Figure 2.
Properties of metaphyseal BMSCs
(A) Hey1-GFP (green) expression in 3-week-old femur is predominantly found in metaphysis. GFP co-localizes with PDGFRβ (red, left) and PDGFRa (red, right) in the metaphysis (arrowheads) but also labels arterial ECs (arrow) in BM. ECs, EMCN+ (blue).
(B) Principal component analysis (PCA) of RNA sequencing data shows 56% variance (PC1) between sorted mpMSCs and dpMSCs and 17% variance (PC2) within sample groups.
(C) Differentially regulated genes in MA plots of mpMSCs relative to dpMSCs. The x axis represents mean normalized counts, and the y axis shows the log2-fold change between the two populations. Differentially regulated genes are represented by red-colored points (false-detection rate [FDR]-adjusted p < 0.01 and absolute log2-fold change < 1). Data points outside of the range of the y axis are represented as triangles.
(D) Heatmap of differentially expressed BMSC marker genes in mpMSCs and BM-derived dpMSCs.
(E) Quantification of colony-forming units (CFU) in mpMSCs compared with dpMSCs. (n = 6; data are presented as mean ± SEM, p values, two-tailed unpaired t test).
(F) Confocal image of spheroid formed by Pdgfra-GFP+ PDGFRβ+ mpMSCs after 15–20 days in culture. Inset marks spheroid shown at high magnification on the right.
(G and H) Osteogenic cells derived from mpMSCs in culture express the osteoblast lineage markers osteopontin (OPN, blue) and osteocalcin (OCN, red) (G). Upregulation of osteogenic markers in differentiated cells relative to mpMSCs (H) (n = 4–8; data are presented as mean ± SEM, p values, two-tailed unpaired t test).
(I) Adipogenic differentiation of mpMSCs in culture indicated by perilipin immunostaining (left) and significantly increased transcription of Adipoq, Cfd, and Pparg relative to control (n = 4; data are presented as mean ± SEM, p values, two-tailed unpaired t test).
(J) Chondrogenic differentiation of cultured mpMSCs indicated by Aggrecan immunostaining (left) and significantly increased transcription of Acan and Col11a2 relative to control (n = 4; data are presented as mean ± SEM, p values, two-tailed unpaired t test).
See also Figure S2