基于CRISPR-Cas9系统高通量筛选间充质干细胞衰老分泌表型的调控因子及其功能的研究

The poor quality and quantity of Mesenchymal stem cells (MSCs) after in vitro expansion limits their widespread use in stem cell therapies for musculoskeletal tissue repair. Cellular senescence is one of the major roadblocks in effective MSCs expansion for clinical transplantation. In previous study, we found that the major components of Senescence-associated Secretory Phenotype (SASP) are upregulated in aged MSCs which accompanied with abnormal lineage differentiation ability and increased clearance of MSCs by immune cells. However, therapeutic inhibition of SASP in MSCs is hindered by incomplete understanding of SASP regulatory network. Thus, we propose to systematic dissect the regulatory network of SASP and its biological effect in MSCs. We will (1) Identify both positive and negative regulators of SASP in MSCs with genome-wide CRISPR/Cas9 screening, and discover regulatory modules and key regulators of SASP through single-cell transcriptomic analysis; (2) Validate the regulatory functions of candidate genes in proliferation, differentiation and senescence of MSCs; (3) Explore the therapeutic potential of small molecules which targeting the candidate genes in cartilage defect repair model, and exam whether the regenerative ability of MSCs is potentiated through SASP inhibition during in vitro expansion. In summary, our study will provide new insights about SASP regulatory network in MSCs, discover new therapeutic targets and strategies for delay MSCs senescence during expansion, which would improve the efficacy and safety of MSCs transplantation in regenerative medicine for musculoskeletal tissue repair.

干细胞数量和质量是确保细胞移植和再生医学疗效的关键因素。间充质干细胞（MSC）在体外扩增过程中容易发生衰老，阻碍其临床应用。本课题组前期研究发现衰老分泌表型（Senescence-associated Secretory Phenotype， SASP）的关键成分在衰老MSC中表达上调，导致MSC分化能力异常且易被免疫细胞识别和清除。但目前SASP分子调控网络知识的缺乏，严重阻碍了特异有效的SASP阻断手段的开发。本项目拟系统性解析SASP的调控网络并研究关键调控因子对MSC再生能力的影响，即（1）运用CRISPR/Cas9系统在全基因组水平筛选SASP的正向和负向调控因子，结合单细胞转录组分析SASP的调控模块及关键调控因子；（2）验证关键调控因子对于MSC的衰老、增殖、分化等功能的调节作用；（3）运用软骨修复模型，研究特异靶向关键调控因子的小分子对于MSC体内再生功能的调控作用。

细胞衰老影响干细胞移植治疗的安全性和有效性，同时参与了衰老相关疾病的发生。细胞衰老相关分泌表型（Senescence-associated Secretory Phenotype，SASP）具有促旁分泌衰老及组织退变的作用，可以作为延缓细胞衰老及衰老相关疾病的治疗靶点。但是，目前对于IL-1α及SASP分泌的分子调控网络了解不甚全面，缺少安全有效的分子靶点。.本项目的主要研究内容为运用CRISPR/Cas9的全基因组基因敲除文库，系统性地筛选调控干细胞 SASP的基因，并通过操纵关键调控基因，抑制SASP的分泌从而达到延缓细胞衰老及骨关节炎进展的作用。.本项目研究发现：.(1) 全基因组高通量筛选SASP调控因子：通过系统性CRISPR/Cas9 的全基因组筛选获得潜在衰老MSC的SASP的正向基因514个，负向调控基因741个。其中我们发现了已知的正向调控通路（DNA repair）和负向调控通路（自噬）,同时也发现新的SASP分泌调控通路如sphingolipid和galactose代谢。.(2) 关键SASP调控因子的鉴定：对衰老MSC进行单细胞转录组测序，我们发现了具有不同转录特征的衰老亚群，根据其特征性应答通路可分为：蛋白质稳态失衡亚型及炎症应答亚型。结合第一部分筛选结果及CRISPR单基因敲除功能验证，我们最终鉴定了两个关键的衰老细胞亚群中上调的IL1A正向调控基因：RCAN1和ADAM19。.(3) 抑制SASP用于抗MSC衰老及体内骨关节炎治疗：通过gRNA或siRNA靶向抑制RCAN1或ADAM19体外可以延缓MSC或软骨细胞衰老。同时，在小鼠骨关节炎模型中，通过siRNA靶向抑制RCAN1或ADAM19可以延缓细胞衰老和软骨基质破坏。而RCAN1调控了细胞脂质代谢产物的形成，提示了脂质代谢可能参与了细胞衰老及SASP分泌的调控。.本研究系统性地解析MSC衰老过程中SASP分泌的正向及负向调控机制并明确其下游生物学效应，为开发用于延缓细胞衰老和骨性关节炎疾病的基因治疗提供了新的思路，且扩大了临床基因治疗的可选择范围。