逐步预处理通过表观遗传修饰增强基于间充质干细胞的软骨再生。
Stepwise preconditioning enhances mesenchymal stem cell-based cartilage regeneration through epigenetic modification.
机构信息
Department of Orthopaedics & Traumatology, Lui Che Woo Institute of Innovative Medicine, Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China; Department of Pharmacology, Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, Guangdong, China; The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, China.
Department of Orthopaedics & Traumatology, Lui Che Woo Institute of Innovative Medicine, Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China; The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, China.
出版信息
Osteoarthritis Cartilage. 2017 Sep;25(9):1541-1550. doi: 10.1016/j.joca.2017.05.008. Epub 2017 May 22.
OBJECTIVE
This study is to investigate the functions and underlying mechanisms of mesenchymal stem cells (MSCs) underwent stepwise preconditioning in chondrogenic medium before expansion, then further explore their therapeutic effects in a surgically induced osteoarthritis (OA) model.
METHODS
MSCs isolated from the adult rats expressing Green Fluorescence Protein (GFP) were incubated in basal medium or primed in chondrogenic medium before expansion. The multipotency including cell proliferation, differentiation, and survivability was compared between chondrogenic manipulated MSCs (M-MSCs) and untreated MSCs. Methylation modification of Nanog and Oct4 were detected by bisulfite genomic sequencing. Loss-of-function phenotype in M-MSCs induced by shNanog was also observed. Then the therapeutic effect of the cells was evaluated in a surgically induced OA rat model by single intraarticular injection. The injected GFP-labeled cells in the joints were monitored in vivo. These rats were sacrificed and subjected to histological examinations and microstructural analysis after 4 weeks.
RESULTS
We found that cell clonogenicity, proliferation, survivability, and chondrogenic property were enhanced after stepwise preconditioning. We then further found that the expression level of Nanog and Oct4 was temporarily increased in the M-MSCs. Results of epigenetic analysis revealed that demethylation happened in Nanog and Oct4 after the stepwise preconditioning. Results of in vivo imaging showed more GFP-labeled cells in the M-MSCs-injected group. And results of histology and micro-CT analysis also indicated a superior therapeutic effect of M-MSCs on the surgically induced-OA.
CONCLUSION
These findings indicated a feasible method to obtain a cell population with high survivability and chondrogenic commitment for the treatment of OA.
目的
本研究旨在探讨间充质干细胞（MSCs）在扩增前经历逐步软骨形成培养基预培养的功能和潜在机制，然后进一步探索其在手术诱导性骨关节炎（OA）模型中的治疗效果。
方法
从表达绿色荧光蛋白（GFP）的成年大鼠中分离的 MSCs 在基础培养基中孵育或在扩增前在软骨形成培养基中预培养。比较软骨形成处理的 MSCs（M-MSCs）和未处理的 MSCs 之间的多能性，包括细胞增殖、分化和存活率。通过亚硫酸氢盐基因组测序检测 Nanog 和 Oct4 的甲基化修饰。还观察了 shNanog 诱导的 M-MSCs 中的功能丧失表型。然后通过单次关节内注射评估细胞在手术诱导的 OA 大鼠模型中的治疗效果。在体内监测关节内注射的 GFP 标记细胞。4 周后处死这些大鼠，并进行组织学检查和微观结构分析。
结果
我们发现细胞集落形成能力、增殖、存活率和软骨形成能力在逐步预培养后增强。我们进一步发现，M-MSCs 中的 Nanog 和 Oct4 表达水平暂时增加。表观遗传学分析结果表明，逐步预培养后 Nanog 和 Oct4 发生去甲基化。体内成像结果显示，M-MSCs 注射组中有更多的 GFP 标记细胞。组织学和 micro-CT 分析结果也表明 M-MSCs 对手术诱导的-OA 具有更好的治疗效果。
结论
这些发现表明了一种获得具有高存活率和软骨形成承诺的细胞群体的可行方法，用于治疗 OA。
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