Figure 4. Variable expression of commonly used reporter genes in skeletal stem cell types.

(A) Microarray data of freshly purified bulk cell populations of the previously defined osteochondrogenic skeletal stem cell (ocSSC) and perivascular SSC (pvSSC) lineage trees from 8-week-old mice showing gene expression of markers commonly known to trace and/or label cell populations enriched for SSCs. Expression is shown as normalized activity as processed by GEXC (Gene Expression Commons). Each cell-type represents a pool of cells derived from three to four mice. (B) Single-cell heatmaps of gene expression of markers commonly used to trace and/or label cell populations enriched for SSCs as observed in single-cell RNA-sequencing (scRNAseq) results of ocSSCs and pvSSCs. (C) Flow cytometric analysis of antibody labeling for leptin receptor (LepR) in different populations of the ocSSC and pvSSC lineage trees in differently aged mice (n = 3). (D) Fibroblast colony-forming unit (CFU-F) assay of freshly isolated ocSSCs and pvSSCs separated by their expression of LepR (ocSSC n = 9; pvSSC n = 6, from two independent experiments). (E) Expression of osteocalcin (OCN, green) in in vitro osteogenically differentiated ocSSCs and pvSSCs in LepR-positive and -negative fractions. (F) Quantification of percentage of cells expressing OCN as determined by antibody labeling at 2 weeks of osteogenic differentiation (n = 5). Significance between groups was assessed by unpaired, two-tailed Student’s t-test. All data are shown as mean + SEM. Scale bars, 30 µm.