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

我正在申请续签我的第一个NSERC DG，这使我能够建立一个研究计划，专注于了解microRNAs(MiRNAs)在干细胞内稳态、干细胞软骨分化和滑膜关节健康生物学中的作用。基于我的长期研究目标和我成功地建立了NSERC资助的以miRNA为重点的研究计划，目前这项提议的具体短期目标和目的是： 目的1：鉴定具有预测价值的miRNA信号，以鉴定高软骨性ECB-MSC培养物。本研究的两个目的是：1)确定miR-203a-3p、miR-146a-5p和miR-183-5p的过表达和敲除对马CB-MSC成软骨潜能的影响。2)确定条件培养液中的miRNAs能否用于预测骨髓间充质干细胞的软骨分化潜能。 目的：鉴定血浆和滑膜关节液中miRNAs或miRNA信号与正常关节稳态和异常关节稳态的关系。这个目标的两个目标是：1)确定选定的血浆和/或滑膜miRNAs是否区分患有正常和异常窒息(股骨关节)的马。2)确定miRNA在健康关节和骨关节炎关节中的表达在不同关节之间是否存在差异，即锁骨关节、腕关节和窒息关节。 目标3：描述ECB-MSC产生的小RNA的进化模式，并确定是否发生显著的稳定变化与易位或与不适当的细胞行为相关的其他细胞变化。这一目标的两个目的是：1)确定条件培养液中的miRNAs是否可用于预测MSCs的细胞遗传学稳定性。2)研究过表达或敲除来自Aim 1的差异表达的miRNAs对马CB-MSC细胞遗传学稳定性的影响。 在接下来的五年里，我们计划继续我们对miRNAs在ECB-MSC软骨形成中的作用及其作为关节健康生物标志物的效用的极其原创性的探索，并开始探索miRNAs与染色体稳定性之间的联系。三名研究生和8-10名本科生将接受最先进的细胞和分子技术培训，如NGS和CRISPR。我们的工作将产生通过miRNAs调节基质细胞的软骨形成能力的基础知识，并发现新的软骨形成能力的标记。通过使用敏感的生物标记物及早发现关节疾病将是一大进步。细胞遗传学异常可以正面或负面地影响细胞功能，我们非常新颖的工作将增加关于体外培养过程中MSCs细胞遗传学成分漂移的潜在机制的迫切需要的知识。

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.