Randomized double-blind study of a single intra-articular injection of allogeneic mesenchymal precursor cells + hyaluronan compared to hyaluronan alone to explore clinical and structural outcomes in patients post anterior cruciate ligament reconstruction

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S16 differentiation. lncRNAs can be derived from both intergenic and overlapping protein-coding gene regions, are expressed in a highly tissue-specific manner and are emerging as key regulators of gene expression. In this study we establish a role for lncRNAs in chondrogenesis and cartilage. Methods: Paired end sequencing was performed by Illumina Genome Analyzer IIx on total RNA isolated from human articular cartilage and human MSCs undergoing chondrogenic differentiation. lncRNAs and novel transcripts were identified by aligning sequence reads to the transcriptome and genome respectively. Novel transcripts were confirmed by rapid amplification of cDNA ends (RACE) and expression validated by real-time RT-PCR in a panel of human tissues. Expression was assessed in the tri-lineage differentiation of MSCs into chondrocytes, osteoblasts and adipocytes. lncRNA expression was depleted with specific siRNAs to establish their role during differentiation. Overexpression of lncRNAs was performed by cloning and lentiviral transduction as well as CRISPR/Cas9-VP16 mediated endogenous promoter activation. Chondrocyte and cartilage development was assessed by gene and protein expression measured with standard molecular biology, biochemical and histological techniques. lncRNA-regulated genes were identified by microarray and lncRNA-regulated signalling pathways in chondrogenesis assessed by Western blotting and reporter activation. Results: RNA sequencing indicates that human adult cartilage contains upwards of 3000 lncRNAs (FPKM u003e1) and that 188 of these are induced during MSC chondrogenic differentiation (u003e2fold). Of particular interest are a group of lncRNAs upstream of the SOX9 locus, found in a gene desert on chromosome 17, which includes SOX9-AS1, LINC01152 and a novel 4 exon version of the predicted 3 exon RNA, ENST00000430908. This novel lncRNA is chondrocyte specific and upregulated during chondrogenic differentiation of MSCs. RNAi targeting novel ENST00000430908 prevented MSC chondrogenesis with a complete loss of cartilage matrix formation and significant reductions in chondrocyte gene expression. Therefore we named this novel lncRNA ROCR (Regulator Of Chondrogenesis RNA). Depletion of ROCR totally blocked the induction of SOX9 during MSC chondrogenesis, consistent with the reduced cartilage-specific gene expression. Microarray analysis indicates that loss of ROCR phenocopies the loss of SOX9 on gene expression changes during the early stages of chondrogenesis. Loss of ROCR may also disrupt chondrogenic cell signalling, in particular activation of the TGF-b pathway. In contrast to the effect during chondrogenesis loss of ROCR enhances osteoblastogenic differentiation and gene expression. Conclusions: The cartilage transcriptome contains numerous noncoding transcripts many of which may have essential functions in cartilage biology. ROCR is essential for cartilage development and appears to promote a chondrocyte cell fate for MSCs over an osteoblast cell fate. Because SOX9-expressing cells are progenitors for numerous tissues identifying chondrocyte-specific regulatory elements might aid understanding of differentiation of chondrocytes from MSCs, potentially useful in chondrocyte tissue engineering applications. 15 SPECIFIC SYNOVIAL JOINT PROGENITOR CELL POPULATIONS UNIQUELY CONTRIBUTE TO ARTICULAR CARTILAGE GROWTH AND REPAIR R.S. Decker y, H.-B. Um y, N.A. Dyment z, N. Cottingham y, E. Koyama y, M. Kronenberg z, P. Maye z, D.W. Rowe z, M. Pacifici y. yChildrenu0027s Hosp. of Philadelphia, Philadelphia, PA, USA; zUniv. of Connecticut, Farmington,