Pre-Doctoral Fellow University of Catania GPP Washington, District of Columbia, United States
Disclosure(s):
Giuseppe Sangiorgio, MD: No financial relationships to disclose
Introduction/Rationale: Thymic stromal cells are essential for T-cell progenitor proliferation and differentiation. While thymic epithelial cells are well studied, non-epithelial stromal populations—particularly neural crest–derived mesenchymal cells—are increasingly recognized for their roles in thymus formation and function. Mesenchymal defects contribute to thymic abnormalities in congenital syndromes such as DiGeorge syndrome (DGS), CHARGE syndrome, and Trisomy 21 (T21), yet human studies are limited by the rarity of these conditions and restricted access to thymic tissue.
Methods: We generated iPSCs from individuals with TBX1, CHD7, HOXA3, and PAX1 mutations, as well as DGS and T21 patients. Control and patient iPSCs, derived from PBMCs or skin biopsies, were differentiated into mesenchymal stem cells (MSCs). Primary thymic mesenchymal cells (ThyMCs) were also isolated from human thymi. All cell populations were analyzed by flow cytometry and bulk RNA-seq. Tri-lineage differentiation assays (adipogenic, chondrogenic, osteogenic) were performed to assess functional pathway defects.
Results: Flow cytometry confirmed robust expression of MSC markers (CD73, CD146, CD105) in all MSC and ThyMC samples, absent in undifferentiated iPSCs. Principal component analysis revealed clear segregation of iPSCs, MSCs, and ThyMCs. Transcriptomic profiling showed that all patient-derived MSCs acquired mesenchymal identity, but each disease group displayed distinct transcriptional changes. MSCs with TBX1, HOXA3, or PAX1 mutations had the highest number of differentially expressed genes, affecting pathways such as extracellular matrix and cartilage development.DGS-derived MSCs and ThyMCs showed marked upregulation of ECM and collagen genes (FBLN5, PCOLCE, EMILIN1, COL3A1, COL1A2).
Conclusion: Our findings reveal disease-specific mesenchymal defects underlying thymic abnormalities in congenital syndromes.This platform enables mechanistic studies of thymic stromal dysfunction and advances understanding of immune deficits in these disorders.
