BM-MSCs have a distinct gene expression and DNA methylation signature. (A) Unsupervised hierarchical clustering dendrograms of gene expression analysis performed using Affymetrix GeneChip Human Gene 2.0 ST arrays of early-passage BM-MSCs and WAT-, UC-, and skin-derived MSCs cultured in pHPL. (B) Heat map representing all significant differentially expressed genes, with red indicating high gene expression and green indicating decreased gene expression of BM-MSCs compared with non-BM-derived (WAT/UC/skin) MSCs. (C) Unsupervised hierarchical clustering dendrograms of differentially methylated regions analyzed using HumanMethylation450 BeadChip arrays performed on the same BM-MSCs and WAT-, UC-, and skin-derived MSCs cultured in pHPL as in panel A. (D) Heat map showing the top 100 differentially methylated regions in BM-MSCs compared with non-BM-derived (WAT/UC/skin) MSCs, with red indicating hypomethylation and green indicating hypermethylation. (E) Identification of genes with concordant changes in messenger RNA expression and DNA methylation. Quadrant plot showing differentially methylated CpGs (only CpGs with the most significant P values are depicted) and expression of associated genes. The x-axis depicts the −log₁₀P value for differentially methylated CpGs; the y-axis depicts the −log₁₀P value of differential expression for associated genes. Vertical dashed lines indicate a false discovery rate of 5%; horizontal dashed lines indicate a P value of .05. The 4 quadrants are (i) hypomethylated and upregulated in BM-MSCs (solid purple circles and solid black circles); (ii) hypermethylated and downregulated in BM-MSCs (solid blue circles); (iii) hypomethylated and downregulated in BM-MSCs (open black circles); and (iv) hypermethylated and upregulated in BM-MSCs (open black circles). Hypomethylated and upregulated genes with known roles in chondrogenesis and osteogenesis (solid black circles) are labeled with arrows. (F) Quantitative reverse transcription–polymerase chain reaction–based validation of the baseline expression in key genes found to be hypomethylated and overexpressed, as well as other transcription factors and lineage-specific genes associated with cartilage and bone formation in various MSC populations. Runt-related transcription factor 2 and 3 (RUNX2 and RUNX3), distal-less homeobox 5 and 6 (DLX5 and DLX6), osteopontin (SPP1), osteocalcin (BGLAP), bone-specific alkaline phosphatase (ALPL), and aggrecan (ACAN) expression was analyzed in MSCs derived from BM (black bars) compared with WAT (dark gray bars), UC (light gray bars), and skin (white bars). Expression differences for each gene compared with the GAPDH housekeeping gene are shown relative to BM levels. (G) Western blot analysis of RUNX3 protein levels in the positive-control U2OS osteosarcoma cell line, as well as in BM-MSCs and WAT-, UC-, and skin-derived MSCs. Bar graphs depict densitometry-derived mean areas under the curve (AUC) ± standard deviation (SD) normalized to β-actin. All densitometry values are in relation to BM-MSCs (n = 3 per MSC source; *P < .0001).