高通量筛选和单细胞水平解析肌腱干细胞表型维持的调控手段及机制

Tendon stem/progenitor cells (TSPC) are potential source of seed cells for tendon tissue engineering and regeneration, but phenotypic loss after in vitro expansion is really a great challenge, and the induction conditions for tenogenic maturation are not ideal at present. In our previous study based on tendon stem cell subpopulation (Science Advances 2016) and stepwise differentiation strategy (Stem cell Trans Med 2016), we have found that single growth factor and their combination can be used to stepwise induce TSPCs differentiation. Due to advantage of small molecule drugs, such as the easy to synthesize, save and quality control, we proposed the hypothesis that we can get stable culture conditions for the expansion and phenotypic maintenance of TSPCs in vitro, and efficient stepwise induction strategies towards teno-lineage specifically by small molecules combination. In the pilot study, we used the SCX-GFP reporter system by visualization to conduct a preliminary high-throughput screening of 1104 small molecules from FDA approved drug library and 122 small molecules from bioactive libraries, and obtained candidate molecules for ex vivo expansion and phenotypic maintenance of TSPCs. Thus, we will develop the stepwise strategy to induce TSPCs differentiation by small molecules, investigate the regulatory mechanisms of tenogenic differentiation by single cell analysis, and demonstrate that the efficacy of the small molecule cocktails approach in tendon injury repair in vivo. The research will establish the optimal culture system of TSPCs in vitro, further clarify the molecular mechanism of teno-lineage differentiation, thus obtain the ideal strategy for the construction of tissue-engineered tendon, as well as provide experimental basis and novel candidates for cell therapy and new drug selection in clinical.

肌腱干细胞是有潜力的肌腱组织工程与再生的种子细胞来源，但目前面临体外扩增培养时表型显著丢失的挑战。鉴于小分子的易合成易储存易量化诸多优点，基于我们前期肌腱干细胞亚群研究（Science Adv. 2016）及利用生长因子实现的分步诱导策略（Stem cell Trans Med 2016），本课题拟通过小分子组合获得肌腱干细胞体外扩增和表型维持的稳定培养条件及高效向腱系特异分化的阶段性诱导方案。在预实验中，我们运用SCX-GFP报告系统对FDA库上千种小分子进行了初步高通量可视化的筛选，得到了肌腱干细胞体外扩增和表型维持的候选小分子。本课题将利用已建立的肌腱分化与再生平台，探索小分子组合分阶段诱导肌腱干细胞向肌腱系分化的策略，在单细胞水平解析腱系分化的调控机制，并验证小分子组合在体内肌腱损伤修复应用中效果。预期本研究将提升肌腱干细胞培养和肌腱修复质量，为临床治疗或新药选择提供依据和新思路。

肌腱干细胞是有潜力的肌腱组织工程与再生的种子细胞来源，但是体外扩增培养时表型显著丢失，尚缺乏适合肌腱干细胞在体外扩增过程中维持表型和定向分化的培养体系。基于小分子药物易于合成、保存和质量控制的优势，以及在诱导细胞分化、转分化和调控细胞命运方面应用的巨大潜力，本课题建立了分阶段诱导肌腱干细胞分化的小分子策略，并结合3D打印技术实现了在体内肌腱损伤修复的应用。本课题的关键研究发现和创新点如下：1）利用高内涵细胞成像分析系统，根据肌腱特异转录因子Scx-GFP报告系统和DAPI染色指标对FDA药物库和生物活性库中的1226种小分子进行高通量筛选，通过多轮筛选验证，获得促进肌腱干细胞体外扩增的6种小分子和促进肌腱干细胞表型维持的9种小分子。2）通过高通量单细胞 qPCR 对单个小分子、以及小分子组合应用于肌腱干细胞培养的增殖相关基因、表型相关基因和骨系、脂系、软骨系基因的表达水平进行了全面评估。3）获得促进肌腱干细胞体外扩增的最佳小分子，以及表型维持并启动腱系分化的最佳小分子组合，确立了肌腱干细胞分阶段腱系诱导策略，第一步利用小分子促进细胞增殖，第二步利用小分子组合进行腱系分化诱导，实现分阶段诱导肌腱干细胞定向肌腱系分化。4）将小分子分阶段腱系诱导的肌腱干细胞与仿生凝胶一同借助3D打印技术构建组织工程肌腱，在体内证明了小分子鸡尾酒在肌腱修复应用中的可行性和有效性。在本项目资助下，研究成果以论文的形式发表，在本领域的国际核心刊物 SCI 上发表 13篇文章，其中 9 篇 IF>10。获得国家发明专利授权1项。本研究创建的肌腱干细胞小分子分阶段腱系诱导策略，有利于进一步明晰腱系分化的分子调控机制，为临床干细胞治疗或新药选择提供实验基础和思路，推动肌腱干细胞的转化应用，具有广阔的临床应用前景。