Thymic stromal cells drive T cell development by supplying key cues for progenitor proliferation and differentiation. Although thymic epithelial cells are well characterized, recent studies highlight crucial functions of non-epithelial stromal populations, especially neural crest–derived mesenchymal cells. Mesenchymal defects underlie thymic abnormalities in mouse models of DiGeorge syndrome (DGS) and contribute to pathology in CHARGE syndrome, trisomy 21 (T21), and other disorders affecting mesoderm-derived structures. Human studies remain limited due to the rarity of these conditions and restricted access to thymic tissue.
To address this, we generated induced pluripotent stem cells (iSPCs) from individuals with mutations in TBX1, CHD7, HOXA3, and PAX1, as well as from DGS and T21 patients. Control and patient iPSCs, generated from peripheral blood mononuclear cells (PBMCs) or skin biopsies, were differentiated into mesenchymal stem cells (MSCs). Primary thymic mesenchymal cells (ThyMCs) were also isolated directly from thymi. All populations were assessed by flow cytometry and bulk RNA sequencing (RNA-seq).
Flow cytometry confirmed robust induction of MSC markers (CD73, CD146, and CD105) in all MSC and ThyMC samples, with no expression in undifferentiated iPSCs. Principal component analysis showed clear segregation of iPSCs, MSCs, and ThyMCs. Transcriptomic profiling revealed that although all patient-derived MSCs achieved a mesenchymal identity, each disease group displayed distinct transcriptional alterations. MSCs with TBX1, HOXA3, or PAX1 mutations exhibited the highest number of differentially expressed genes, consistent with their upstream roles in thymus development. Dysregulated genes mapped to several clinically relevant pathways, including extracellular matrix (ECM) and cartilage development. Particularly, DGS-derived MSCs and ThyMCs showed marked upregulation of ECM and collagen genes such as FBLN5, PCOLCE, EMILIN1, COL3A1, and COL1A2.
Ongoing tri-lineage (adipogenic, chondrogenic, and osteogenic) differentiation assays continue to uncover pathway defects relevant to T21 and CHARGE syndromes. Overall, these findings provide new insight into thymic development and immune dysfunction in congenital syndromes and establish a platform for probing thymic stromal defects.
