Unique patterns of gene expression in articular chondrocytes: important insight for joint surface lesion repair and cell-based therapies

Rationale: Degenerative joint disease and osteoarthritis are the major cause of lameness and loss of athletic performance in horses. New cell-based strategies to repair joint surface lesions are generating a high level of interest, but have yet to achieve full restoration of articular cartilage structure and function. The body contains several different types of cartilage. By extension, chondrocytes from within these cartilaginous tissues are also different. An understanding of chondrocyte cell biology, especially the molecular phenotype of articular chondrocytes, is an important knowledge gap limiting progress. Developmental biology studies have identified a tissue, termed interzone, that is a progenitor of articular cartilage and other joint tissues. Experiments in the mentor's laboratory using salamanders have demonstrated that interzone tissue facilitates the complete repair of large cartilage defects in joints. More astonishingly, this same interzone tissue transplanted into a tibial gap fracture creates an entirely new joint de novo in the middle of the diaphysis. Interzone cells in mammals are confined to a short developmental window in the early fetus, but then disappear. This year, however, we have been able to isolate equine interzone tissue and derive primary cell lines for tissue culture. The rationale for this project is that the interzone may contain a population of progenitor cells already biologically committed to form articular cartilage and other synovial joint tissues. As such, they may provide a far superior candidate for cell-based regenerative therapies to repair articular surface lesions. Hypotheses & Objectives: Proposed experiments are designed to test two hypotheses. The first is that different types of cartilage have unique tissue-restricted patterns of gene expression. The second is that chondrocytes generated from interzone cells in culture will express mRNAs that are characteristic of normal articular cartilage. Project objectives are: 1) analyze and compare the mRNA transcriptomes of different adult, neonatal, and fetal cartilaginous tissue types, 2) define the unique, tissue-restricted mRNAs of normal adult articular cartilage, and 3) compare chondrocytes generated in culture from interzone cells to those from other cell types currently used for joint therapies. Study Design: Messenger RNA sequencing (2x1 00 paired-end lllumina reads) will be used to compare relative gene expression on a transcriptome level between different cartilaginous tissues, specifically articular cartilage and nasal septum in adult horses (n=5), articular and epiphyseal cartilage in neonatal foals (n=7), and interzone-rich tissue and adjacent cartilage anlage from early equine fetuses (n=7). These data will be used to define mRNA transcripts and exon splices restricted to each tissue. Different cells types, both fetal and adult, will be placed under conditions to stimulate chondrogenic differentiation in culture. Gene expression profiles in the resulting derived "chondrocytes" will be compared on qualitative and quantitative levels, and also relative to the tissue biomarker panels. Preliminary Data: All of the tissue samples required for this study have already been collected. By age category (fetal, neonatal, and adult), the tissues are paired in biological replicates. In addition, primary cell lines from equine interzone tissue have been successfully derived and shown to retain expression of major interzone biomarkers including growth and differentiation factor 5 (GDF5). Expected Results: Experimental, analytical, and computational methods required for the processing of samples, mRNA sequencing, and tissue culture are well established in the laboratory. No major technical problems are anticipated. The data are expected to identify at least 50 tissue-restricted transcripts and exon splice junctions for each tissue type analyzed. The resulting "mRNA fingerprint" panels for different cartilage tissue types will be a valuable resource for the scientific community. The extent and magnitude of gene expression variance between induced "chondrocytes" generated in culture from biological replicates of the same progenitor cell type and entirely different progenitor cell populations will be determined. Budget & Timeline: Funding will be used for stipend support of the trainee, with an effort distribution of 100% research. RNA sequencing and transcriptome analyses of different cartilaginous tissues will be completed in year 1. Tissue culture and the analysis of gene expression patterns following stimulation of chondrogenic differentiation will be completed in year 2. Impact: Critically important baseline data that defines the molecular phenotype (gene expression profile) of chondrocytes from equine articular cartilage will be generated. The interzone, a novel and promising developmental tissue for cell-based regenerative therapies of articular lesions and degenerative joint disease in the horse, will be evaluated.