ABSTRACT The goals of this study are to determine the function and mechanism of specific human long non-coding RNAs (lncRNAs) in regulating chondrocyte differentiation and cartilage formation. LncRNAs are transcripts of greater than 200 nucleotides with no protein coding potential that can function to regulate gene expression or protein function to coordinate cellular processes critical for tissue-specific differentiation. While there is compelling data on the role of lncRNAs in other systems, there is a significant gap in our understanding of how lncRNAs function in cartilage biology. To date, there is vert little information on how lncRNAs affect human chondrocyte differentiation either functionally or mechanistically. We have generated a database containing expression patterns of lncRNAs during chondrocyte differentiation of human primary bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs). Detailed analysis of this RNA-Seq data has shown that many lncRNAs exhibit dynamic expression patterns. Five candidate lncRNAs were subsequently chosen for further study based on significant changes in their expression levels during early phases of chondrogenesis in addition to correlation with SOX5/6/9 expression. We showed that knock-down of one of these lncRNAs (LINC01503) inhibits BM-MSC chondrogenesis. Based on this result, we hypothesize that LINC01503, as well as the other dynamically-expressed candidate lncRNAs, play an essential role in regulating chondrogenesis. By determining their function during chondrogenesis, insight into the molecular mechanisms controlling chondrogenic programming of human BM-MSCs will be gained. To address our hypothesis, two specific aims are proposed. Specific Aim 1 is designed to determine the cellular localization of the lncRNA of interest as well as its role in modulating BM-MSC chondrogenesis via loss-of-function or gain- of-function approaches. RNA-Seq will then be carried out to elucidate the genes / cellular pathways altered by modulating the lncRNA. Specific Aim 2 is designed to decipher lncRNA interactomes by using state-of-the art technologies to determine protein and RNA binding partners as well as chromosomal regions contacted by the lncRNA, whenever applicable. These approaches will aid in determining the mechanism by which the lncRNA of interest controls chondrogenesis. Overall, these studies will provide new knowledge on lncRNA regulation of human progenitor cell differentiation toward the chondrocyte lineage. Obtaining such information will be important toward the future design of therapeutic strategies to induce human articular cartilage repair or regeneration, thereby advancing the field of cartilage tissue engineering. From a clinical standpoint, these studies are significant given the fact that articular cartilage has no intrinsic repair capabilities, and there are currently no effective treatments to attenuate, inhibit, or reverse articular cartilage degradation that occurs in osteoarthritis, the most common cartilage degenerative disease in humans.