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.

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.

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.

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.

The progression of chronic kidney disease (CKD) is closely associated with the disruption of renal spatial immune microenvironment. This study aimed to develop an integrated experimental and analytical system for dissecting the immune microenvironment of CKD using imaging mass cytometry (IMC), thereby enabling panoramic analysis of single-cell spatial immune microenvironment in CKD renal tissues.

① Antigenic markers targeting renal structural units, stromal cells, and immune cells were screened to construct a mass cytometry imaging antibody panel; all metal-conjugated antibodies were validated for specificity by immunohistochemistry. ②Two normal kidney biopsy specimens adjacent to ureteral carcinoma, 2 diabetic nephropathy (DN), and 2 IgA nephropathy (IgAN) renal biopsy specimens were collected, stained with the aforementioned antibodies, and subjected to image data acquisition using the Hyperion imaging system to obtain multiplexed spatial data. ③ An integrated Ilastik and CellProfiler workflow was established for single-cell segmentation pipeline tailored for renal tissues, and cell identity annotation was achieved using a combination of manual gating and Phenograph unsupervised clustering. ④ Spatial proximity relationships between cells were defined, and permutation testing was employed to simulate random distribution models to examine spatial relationships between tissue cells and immune cells. ⑤ k-nearest neighbors (kNN) algorithm was applied for spatial clustering, and cellular neighborhoods (CNs) were used to delineate the spatial functional units within renal tissue, followed by analysis of the immunological characteristics and relative abundance of these units.

① A 37-color antibody panel was constructed and optimized, with all antibodies demonstrating high specificity and low background noise; this panel enabled accurate identification of renal structural cells, stromal cells, and infiltrating immune cells. ② High-dimensional IMC imaging data were successfully obtained from normal and CKD renal tissues using the Hyperion imaging system. ③ With the aid of the single-cell segmentation pipeline, a high-resolution single-cell atlas of human kidney was systematically mapped, comprising 21 structural cell types and 17 immune cell subtypes. Immune cell populations such as CD15⁺ macrophages, GzmK⁺ CD4⁺ effector T cells, and 3 distinct double-positive renal tubular cells were identified, revealing differential infiltration of immune cells in DN and IgAN tissues. ④ A single-cell spatial interaction analysis framework was established, demonstrating a disease-specific spatial interaction network of immune cells in CKD tissues, and identifying myofibroblasts as a critical spatial hub connecting tissue injury and immune cell infiltration. ⑤ Spatial neighborhood analysis delineated 9 functional units of renal tissue, including glomerular, proximal tubular, ascending limb of the loop of Henle, vascular and fibrotic, and immune cell-enriched regions. In diseased tissues, the proportion of normal functional regions was decreased, whereas the proportions of fibrotic and immune-enriched regions were increased significantly. Moreover, immune cell-enriched zones maintained close colocalization with various functional regions, while vascular and fibrotic regions were predominantly adjacent to collecting duct and ascending limb of the loop of Henle.

An IMC methodology tailored for the spatial analysis of renal tissue is successfully developed. This platform enables high-resolution, panoramic visualization of spatial distribution of immune cells in chronic renal tissues, dissecting spatial interactions between immune and structural cells and characteristics of local immune microenvironment. This technology holds promise to advance the development of spatially informed precision diagnostics and targeted therapeutic strategies for renal diseases.