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Journal of Integrative Agriculture
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Establishment and analysis of immortalized chicken skeletal muscle satellite cell lines
Yanxing Wang1, Haigang Ji1, Liyang He1, Yufang Niu1, Yushi Zhang1, Yang Liu1, Yadong Tian1, 2, Xiaojun Liu1, 2, Hong Li1, 2, Xiangtao Kang1, 2, Yanling Gao3#, Zhuanjian Li1, 2#

1 College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China

2 Key Laboratory of Livestock and Poultry Resources (Poultry) Evaluation and Utilization, Ministry of Ag-riculture and Rural Affairs, Zhengzhou 450046, China

3 Henan Vocational College of Agriculture, Zhengzhou 451450, China

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摘要  骨骼肌卫星细胞作为肌源性干细胞,具有增殖、分化和自我更新的能力。然而,原代骨骼肌卫星细胞在体外的增殖能力有限、分离步骤繁琐等因素限制了其在骨骼肌发育研究中的应用。细胞永生化能够使原代细胞越过细胞衰老期获得持续增殖的能力,永生化鸡骨骼肌卫星细胞系(ICMS)的建立将为鸡骨骼肌相关基因的功能研究提供可靠的细胞模型。在本研究中,我们利用慢病毒包装鸡端粒酶逆转录酶(chTERT)感染原代细胞,筛选出慢病毒的最适MOI和杀稻瘟菌素的最适浓度;随后,对感染的细胞进行药物筛选,获得阳性细胞后连续传代培养,通过端粒酶活性的重建以实现鸡骨骼肌卫星细胞的永生化。研究结果显示,传代至第10代、第15代、第22代的永生化鸡骨骼肌卫星(P10 ICMSP15 ICMSP22 ICMS)细胞形态与鸡原代骨骼肌卫星细胞(CPMSCs)保持一致,均呈纤维样状;与CPMSCs相比,P10 ICMS生长曲线CPMSCs相似;P10 ICMS具有正常的细胞增殖周期,且处于S期的细胞数量极显著高于CPMSCsP10 ICMS复苏后的细胞活性较好;当血清浓度增加到15%时,P10P22 ICMS可以增殖,说明ICMS具有一定的血清依赖性;软琼脂试验表明,培养14 d后,P10 ICMS依旧为单独的细胞,没有细胞克隆团的形成,可见ICMS没有发生恶性转化;经2%马血清的诱导,P22 ICMS的增殖能力较强、分化能力较弱。综上所述,本研究首次建立了鸡永生化骨骼肌卫星细胞系,其保持了与原代细胞相似的生物学特性,无致瘤性转化。鸡永生化骨骼肌卫星细胞系的建立为chTERT构建家禽细胞的永生化提供了参考,为家禽骨骼肌发育的研究提供了理想的体外细胞模型

Abstract  Skeletal muscle satellite cells are stem cells that are known for their multipotency and ability to proliferate in vitro. However, primary skeletal muscle satellite cells have limited proliferative capacity in vitro, which hinders their study in poultry skeletal muscle. The emergence of immortalization techniques for cells has provided a useful tool to overcome this limitation and explore the functions of skeletal muscle satellite cells. In this study, we achieved the immortalization of chicken skeletal muscle satellite cells by transducing primary cells with TERT (Telomerase reverse transcriptase) amplified from chicken (chTERT) using a lentiviral vector through reconstitution of telomerase activity. The cells successfully bypassed replicative senescence but did not achieve true immortalization. Preliminary functional characterization of the established cell line revealed that the proliferative characteristics and cell cycle profile of the immortalized chicken skeletal muscle satellite cell lines (ICMS) were similar to those of chicken primary muscle satellite cells (CPMSCs). Serum dependency analysis and soft agar assays indicated that ICMS did not undergo malignant transformation. Induced differentiation results demonstrated that ICMS retained their capacity for differentiation. The cell lines established in this study provide an important basis for the establishment of immortalized poultry cell lines and a cell model for the study of poultry skeletal muscle-related functional genes.
Keywords:  chicken       skeletal muscle satellite cell              immortalization              chTERT              proliferation              differentiation  
Online: 06 March 2024  
About author:  Yanxing Wang, E-mail: 18700258066@163.com; #Correspondence Zhuanjian Li, Tel: +86-18037465363, E-mail: lizhuanjian@163.com; Yanling Gao, Tel: +86-13838069436, E-mail: hnivdc@163.com

Cite this article: 

Yanxing Wang, Haigang Ji, Liyang He, Yufang Niu, Yushi Zhang, Yang Liu, Yadong Tian, Xiaojun Liu, Hong Li Xiangtao Kang, Yanling Gao, Zhuanjian Li. 2024. Establishment and analysis of immortalized chicken skeletal muscle satellite cell lines. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.01.034

Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A, Partridge T, Buckingham M. 2005. Direct isolation of satellite cells for skeletal muscle regeneration. Science, 309, 2064-2067.

Wang Y X, Rudnicki M A. 2011. Satellite cells, the engines of muscle repair. Nature Reviews. Molecular Cell Biology, 13, 127-133.

Sousa-Victor P, García-Prat L, Muñoz-Cánoves P. 2022. Control of satellite cell function in muscle regeneration and its disruption in ageing. Nature Reviews. Molecular Cell Biology, 23, 204-226.

Crandell R A, Hierholzer J C, Krebs J W, Jr., Drysdale S S. 1978. Contamination of primary embryonic bovine kidney cell cultures with parainfluenza type 2 simian virus 5 and infectious bovine rhinotracheitis virus. Journal of Clinical Microbiology, 7, 214-218.

Ramboer E, De Craene B, De Kock J, Vanhaecke T, Berx G, Rogiers V, Vinken M. 2014. Strategies for immortalization of primary hepatocytes. Journal of Hepatology, 61, 925-943.

Xing X I E, Fei H a O, Hai-Yan W, Mao-Da P, Yuan G a N, Bei-Bei L I U, Lei Z, Yan-Na W E I, Rong C, Zhen-Zhen Z, Wen-Bin B a O, Yun B a I, Guo-Qing S, Qi-Yan X, Zhi-Xin F. 2022. Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection. Journal of Integrative Agriculture, 21, 504-520.

Liu T M, Ng W M, Tan H S, Vinitha D, Yang Z, Fan J B, Zou Y, Hui J H, Lee E H, Lim B. 2013. Molecular basis of immortalization of human mesenchymal stem cells by combination of p53 knockdown and human telomerase reverse transcriptase overexpression. Stem Cells and Development, 22, 268-278.

Guo D, Zhang L, Wang X, Zheng J, Lin S. 2022. Establishment methods and research progress of livestock and poultry immortalized cell lines: A review. Frontiers in Veterinary Science, 9, 956357.

Takenouchi T, Masujin K, Miyazaki A, Suzuki S, Takagi M, Kokuho T, Uenishi H. 2022. Isolation and immortalization of macrophages derived from fetal porcine small intestine and their susceptibility to porcine viral pathogen infections. Frontiers in Veterinary Science, 9, 919077.

Hansen K D, Sabunciyan S, Langmead B, Nagy N, Curley R, Klein G, Klein E, Salamon D, Feinberg A P. 2014. Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization. Genome Research, 24, 177-184.

Schütze D M, Kooter J M, Wilting S M, Meijer C J L M, Quint W, Snijders P J F, Steenbergen R D M. 2015. Longitudinal assessment of DNA methylation changes during HPVE6E7-induced immortalization of primary keratinocytes. Epigenetics, 10, 73-81.

Püttmann S, Senner V, Braune S, Hillmann B, Exeler R, Rickert C H, Paulus W. 2005. Establishment of a benign meningioma cell line by hTERT-mediated immortalization. Laboratory Investigation; A Journal of Technical Methods and Pathology, 85, 1163-1171.

Zhang Z, Han Z, Guo Y, Liu X, Gao Y, Zhang Y. 2021. Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells. International Journal of Molecular Sciences, 22, 12540.

Gadalla S M, Savage S A. 2011. Telomere biology in hematopoiesis and stem cell transplantation. Blood Reviews, 25, 261-269.

Hathcock K S, Jeffrey Chiang Y, Hodes R J. 2005. In vivo regulation of telomerase activity and telomere length. Immunological Reviews, 205, 104-113.

Nicholls C, Li H, Wang J-Q, Liu J-P. 2011. Molecular regulation of telomerase activity in aging. Protein & Cell, 2, 726-738.

Petkov S, Kahland T, Shomroni O, Lingner T, Salinas G, Fuchs S, Debowski K, Behr R. 2018. Immortalization of common marmoset monkey fibroblasts by piggyBac transposition of hTERT. PloS One, 13, e0204580.

Sun S, Zhao K, Lu H, Liu X, Li Y, Li Q, Song D, Lan Y, He W, Gao F, Li Z, Guan J. 2022. Establishment of a sheep immortalization cell line for generating and amplifying Orf virus recombinants. Frontiers in Veterinary Science, 9, 1062908.

Wang W, Zhang T, Wu C, Wang S, Wang Y, Li H, Wang N. 2017. Immortalization of chicken preadipocytes by retroviral transduction of chicken TERT and TR. PloS One, 12, e0177348.

Yin H, Zhao J, He H, Chen Y, Wang Y, Li D, Zhu Q. 2020. Gga-miR-3525 Targets PDLIM3 through the MAPK Signaling Pathway to Regulate the Proliferation and Differentiation of Skeletal Muscle Satellite Cells. International Journal of Molecular Sciences, 21, 5573.

Zhang Z, Lin S, Luo W, Ren T, Huang X, Li W, Zhang X. 2022. Sox6 Differentially Regulates Inherited Myogenic Abilities and Muscle Fiber Types of Satellite Cells Derived from Fast- and Slow-Type Muscles. International Journal of Molecular Sciences, 23, 11327.

Agarwal M, Sharma A, Kumar P, Kumar A, Bharadwaj A, Saini M, Kardon G, Mathew S J. 2020. Myosin heavy chain-embryonic regulates skeletal muscle differentiation during mammalian development. Development, 147, dev184507.

Lei Q-J, Pan Q, Ma J-H, Zhou Z, Li G-P, Chen S-L, Hua J-L. 2017. Establishment and characterization of immortalized bovine male germline stem cell line. Journal of Integrative Agriculture, 16, 2547-2557.

Tiscornia G, Singer O, Verma I M. 2017. Production and purification of lentiviral vectors. Nature Protocols, 1, 241-245.

Sosa P, Alcalde-Estévez E, Asenjo-Bueno A, Plaza P, Carrillo-López N, Olmos G, López-Ongil S, Ruiz-Torres M P. 2021. Aging-related hyperphosphatemia impairs myogenic differentiation and enhances fibrosis in skeletal muscle. Journal of Cachexia, Sarcopenia and Muscle, 12, 1266-1279.

Kennedy J M, Sweeney L J, Gao L Z. 1989. Ventricular myosin expression in developing and regenerating muscle, cultured myotubes, and nascent myofibers of overloaded muscle in the chicken. Medicine and Science in Sports and Exercise, 21, S187-197.

Choi K-H, Yoon J W, Kim M, Lee H J, Jeong J, Ryu M, Jo C, Lee C-K. 2021. Muscle stem cell isolation and in vitro culture for meat production: A methodological review. Comprehensive Reviews in Food Science and Food Safety, 20, 429-457.

Agrez M V, Kovach J S, Lieber M M. 1982. Cell aggregates in the soft agar "human tumour stem-cell assay". British Journal of Cancer, 46, 880-887.

San Francisco I F, Dewolf W C, Peehl D M, Olumi A F. 2004. Expression of transforming growth factor-beta 1 and growth in soft agar differentiate prostate carcinoma-associated fibroblasts from normal prostate fibroblasts. International Journal of Cancer, 112, 213-218.

Smith S L, Shioda T. 2009. Advantages of COS-1 monkey kidney epithelial cells as packaging host for small-volume production of high-quality recombinant lentiviruses. Journal of Virological Methods, 157, 47-54.

Numa F, Hirabayashi K, Tsunaga N, Kato H, O'rourke K, Shao H, Stechmann-Lebakken C, Varani J, Rapraeger A, Dixit V M. 1995. Elevated levels of syndecan-1 expression confer potent serum-dependent growth in human 293T cells. Cancer Research, 55, 4676-4680.

Zander L, Bemark M. 2008. Identification of genes deregulated during serum-free medium adaptation of a Burkitt's lymphoma cell line. Cell Proliferation, 41, 136-155.

Guo L, Wang Z, Li J, Li J, Cui L, Dong J, Meng X, Qian C, Wang H. 2022. Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells. BMC Veterinary Research, 18, 181.

Kim E, Wu F, Wu X, Choo H J. 2020. Generation of craniofacial myogenic progenitor cells from human induced pluripotent stem cells for skeletal muscle tissue regeneration. Biomaterials, 248, 119995.

Shi R, Bai Y, Li S, Wei H, Zhang X, Li L, Tian X C, Jiang Q, Wang C, Qin L, Cai J, Zhang S. 2015. Characteristics of spermatogonial stem cells derived from neonatal porcine testis. Andrologia, 47, 765-778.

Wakitani S, Saito T, Caplan A I. 1955. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle & Nerve, 18, 1417-1426.

Svoradova A, Zmrhal V, Venusova E, Slama P. 2021. Chicken Mesenchymal Stem Cells and Their Applications: A Mini Review. Animals, 11, 1883.

Luan N T, Sharma N, Kim S-W, Ha P T H, Hong Y-H, Oh S-J, Jeong D-K. 2014. Characterization and cardiac differentiation of chicken spermatogonial stem cells. Animal Reproduction Science, 151, 244-255.

Dumont N A, Wang Y X, Rudnicki M A. 2015. Intrinsic and extrinsic mechanisms regulating satellite cell function. Development, 142, 1572-1581.

Ten Broek R W, Grefte S, Von Den Hoff J W. 2010. Regulatory factors and cell populations involved in skeletal muscle regeneration. Journal of Cellular Physiology, 224, 7-16.

Wagers A J, Conboy I M. 2005. Cellular and molecular signatures of muscle regeneration: current concepts and controversies in adult myogenesis. Cell, 122, 659-667.

Zammit P S. 2017. Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis. Seminars in Cell & Developmental Biology, 72, 19-32.

Hernández-Hernández J M, García-González E G, Brun C E, Rudnicki M A. 2017. The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration. Seminars in Cell & Developmental Biology, 72, 10-18.

Schiaffino S. 2018. Muscle fiber type diversity revealed by anti-myosin heavy chain antibodies. The FEBS Journal, 285, 3688-3694.

Zhu H, Wu Z, Ding X, Post M J, Guo R, Wang J, Wu J, Tang W, Ding S, Zhou G. 2022. Production of cultured meat from pig muscle stem cells. Biomaterials, 287, 121650.

Zammit P S, Golding J P, Nagata Y, Hudon V, Partridge T A, Beauchamp J R. 2004. Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? The Journal of Cell Biology, 166, 347-357.

Li W-Y, Ma H-X, Wang Y-X, Zhang Y-S, Liu Y, Han R-L, Li H, Cai H-F, Liu X-J, Kang X-T, Jiang R-R, Li Z-J. 2023. The VGLL2 gene participates in muscle development in Gushi chickens1. Journal of Integrative Agriculture.

Piette J, Huchet M, Duclert A, Fujisawa-Sehara A, Changeux J P. 1992. Localization of mRNAs coding for CMD1, myogenin and the alpha-subunit of the acetylcholine receptor during skeletal muscle development in the chicken. Mechanisms of Development, 37, 95-106.

Velleman S G, Coy C S, Abasht B. 2022. Effect of expression of PPARG, DNM2L, RRAD, and LINGO1 on broiler chicken breast muscle satellite cell function. Comparative Biochemistry and Physiology, 268, 111186.

 

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