Figure 2

Clonogenic fibroblast-like cells originate from both CD105⁺CD90⁺CD13⁻ and CD105⁺CD90⁺CD13⁺ populations. (A–C) Gating strategy for sorting of mesenchymal cell populations for CFU-f assay. (A) Exclusion of dead cells (7AAD⁺) and non-mesenchymal lineage (lin⁻) cells (hematopoietic cells: CD45⁺, endothelial cells: CD31⁺, epithelial cells: EpCAM⁺) after forward-scatter/side-scatter gating. (B) Gates set for sorting of lin⁻CD105⁻ (black) and lin⁻CD105⁺ (yellow) cells. (C) Gates set for sorting of lin⁻CD105⁺CD90⁻CD13⁻ (black), lin⁻CD105⁺CD90⁺CD13⁻ (blue) and lin⁻CD105⁺CD90⁺CD13⁺ (red) cells. (D) CFU-f frequency in sorted lin⁻CD105⁺ cells compared to lin⁻CD105⁻ and unsorted lung cells (fresh and cryopreserved). (E) CFU-f frequency in sorted lin⁻CD105⁺CD90⁻CD13⁻, lin⁻CD105⁺CD90⁺CD13⁻ and lin⁻CD105⁺CD90⁺CD13⁺ cell populations, with lin⁻CD105⁺ cells as comparison. CFU-f frequency data (healthy donors, n = 3; non-tumorous lung cancer tissue, n = 1) is presented as mean (with SD). One-way ANOVA with Geisser–Greenhouse correction and Holm–Sidak correction for multiple comparisons was used for statistical analysis (*p < 0.05). (F) The plot shows forward-scatter and side-scatter characteristics for 1532 index-sorted cells (749 CD13⁻ and 783 CD13⁺) combined from 4 healthy donors. The 35 CD13⁻ cells (blue dots) and 97 CD13⁺ cells (red dots) formed colonies in CFU-f assays. Sorted cells that did not generate colonies are represented by grey dots. (G,H) Images of crystal violet stained colonies derived from CD13⁻ cells (G) and CD13⁺ cells (H). Scale bars represents 500 µm. (I) Representative histograms showing the distribution of colony size in CD13⁻ and CD13⁺ derived CFU-fs (crystal violet stained) from one healthy donor. Normalized colony count is shown on the y-axis and colony size is on the x-axis. Solid lines represent density plots. Vertical dashed lines represent median colony area. (J) The median colony size of CD13⁻ and CD13⁺ derived CFU-fs (healthy donors, n = 3; non-tumorous lung cancer tissue, n = 1). Bars represent mean (with SD) and dots represent median for individual subjects. Paired two-tailed t-test was used for statistical analysis of colony area data (**p < 0.01). FSC-A forward scatter-area, SSC-A side scatter-area, CFU-f colony forming unit-fibroblast.