人与恒河猴下颌骨骨膜干细胞的分离、鉴定和跨物种单细胞分析

Isolation and characterization of normal mandibular periosteal stem cells from human and macaca mulatta and cross-species single-cell analysis

目的探究人与非人灵长类动物(恒河猴)的下颌骨骨膜中是否存在有别于传统间充质干细胞的干细胞群体及其在膜内成骨过程中的特性,并提供区别于其他解剖区域骨膜干细胞(PSCs)的下颌骨PSCs的稳定分离、培养、扩增的标准化流程。方法分别从上海交通大学医学院附属第九人民医院的18~24岁行第三磨牙拔除术3例患者翻瓣去骨过程中的骨块表面和3只6岁龄恒河猴下颌骨的前磨牙区颊侧获取骨膜,使用Illumina平台Novaseq 6000测序仪进行单细胞测序,并通过同源基因匹配进行跨物种单细胞转录组测序的结果比较。使用37℃和低温两种消化方式从原代组织中分离PSCs,经流式细胞术分析鉴定其表面标志物(CD200、CD31、CD45和CD90)并通过免疫荧光鉴定组织蛋白酶K(CTSK)与CD200的共定位情况。接着评估了体外扩增至第三代的不同物种PSCs的细胞增殖能力和三系分化能力。最后比较了PSCs和骨髓间充质干细胞(BMSCs)在成骨方面的特性异同。结果单细胞测序结果提示直系同源基因匹配降维后获得了18个聚类的细胞群,基于各细胞标志物数据库对各聚类进行细胞注释。低温消化方案可以稳定地从人、恒河猴下颌骨骨膜中分离出PSCs,细胞呈成纤维细胞状。细胞计数法结果显示人与恒河猴的PSCs增殖能力差异无统计学意义,流式细胞术分析鉴定结果显示,从骨膜分离的细胞表面抗原表达CD200^(+)、CD31^(-)、CD45^(-)和CD90^(-),免疫荧光提示CTSK与CD200共定位于此细胞中。茜素红染色、油红O染色和阿尔率蓝染色结果显示,人和恒河猴的PSCs均具备成骨、成脂、成软骨的三系分化能力。与BMSCs的成骨能力相比,PSCs增殖能力略优,分化过程中,PSCs在早期有良好的成骨表现。结论本研究成功稳定分离并鉴定出人和非人灵长类动物(恒河猴)的正常下颌骨PSCs,为探索下颌骨膜内成骨的机制、建立理想的非人灵长类动物模型以及下颌骨缺损修复新型策略提供了细胞学基础。

Objective To investigate the presence of a distinct stem cell populations different from mesenchymal stem cells in the mandibular periosteum of both human and non-human primates(macaca mulatta),to explore its properties during intramembranous osteogenesis and to establish standard protocols for the isolation,culturing and expanding of mandibular periosteal stem cells(PSC)distinguished from other PSCs in other anatomical regions.Methods Periosteum was harvested from the bone surface during flap bone removal in patients aged 18-24 years undergoing third molar extraction and from the buccal side of the mandibular premolar region of 6-year-old macaca mulatta respectively,and then subjected to single-cell sequencing using the Illumina platform Novaseq 6000 sequencer.Cross-species single-cell transcriptome sequencing results were compared using homologous gene matching.PSC were isolated from primary tissues using two digestion methods with body temperature and low temperature,and their surface markers(CD200,CD31,CD45 and CD90)were identified by cell flow cytometry.The ability of cell proliferation and three-lineage differentiation of PSC expanded to the third generation in vitro in different species were evaluated.Finally,the similarities and differences in osteogenic properties of PSC and bone marrow mesenchymal stem cells(BMSC)were compared.Results The single-cell sequencing results indicated that 18 clusters of cell populations were identified after homologous gene matching for dimensionality reduction,and manual cellular annotation was conducted for each cluster based on cell marker databases.The comparison of different digestion protocols proved that the low-temperature overnight digestion protocol can stably isolate PSC from the human and m.mulatta mandibular periosteum and the cells exhibited a fibroblast-like morphology.This research confirmed that PSC of human and m.mulatta had similar proliferation capabilities through the cell counting kit-8 assay.Flow cytometry analysis was then used to identify the cells isolated from the periosteum expressed CD200(+),CD31(-),CD45(-),CD90(-).Then,human and m.mulatta PSC were induced into osteogenesis,adipogenesis,and chondrogenesis to demonstrate their corresponding multi-lineage differentiation capabilities.Finally,comparison with BMSC further clarified the oesteogenesis characteristics of PSC.The above experiments proved that the cells isolated from the periosteum were peiosteal cells with characteristics of stem cells evidenced by their cell morphology,proliferation ability,surface markers,and differentiation ability,and that this group of PSC possessed characteristics different from traditional mesenchymal stem cells.Conclusions In this study,normal mandibular PSC from humans and m.mulatta were stably isolated and identified for the first time,providing a cellular foundation for investigating the mechanism of mandibular intramembranous osteogenesis,exploring ideal non-human primate models and establishing innovative strategies for clinically mandibular injury repair.