Isolation and characterization of normal mandibular periosteal stem cells from human and macaca mulatta and cross-species single-cell analysis
10.3760/cma.j.cn112144-20240205-00066
- VernacularTitle: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
- Author:
Zishuo WANG
1
;
Yangyang LI
;
Haitao WANG
;
Duohong ZOU
;
Zhiyuan ZHANG
Author Information
1. 上海交通大学医学院附属第九人民医院口腔外科 上海交通大学口腔医学院 国家口腔医学中心 国家口腔疾病临床医学研究中心 上海市口腔医学重点实验室 上海市口腔医学研究所 中国医学科学院口腔颌面再生医学创新单元，上海 200011
- Keywords:
Primate;
Mandible;
Periosteal stem cells;
Osteogenic differentiation;
Intramembranous osteogenesis;
Single-cell sequencing;
Cross-species analysis
- From:
Chinese Journal of Stomatology
2024;59(7):696-705
- CountryChina
- Language:Chinese
-
Abstract:
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.
