Organoid-Derived β-Catenin-Overexpressing Dendritic Cells Alleviate Sepsis- Induced Acute Lung Injury

Abstract Background Sepsis-induced acute lung injury (ALI) is characterized by pulmonary immune dysregulation, while the limited availability and functional stability of conventional dendritic cells (DCs) constrain their therapeutic application. We investigated whether β-catenin engineering at the mouse embryonic stem cell (mESC) stage could enhance blood vessel organoid (BVO)-based DC generation and improve DC-mediated immune regulation in sepsis-induced ALI. Methods β-Catenin-overexpressing (β-cat OE ) mESCs were sequentially differentiated through a three-dimensional BVO–hematopoietic progenitor–DC pipeline. Hematopoietic differentiation, DC phenotype and function, ovalbumin (OVA)-containing OT-II/OT-I T-cell responses, and transcriptomic profiles were evaluated. Therapeutic effects were assessed following intranasal administration of β-cat OE BVO-derived DCs (BVO-DCs) in a cecal ligation and puncture (CLP)-induced sepsis model by pulmonary bioluminescence imaging, lung injury and inflammation assessment, pulmonary T-cell analysis, and 96-h survival monitoring. Statistical comparisons were performed using t-tests or analysis of variance as appropriate, with survival analyzed by the log-rank test. Results β-Catenin overexpression increased hematopoietic progenitor frequency to 11.96 ± 1.55% and total cell yield to (12.52 ± 1.43) × 10⁷ cells. Following lipopolysaccharide stimulation, β-cat OE BVO-DCs showed 47.73 ± 7.77% major histocompatibility complex class II (MHC-II) positivity and induced CD40 ligand (CD40L) expression in OT-II CD4⁺ T cells (35.37 ± 3.45%), OT-I CD8⁺ T-cell proliferation (77.01 ± 7.46%), and CD25 expression (51.84 ± 4.61%) in full-length OVA-containing co-cultures. Blockade experiments supported the involvement of the MHC-II/CD4⁺ T-cell/CD40L axis. Transcriptomic analysis showed enhanced Wnt signaling, DC differentiation, and MHC-mediated antigen-presentation programs. In CLP-induced sepsis, β-cat OE BVO-DC treatment increased pulmonary bioluminescence at 24 and 48 h, attenuated lung injury and inflammatory responses, and improved 96-h survival to 53.3% compared with 13.3% in untreated CLP mice. Treatment also increased pulmonary CD8⁺ T-cell abundance and reduced the bronchoalveolar lavage fluid (BALF) CD4⁺/CD8⁺ ratio to 2.12 ± 0.40 versus 4.01 ± 0.69 in untreated CLP mice. Conclusions β-Catenin engineering at the mESC stage enhances hematopoietic output from BVOs and generates functionally enhanced BVO-derived DCs that improve pulmonary immune balance and outcomes in experimental sepsis-induced ALI. These findings support further development of organoid-based DC strategies for sepsis-associated immunosuppression.