Domestic animal stem cell biology for understanding tissue development, maintenance and therapeutic interventions 2.0

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I am applying to renew my first NSERC DG, which allowed me to establish a research program focused on understanding the role of microRNAs (miRNAs) in stem cell homeostasis, stem cell cartilage differentiation and synovial joint health biology. Based on my long-term research goals and my successful establishment of a NSERC funded miRNA-focused research program, the specific short-term objectives and aims for this current proposal are: Objective 1: To identify miRNA signatures with predictive value to identify highly chondrogenic eCB-MSC cultures. The two aims within this objective are: 1)To determine the effect on chondrogenic potential of equine CB-MSC by overexpression and knockout of miR- 203a-3p, miR-146a-5p, and miR-183-5p. 2) To determine if miRNAs in conditioned equine CB-MSC culture medium can be used to predict chondrogenic potency of the MSCs. Objective 2: To identify miRNAs or miRNA signatures in blood plasma and synovial joint fluid associated with normal healthy joint homeostasis and abnormal joint homeostasis. The two aims within this objective are: 1) To determine if select plasma and or synovial miRNAs discriminate between horses with normal and abnormal stifle (femoropatellar) joints. 2) To determine if miRNA expression in healthy and osteoarthritic joints differ between different joints, i.e. fetlock versus carpal versus stifle joints. Objective 3: To characterise the evolving pattern of small RNAs produced by eCB-MSC and determine whether significant stable changes occur in conjunction with translocation or other cellular changes that are associated with unsuitable cellular behaviour. The two aims within this objective are: 1) To determine if miRNAs in conditioned equine CB-MSC culture medium can be used to predict cytogenetic stability of the MSCs. 2) To determine the effect on cytogenic stability of equine CB-MSC by overexpression or knockout of candidate differentially expressed miRNAs from aim 1. Over the next five years, we plan to continue our extremely original exploration of miRNAs' roles in eCB-MSC chondrogenesis and their utility as biomarkers of joint health and initiate work exploring associations between miRNAs and chromosomal stability. Three graduate students and 8-10 undergraduate students will receive advanced cell and molecular training in state-of-the-art technologies such as NGS and CRISPR. Our work will generate fundamental knowledge in the regulation of chondrogenic potency of stromal cells by miRNAs and discover novel markers of chondrogenic capacity. Early detection of joint disease through the use of sensitive biomarkers would be a major advance. Cytogenetic aberrations can positively or negatively affect cell functions, and our very novel work will add much needed knowledge about the underlying mechanisms of drifting cytogenetic composition of MSCs during in vitro culturing.