Fig. 1: Identification of a subpopulation of resident hVCFs expressing dual mesenchymal and lymphoid cell surface markers with multidimensional single-cell mass cytometry.

Immunophenotyping of hVCFs derived from human subjects (2 males (ID# 62122, ID# 1281202) and 1 female (ID#534282)) was performed by staining 3 × 10⁶ cells with heavy metal-tagged antibodies, followed by mass cytometry to identify frequencies of populations in human cardiac fibroblasts. a Schematic summary of mass cytometry analysis. hVCFs were immunostained with epitope-specific antibodies conjugated to transition element isotope reporters of different masses. The cells were nebulized into single-cell droplets, followed by the acquisition of an elemental mass spectrum. Conventional flow cytometry methods were used to analyze integrated elemental reporter signals for each cell. This diagram was drawn exclusively by the authors using BioRender (Toronto, Canada) under the aegis of an academic license with Brown University. b viSNE graphs of manually gated live, nucleated, hVCF cell clusters expressing varying marker intensities and distribution for a representative donor showed a subpopulation of cells (island) distinct from the majority of cardiac fibroblasts marked by the expression of Vimentin and αSMA⁺. c The hVCFs expressing cluster of differentiation (CD) CD4, CCR6, and CD183 associated with lymphoid cells on hVCF. Protein expression levels are demonstrated on the secondary y-axis scale with blue showing no expression, green, the least expression, and red, the highest expression. Each dot represents the expression profile of a single cell. The red box shows the newly identified population separated from the rest of the cell cluster. Bottom panel of (b) and (c) are the viSNE of viSNE after gating the newly identified population. d UMAP-based clustering of the cells showing the distinct population of cells isolated from the main cluster of homogenous cardiac fibroblast cells. e Representative flow cytometry histograms showing frequencies of CD4⁺ subset of αSMA⁺Vim⁺ hVCFs for all the human donor cells. f Quantification of CD4⁺ CD3⁻ cells gated from Vimentin⁺αSMA⁻, Vimentin⁻αSMA⁺ and Vimentin⁺ αSMA⁺ populations is represented as a scatter plot of Mean ± SD (n = 3 biological replicates, 2 males and 1 female); *P < 0.05, as determined using one-way ANOVA and Tukey’s post-hoc multiple comparison. g Representative hVCF cells immunostained CD4, Vimentin and DAPI antibodies showing co-expression of Vimentin and CD4. Scale bars are 20 µm.