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

用于了解组织发育、维持和治疗干预的家畜干细胞生物学 2.0

• 批准号: RGPIN-2020-05170
• 负责人: Koch, Thomas
• 金额: $ 2.4万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717193
• 关键词: Domestic; animal; stem; cell; biology

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.

我正在申请更新我的第一个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建立的NSERC资助的以miRNA为重点的研究计划，本提案的具体短期目标和目的是： 目的1：鉴定具有预测价值的miRNA特征，以鉴定高度软骨形成的eCB-MSC培养物。该目标中的两个目的是：1）确定通过过表达和敲除miR-203 a-3 p、miR-146 a-5 p和miR-183- 5 p对马CB-MSC的软骨形成潜力的影响。2)确定马CB-MSC条件培养基中的miRNAs是否可用于预测MSC的软骨形成能力。 目标二：鉴定血浆和关节滑液中与正常健康关节稳态和异常关节稳态相关的miRNA或miRNA特征。该目的内的两个目的是：1）确定选择的血浆和/或滑膜miRNA是否区分具有正常和异常后膝（股髌）关节的马。2)确定健康和骨关节炎关节中的miRNA表达在不同关节之间是否不同，即球节与腕骨关节与后膝关节。 目标三：研究eCB-MSC产生的小RNA的演变模式，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。该目标中的两个目的是：1）确定条件马CB-MSC培养基中的miRNA是否可用于预测MSC的细胞遗传学稳定性。2)确定通过过表达或敲除来自目标1的候选差异表达的miRNA对马CB-MSC的细胞遗传稳定性的影响。 在接下来的五年里，我们计划继续对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进行非常原始的探索，并开始探索miRNAs与染色体稳定性之间的关联。3名研究生和8-10名本科生将接受NGS和CRISPR等最先进技术的高级细胞和分子培训。我们的工作将产生通过miRNA调节基质细胞成软骨能力的基础知识，并发现成软骨能力的新标志物。通过使用敏感的生物标志物早期检测关节疾病将是一个重大进展。细胞遗传学畸变可以积极或消极地影响细胞功能，我们非常新颖的工作将增加有关MSC在体外培养期间漂移细胞遗传学组成的潜在机制的知识。