Fig 2: Gene expression of markers identified for stemness. (A) Feature plots of Epcam, Itga6, and Ly6a, which represents for the proteins Epcam, Cd49f, and Sca-1 (B) Feature plot expression of the key gene markers after filtering for cells expressing a minimal threshold of 0.3 for all markers. (C) Pie plot visualizing population broken down by cluster. Between the entire Comma-1D population and P8, there is increased occupancy of luminal 1 and myoepithelial 4 with a significant decrease of myoepithelial two and loss of the fibroblast cell population.

Fig 3: Phenotypic assay overview. (A) A subsection of a beacon chip across five time points of a culture period. The bar on the bottom indicates heterogeneity in observed growth response. Red indicates dying cells, white is no significant observed response, and green is for cells with detected proliferation (B) The top graph is plotting the growth ratio across identified subpopulations. The bottom graph indicates growth differences depending on initial cell concentration. 5% error bars are visualized for each plot. (C) Sample pen images of various cells across imaging channels. The colored outline is to highlight expression (green) or lack of (red). The top row is a known P8 population imaged at three channels with expression detected for all three markers. UA and UB indicate sample images of unknown cell types with marker expression pattern resolving subtypes P8 and P5, respectively (D) Overview of MATLAB script pipeline to identify cell populations by protein expression, automating observations described in (C). (Di) Illustrates regions of interest (ROIs) representing each nanopen. (Dii) Shows possible cell detection locations based on circle detection. (Diii) Visualizes filtered cell detections leveraging OEP fluorescence thresholds. Fluorescence intensity above 65,535 is set as the threshold for true positive cell detection. (Div) Visualizes normalized expression scores for two cell detection locations in the TRED channel. Normalized scoring accounts for variability in background fluorescence between FOVs and between experimental chips. Readings are imported into a matrix for each detected cell for each marker and channel. (Dv)A sample readout of cells subtype based on the measured expression. Numbers “5” and “7” indicate populations P5 and P7, respectively. (Dvi)A scatter plot of all the detected cells plotted based on their expression of Cd49f and Epcam and the subtype they are predicted to belong to. 88.3% of FACS sorted cells pseudo-bin to same population on-chip. Each color represents the pseudo-binned subtype for that cell, as indicated in the legend. Nanopens with multiple cells at t0 are sources of noise and background fluorescence that misidentify some cells. (E) ROI images of randomly selected P8 identified cells from each imaging channel. The heatmap on the bottom compares expression across channels and cells. This functionality provides verification to the pseudo binning results, allows for clustering and threshold adjustment, and provides easy visualization for every cell subpopulation of interest.

Fig 4: Predicting cell types and functions by cluster. (A) Feature plots of key canonical markers of fibroblastic cells (B) Violin plots of key canonical markers of fibroblastic cells. Identified fibroblast cluster exclusively expressed all markers. (C) Feature plots depicting expression of markers derivative of luminal and myoepithelial subtypes, EPCAM and ACTA2, respectively. (D) Violin plots outlining distinct expression of Krt18 expression within the luminal subtypes indicating possible alveolar cell function. Krt8 expression was significantly isolated to the predicted luminal progenitor population within the luminal subtype. Almost exclusive expression of Krt8 indicates luminal progenitor cluster as a progenitor population within luminal cells and with luminal 1 as slightly differentiated cells with secretory/alveolar function. (E) Differential genes between the two clusters in the luminal partition. (F) Differential genes within the local myoepithelial partition. (G) Ridge plot of gene markers conserved within luminal cells, but not expressed significantly outside the luminal population. (H) Ridge plot of gene markers conserved within myoepithelial cells, but not expressed significantly outside the myoepithelial population.

Fig 5: K19 identifies embryonic multipotent mTEC progenitors (mmTECp).a K19Cre was induced in Krt19CreERTtdTom embryos at E15.5 via the administration of tamoxifen to pregnant mice, and fate-mapped thymi were harvested at PNd0 (n = 14, from 3 independent experiments). Fate-mapped cells were detected within EpCAM1+ cells by flow cytometry and quantitated. b Representative FACS plots and quantitation of K19-tdTom fate-mapped cells within EpCAM1+UEA1+ and EpCAM1+Ly51+ TEC. c Expression of Ly51 and UEA1 by total EpCAM1+ cells, K19-tdTom- EpCAM1+ cells (non-fate-mapped) and K19-tdTom+EpCAM1+ cells (fate-mapped). d Bar charts show proportions of Ly51+UEA1- and Ly51-UEA1+ cells, and mTEC:cTEC ratio within non-fate-mapped and fate-mapped TEC. e Bar chart shows fold change in Ly51+UEA1- and Ly51-UEA1+ cells within non-fate-mapped and fate-mapped TEC. f Immunofluorescence of PNd0 thymi following K19-fate-mapping at E15.5. K19-tdTom (red) and K5 (turquoise). ‘C’, and ‘M’ indicate cortex and medullary areas respectively. Scale bar denotes 20 μm. Image representative of 4 thymi. g Expression of MHCII by EpCAM1+Ly51+ fate-mapped (FM) or non-fate-mapped (Non-FM) TEC. h Representative FACS plots illustrating the phenotype of fate-mapped mTEC subsets. mTEChi (MHCIIhiCD80hi, n = 14), mTEClo (MHCIIloCD80lo, n = 14), Aire+ (MHCIIhiCD80hiAire+, n = 14), CCL21+ (n = 6), tuft cells (MHCIIloCD80loDCLK1+, n = 8), and corresponding quantitation. i, j Immunofluorescence of PNd0 thymi where K19Cre was induced at E15.5. Scale bar denotes 10 μm. Image representative of 4 thymi, from 3 independent experiments. K19-tdTom (red), EpCAM1 (blue), f Aire (turquoise), g DCLK1 (turquoise). Data analysed using a Student’s t test. The data are shown as mean ± SEM.