FIG. 1.

Highly enriched CD271^pos/CD140a^low/neg BMSCs display typical stromal cell characteristics. (A) Cytospin preparations of sorted human bone marrow (BM) CD271^pos/CD140a^low/neg cells showed a typical primary BMSC morphology, that is, large immature nuclei with an open chromatin pattern and cytoplasmic vacuoles (May-Grunwald/Giemsa staining, scale bar represents 20 μm). (B) Flow cytometric surface marker co-expression analysis on primary CD271^pos/CD140a^low/neg BMSCs. 2 × 10⁶ events were acquired and plotted for CD271 versus the marker listed on the top after FSC/SSC gating, doublet, and dead cell exclusion (the gating strategy is illustrated in Supplementary Fig. S1A). A representative dataset is shown. (C, D) Single-cell gene expression analysis was performed on sorted CD271^pos/CD140a^low/neg from three donors. The results are shown as heatmap, in which each of the 39 columns represents an individual cell (C) and as violin plots illustrating the expression level of the genes across the samples based on ANOVA (D). Genes are listed according to function and cell type. Group I: hematopoietic supporting genes, group II: commonly expressed MSC genes, group III: differentiation-related genes, group IV: mesodermal markers, group V: neural crest markers, and group VI: signaling pathway genes. (E) Gene expression analysis of sorted single lin^neg/CD45^neg/CD271^pos/CD140a^low/neg cells compared with non-CFU-F-containing lin^neg/CD45^neg/CD271^neg/CD140a^neg cells from the same donors. The results are shown as Principal Component Analysis (PCA).