Abstract
A method for the in vitro isolation, purification, identification, and induced differentiation of satellite cells from adult tree shrew skeletal muscle was established. The mixed enzyme digestion method and differential adhesion method were used to obtain skeletal muscle satellite cells, which were identified and induced to differentiate to verify their pluripotency. The use of a mixture of collagenase II, hyaluronidase IV, and DNase I is an efficient method for isolating adult tree shrew skeletal muscle satellite cells. The P3 generation of cells had good morphology, rapid proliferation, high viability, and an “S”-shaped growth curve. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining indicated that marker genes or proteins were expressed in skeletal muscle satellite cells. After myogenic differentiation was induced, multiple-nucleated myotubes were observed, and the MyHC protein was expressed. The expression of myogenic marker genes changed with the differentiation process. After the induction of adipogenic differentiation, orange-red lipid droplets were observed, and the expression of adipogenic marker genes increased gradually with the differentiation process. In summary, satellite cells from adult tree shrew skeletal muscle were successfully isolated using a mixed enzyme digestion method, and their potential for differentiation into myogenic and adipogenic cells was confirmed, laying a foundation for further in vitro study of tree shrew muscle damage.
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Acknowledgements
We thank the Kunming Zoology Institute, Chinese Academy of Science, for providing experimental animal sources and the Experimental Animal Center of Guangxi Medical University for providing technical support for animal feeding. We thank Jiawei Wang, Xunwei Feng, Qiuni Luo, and Pengcheng Zhao for technical support during the initial isolation process. We thank Yingying Zhu from Beijing University Chinese Medicine for her support in experimental technical issues. We thank members of our research team for their technical assistance and participation in this work.
Funding
This work was supported by grants from the Guangxi Natural Science Fund (Grant No. 2020GXNSFAA297235), the Central Guidance on Local Science and Technology Development Fund of Guangxi Province (Grant No. GuikeZY23055031), the Natural Science Foundation of China (Grant No. 82160217), the Guangxi Clinic Medicine Research Center of Nasopharyngeal Carcinoma (Grant No. 2020AC03007), the Guangxi Scholarship Fund of Guangxi Education Department of China and Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumors (Guangxi Medical University), and the Ministry of Education (No. GKE-ZZ202231, No. GKE-ZZ202302).
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Shenghui Ke and Guangyao He conceptualized the study. Shenghui Ke, Liying Luo, Wanzhao Qin, Huayu Liu, Jingchong Nie, Beijiang Liang, and Hongjie Ma conducted the experiments. Shenghui Ke, Mao Xie, Jingyu Li, Guojian Li, and Zhijie Niu processed the samples. Shenghui Ke and Yiwei Feng performed the data analysis. Shenghui Ke and Yiwei Feng drafted, revised, and edited the manuscript. Anzhou Tang, Wei Xia, and Guangyao He provided executive supervision, project management, and funding acquisition. All the authors have read the manuscript and approved the final submitted manuscript for publication.
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Ke, S., Feng, Y., Luo, L. et al. Isolation, identification, and induced differentiation of satellite cells from skeletal muscle of adult tree shrews. In Vitro Cell.Dev.Biol.-Animal 60, 36–53 (2024). https://doi.org/10.1007/s11626-023-00836-5
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DOI: https://doi.org/10.1007/s11626-023-00836-5