Residual cell types, molecular expression profiles and quality assessment of in vitro cultured human thymic slices

Objective‍ ‍‍To analyze the composition and function of residual cells in pre-transplantation human thymic slices by single-cell transcriptomics sequencing (scRNA-seq), and established a quality assessment method for thymic slices based on the expression levels of molecular markers in the culture supernatant. Methods‍ ‍The discarded thymus from 18 patients with congenital heart disease undergoing surgical treatment in Department of Cardiothoracic Surgery of Children’s Hospital Affiliated to Chongqing Medical University from May 2023 to January 2024 were collected and prepared into thymic slices. After the slices were cultured in vitro for 14 d, scRNA-seq was employed to identify the residual cell types, and gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis was performed to analyze the biological function of the residual cells. Then based on the literature concerning thymic slice culture, the molecular markers indicating thymocyte function were screened out. ELISA was applied to detect the changes in protein levels of molecular markers in the supernatant. Receiver operating characteristic (ROC) curve was plotted and assess the value of the molecular markers in the supernatant in evaluating the quality of thymic slices with area under the curve (AUC). Then, the qualified and unqualified thymic slices determined by our obtained molecular markers were transplanted subcutaneously into male nude mice (6~8 weeks old, weighing 14~17 g), respectively, and the male nude mice without transplantation of the thymic slices served as control group. Flow cytometry and histologic analysis were utilized to observe the immune reconstitution after transplantation. Results‍ ‍① scRNA-seq identified 11 cell types in thymic slices, dominated with epithelial cells, fibroblasts, and T cells. GO and KEGG enrichment analysis showed that epithelial cells were involved in enrichment entries related to chemotaxis, epithelial cell development, cell matrix adhesion and tight junction; fibroblasts were involved in enrichment entries related to extracellular matrix, epithelial cell proliferation, negative regulation of cell migration, and regulation of actin cytoskeleton; T cells were mainly related to T cell differentiation, regulation of T cell activation, T cell apoptosis, and T cell receptor signaling. ② Molecular markers, CCL19, CCL21, CXCL12, CXCL16, IL16 and SELL were identified to indicate thymocyte function. Compared with the levels of the first day, the protein secretions of CCL19, CCL21, CXCL12 and CXCL16 were significantly increased during in vitro culture (P<0.05), while the protein secretions of IL16 and L-selectin (protein form of SELL) were significantly decreased (P<0.05). The combined predictor Pre1 from subset of cytokines (IL16 and L-selectin) had the highest value in the quality assessment of thymic slices after 1 d of culture (AUC=0.883), and the combined predictor Pre2 from subset of cytokines (CCL19, CCL21, CXCL12 and CXCL16) had the highest value in the quality assessment after 14 d of culture (AUC=0.948). ③ Transplantation in nude mice indicated that the qualified thymic slices could develop to thymus structure in vivo, and effectively increase the proportion of T cells in peripheral blood (P<0.01), while the unqualified thymic slices could not obtain the reconstitution of T cell development. Conclusion‍ ‍The main residual component cells in thymic slices are epithelial cells, fibroblasts and T cells. IL16 and L-selectin can be used as potential indicators to determine the quality of donor thymic samples. CCL19, CCL21, CXCL12 and CXCL16 can effectively evaluate the quality of thymic slices before transplantation.