-related genes induced by TGF-β1 in vitro and improved pathological fibrosis in RIPF mice in vivo (all p < 0.05). SARS-CoV-2-S-RBD-modified and miR-486-5p-engineered MSC exosomes (miR-486-RBD-MSC-Exo) were also constructed, and the distribution of DiR dye-labeled miR-486-RBD-MSC-Exo in hACE2CKI/CKI Sftpc-Cre+ mice demonstrated long-term retention in the lung (p < 0.05). MiR-486-RBD-MSC-Exo significantly improved the survival rate and pathological changes in hACE2CKI/CKI Sftpc-Cre+ RIPF mice (all p < 0.05). Furthermore, miR-486-MSC-Exo exerted anti-fibrotic effects via targeted SMAD2 inhibition and Akt phosphorylation activation (p < 0.05). Engineered MSC exosomes with SARS-CoV-2-S-RBD- and miR-486-5p-modification were developed. MiR-486-RBD-MSC-Exo suppressed ferroptosis and fibrosis of MLE-12 cells in vitro, and alleviated RILI and long-term RIPF in ACE2 humanized mice in vivo. MiR-486-MSC-Exo exerted anti-fibrotic effects via SMAD2 inhibition and Akt activation. This study provides a potential approach for RIPF intervention.

Introduction: With the growing interest in exosomes for research and therapeutic applications, there's a critical need for effective enrichment strategies that can isolate exosomes in large quantities without contaminants. Despite various methods being developed, none have satisfactorily achieved this goal without contamination issues. This study introduces a novel approach for exosome separation - ion-exchange chromatography (IEC) - that leverages the negatively charged phospholipids on the exosomal surface for more effective isolation. Methods: The study developed a novel exosome separation strategy using ion-exchange chromatography (IEC) and compared it with the traditional ultracentrifugation (UC) method. The comparison focused on yield, purity, and the biological effects of the exosomes enriched by both methods. The new IEC method was tested alongside classical separation techniques like ultrafiltration for its efficiency in large-scale exosome extraction. Results: Both UC and IEC efficiently enriched exosomes with high abundance, but IEC was superior in terms of lower protein contamination and better particle dispersion. Furthermore, exosomes enriched with IEC exhibited a stronger clonogenic effect on murine lung epithelial cells compared to those enriched by UC. In wound healing assays, exosomes isolated by both IEC and UC significantly improved the healing ratio in lung epithelial cells, showcasing the potential therapeutic benefits of the exosomes isolated by these methods. Conclusion: The novel ion-exchange chromatography (IEC) method developed for exosome separation demonstrated significant advantages in terms of yield, purity, and biological effects when compared to traditional ultracentrifugation. The lower protein contamination and better particle dispersion achieved with IEC, along with its superior clonogenic and healing effects on lung epithelial cells, suggest that IEC can play a crucial role in future exosome separation strategies, particularly for large-scale applications.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are biologically active substances secreted by MSCs into the extracellular matrix that play an immunomodulatory role in skin damage repair. To investigate the mechanism of MSC-EVs in reducing inflammation, promoting angiogenesis, promoting the proliferation and migration of epithelial cells and fibroblasts, and extracellular matrix remodeling during wound healing, we focused on the effects of EVs on multiple cell types at various stages of skin injury. A literature review was conducted to explore related research on the influence of MSC-EVs on the types of cells involved in wound healing. MSC-EVs show a strong regulatory ability on immune cells involved in the regulation of inflammation, including macrophages, neutrophils, and T cells, and other cells involved in tissue proliferation and remodeling, such as fibroblasts, keratinocytes, and endothelial cells, during wound healing in in vitro and in vivo experiments, which substantially promoted the understanding of wound healing in the field of trauma medicine. MSC-EVs have potential applications in combating poor skin wound healing. Elucidating the mechanism of action of EVs in the wound-healing process would greatly advance the understanding of therapeutic wound healing.

Background: Renal clear cell carcinoma (RCC) has negative prognosis and high mortality due to its early diagnosis difficulty and early metastasis. Although previous studies have confirmed the negative progression of RCC is closely related to M2 macrophages in tumor-associated macrophages (TAMs), the specific mechanism is still unknownMethods: We used immunofluorescence labeling and flow cytometry to detect the proportion of M2 macrophages in RCC tissues. And bioinformatics technique was used to obtain 9 M2 macrophage-related model genes, including SLC40A1, VSIG4, FUCA1, LIPA, BCAT1, CRYBB1, F13A, TMEM144, COLEC12. Using these genes, model formulas are constructed to devide samples into high and low risk groups, and then the overall survival (OS), progression-free survival (PFS) and Gene set enrichment analysis (GSEA) of the high and low risk groups were analyzed. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of model genes between normal kidney tissue and RCC tissue, as well as between HK-2 cell and 786-O cell. Besides, we induced the M2 differentiation of THP-1 cell, and then co-cultured with the RCC cell 786-O in transwell to observe what effect M2 macrophages will cause on the invasion, migration and the expression of model genes of RCC.Results: Our study demonstrated M2 macrophages in RCC was about 2 folds that of normal renal tissue (Pin vitro experiments showed that in RCC tissues and 786-O cells, the model gene FUCA1 was down-regulated, and SLC40A1, VSIG4, CRYBB1 and LIPA were up-regulated. Besides, the results of co-culture showed that after 786-O co-culture with M2 macrophages, the ability of migration and invasion was promoted and the expressions of FUCA1, SLC40A1, VSIG4, CRYBB1, LIPA and TMEM144 were all up-regulated.Conclusion: The proportion of tumor-associated M2 macrophages in RCC tissues is upregulated, and M2 macrophages promote the progression of RCC by regulating the expression of SLC40A1, VSIG4, FUCA1, LIPA, BCAT1, CRYBB1, F13A, TMEM144, COLEC12 genes, thereby affecting the prognosis of patients with RCC.

Purpose: Mesenchymal stem cells (MSCs) have become novel therapeutic agents for the treatment of inflammatory bowel diseases (IBDs). However, the precise cellular and molecular mechanisms by which MSCs restore intestinal tissue homeostasis and repair the epithelial barrier have not been well elucidated. This study aimed to investigate the therapeutic effects and possible mechanisms of human MSCs in the treatment of experimental colitis. Methods: We performed an integrative transcriptomic, proteomic, untargeted metabolomics, and gut microbiota analyses in a dextran sulfate sodium (DSS)-induced IBD mouse model. The cell viability of IEC-6 cells was determined by Cell Counting Kit-8 (CCK-8) assay. The expression of MUC-1 and ferroptosis-related genes were determined by immunohistochemical staining, Western blot, and real-time quantitative polymerase chain reaction (RT-qPCR). Results: Mice treated with MSCs showed notable amelioration in the severity of DSS-induced colitis, which was associated with reduced levels of proinflammatory cytokines and restoration of the lymphocyte subpopulation balance. Treatment with MSC restored the gut microbiota and altered their metabolites in DSS-induced IBD mice. The 16s rDNA sequencing showed that treatment with MSC modulated the composition of probiotics, including the upregulation of the contents of Firmicutes, Lactobacillus, Blautia, Clostridia, and Helicobacter bacteria in mouse colons. Protein proteomics and transcriptome analyses revealed that pathways related to cell immune responses, including inflammatory cytokines, were suppressed in the MSC group. The ferroptosis-related gene, MUC-1, was significantly upregulated in the MSC-treated group. MUC-1-inhibition experiments indicated that MUC-1 was essential for epithelial cell growth. Through overexpression of MUC-1, it showed that upregulation of SLC7A11 and GPX4, and downregulation of ACSL4 in erastin and RSL3-treated IEC-6 cells, respectively. Conclusion: This study described a mechanism by which treatment with MSCs ameliorated the severity of DSS-induced colitis by modulating the gut microbiota, immune response, and the MUC-1 pathway.

Background: Several studies have shown that MSCs can significantly improve the survival of sepsis animals. CD146+ mesenchymal stem cells (MSCs) correlate with high therapeutic potency. However, their therapeutic effect on sepsis and detail mechanisms have not been explored. Methods: The effect of CD146±MSCs on differentiation of Treg, Th1, Th17 subsets was evaluated by flow cytometry. The effects of CD146±MSCs on RAW264.7 phagocytosis and LPS-stimulated polarization were studied using a co-culture protocol. Luminex bead array and RNA sequencing were employed to determine the mechanisms of MSCs on LPS-stimulated RAW264.7. The Arg1 protein was detected by Western blot. CD146±MSCs were injected into LPS-induced sepsis mice by tail vein. The therapeutic effect was assessed by organ HE staining, T-cell subsets, cytokine in plasma, peritoneal macrophages, infiltrating monocytes subpopulations. Results: In vitro, CD146+MSCs could significantly increase the proportion of Treg cells. Co-culture with CD146+MSCs increase the phagocytic rate of RAW264.7. CD146+MSCs regulate M2-type macrophages production more rapidly. The transcript profile differences between the CD146+MSCs and CD146-MSCs groups were clustered in arginine metabolism pathways. CD146+MSCs decreased NO production and increased ARG1 expression. CD146+MSCs secreted higher level of IL15,IFNγ, VEGF and lower level of IL1β, IL8 under LPS stimuli. In vivo, The level of IL10 at 24h and CXCL1, IFNγ at 12h in CD146+MSCs group was the highest. CD146+MSCs treatment enhances the phagocytic capacity of peritoneal macrophages. CD146+MSCs also increases the ratios of CD11b+Ly6C^lo reparative monocytes and CD11b+Ly6C^hi inflammatory monocytes until 24h. Conclusion: Compared with CD146-MSCs, CD146+MSCs can accelerate the end of the inflammatory response and have robust anti-inflammatory effects, by increasing the Treg cells, promoting macrophage phagocytosis, enhancing the reparative macrophage, secreting more VEGF, etc.