Methods S1 Ultra-deep sequencing analysis. (DOC) Gene Expression Profile during Chondrogenesis in Human Bone Marrow derived Mesenchymal Stem Cells using a cDNA Microarray Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into multiple connective tissue lineages, which include cartilage, bone, and fat. Cartilage differentiation and chondrocyte maturation are required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. This study was designed to identify novel genes that might help clarify the molecular mechanisms of chondrogenesis. Chondrogenesis was induced by culturing human bone marrow (BM) derived MSCs in micromass pellets in the presence of defined medium for 3, 7, 14 or 21 days. Several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (RT-PCR). Using an ABI microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and BM-MSCs. Normalization of this data resulted in the identification of 1,486 differentially expressed genes. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes were confirmed by RT-PCR. Gene expression patterns of 9 genes (Hrad6B, annexinA2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) in RT-PCR were similar to the microarray gene expression patterns. These findings provide novel information concerning genes involved in the chondrogenesis of human BM-MSCs. Mesenchymal stem cells (MSCs) are present in a variety of tissues during human development, and in particular, are prevalent in adult bone marrow (1) . MSCs isolated from bone marrow (BM) and expanded in vitro in their undifferentiated phenotype, retain an extensive capacity for multi-lineage differentiation into chondrocytes, adipocytes, osteoblasts, and tenocytes under appropriate environmental cues (2) . The presence of specific, distinct antigens identified by the monoclonal antibodies SH2, SH3, and SH4, on the surfaces of marrow-derived MSCs, that are not present on osteocytes and osteoblasts, suggests that these epitopes are developmentally regulated. The antigen which bound to SH2 antibody was identified as endoglin (CD105), a receptor for TGF-β3, which potentially plays a role in mediating the chondrogenic differentiation of MSCs and in their interactions with hematopoietic cells (3) .