Extended Data Fig. 5: Validation of mouse ThyMC markers by flow cytometry and fibroblast colony-forming capacity.
From: Mesenchymal thymic niche cells enable regeneration of the adult thymus and T cell immunity
(A) UMAP embedding of mouse thymus cell populations including Cd3e + Cd4 + Cd8b+ Ptprc+ hematopoietic cells with detailed annotation (left). ThyMC marker genes Cd99l2, Itgb5, Pdgfra and Cd248 presented as UMAP graphs (right). (n = 4) Four independent experiments. (B) FACS plot validating the presence of both CD99l2 and Itgb5 at the protein level on non-hematopoietic, non-epithelial, non-endothelial thymic cells. (C) Flow cytometric plots demonstrating the overlap between CD99l2 and Itgb5 and other cell type defining markers. Labels to the right of the flow plots refer to parent gate and the percentages are of the parent gate. (D) Image of fixed and Giemsa stained CD45-Ter119-CD31- EpCam- CD99l2+ Itgb5+ ThyMCs grown in aMEM with 20% FBS under hypoxic conditions for 6 days (n = 3). Three independent experiments. (E) Bar graphs displaying colony forming ability of individually sorted CD45-Ter119-CD31- EpCam- CD99l2+ Itgb5+ and CD45-Ter119-CD31- EpCam- CD99l2- Itgb5- cells grown of 7 days in aMEM with 20% FBS under hypoxic conditions. (F) Bar graph representation of bone marrow and thymic ThyMCs fibroblast colony forming units (CFU-F) after 6 days of hypoxic culture in aMEM with 20% FBS (n = 6 per group, mean ± SEM). Two independent experiments. (G) Flow cytometric analysis confirmation at the protein level of CD248 and Pdgfra. (H) Validation of increased Postn expression by qPCR on sorted CD248+ ThyMC compared to CD248- ThyMC. Statistical significance was calculated by a two-sided, unpaired student’s t-test. n = 4, mean ± SEM, from two independent experiments. CD248+ ThyMC vs. CD248- ThyMC * p = 4.8×10−2.
