Development of a biologically immortalized equine stem cell line.

IF 1.1 4区 农林科学 Q2 Veterinary Canadian journal of veterinary research = Revue canadienne de recherche veterinaire Pub Date : 2021-10-01
Rodolfo Nino-Fong, Blanca P Esparza Gonzalez, Juan Carlos Rodriguez-Lecompte, William Montelpare, Laurie McDuffee
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Abstract

Bone repair in horses implies invasive surgeries and increased cost. Research on musculoskeletal disorders therapy in horses includes cell-based therapy with mesenchymal stromal cells (MSCs). Mesenchymal stromal cells can be obtained from bone marrow (BMMSCs). Unfortunately, BMMSCs have limited cell replication in vitro. The objective of this study was to develop a biologically immortalized equine stem cell line derived from bone marrow, with unlimited in-vitro proliferation and the ability to differentiate into bone cells. Equine BMMSCs were transfected and immortalized with human telomerase reverse transcriptase (hTERT) gene. Cell passages from equine immortal BMMSCs were characterized by the presence of stemness CD markers and expression of multi-potent differentiation genes (OCT-4, SOX2, and NANOG). Equine immortal BMMSCs were incubated in osteogenic medium and bone cell differentiation was determined by alkaline phosphatase and von Kossa staining, and osteogenic gene expression (osteocalcin, Runx2, and osterix). Telomerase activity was determined by telomeric repeat amplification technique. Results showed that equine immortal BMMSCs were able to replicate in-vitro up to passage 50 and maintain stem cell characteristics by the presence of CD90 and expression of multi-potent genes. Equine immortal BMMSCs were able to differentiate into bone cells, which was confirmed by the positive osteogenic staining and gene expression. Equine BMMSCs were successfully immortalized and maintained characteristics of stem cells and readily differentiated into osteogenic cells. Extending the life span of equine BMMSCs by transfection of the hTERT gene will revolutionize the clinical use of MSCs by making them available to orthopedic surgeons "off the shelf."

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生物学上永生化的马干细胞系的发展。
马骨修复意味着侵入性手术和成本增加。马肌肉骨骼疾病的治疗研究包括间充质基质细胞(MSCs)的细胞治疗。间充质间质细胞可以从骨髓中获得(BMMSCs)。不幸的是,BMMSCs在体外的细胞复制有限。本研究的目的是开发一种从骨髓中提取的生物永生的马干细胞系,具有无限的体外增殖和分化成骨细胞的能力。用人端粒酶逆转录酶(hTERT)基因转染马骨髓间充质干细胞并使其永生化。来自马永生BMMSCs的细胞传代通过存在干性CD标记和多能分化基因(OCT-4, SOX2和NANOG)的表达来表征。在成骨培养基中培养马永生bmmmscs,通过碱性磷酸酶和von Kossa染色检测骨细胞分化,以及成骨基因(骨钙素、Runx2和osterix)的表达。端粒酶活性测定采用端粒重复扩增技术。结果表明,通过CD90的存在和多效基因的表达,马永生bmmmscs能够在体外复制至传代50,并保持干细胞特性。马永生骨髓间充质干细胞能够分化为骨细胞,成骨染色和基因表达阳性证实了这一点。马骨髓间充质干细胞成功永生化,保持了干细胞的特性,并容易分化为成骨细胞。通过转染hTERT基因来延长马骨髓间充质干细胞的寿命,将彻底改变骨髓间充质干细胞的临床应用,使其成为骨科医生的“现成”产品。
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来源期刊
CiteScore
1.80
自引率
0.00%
发文量
58
审稿时长
>24 weeks
期刊介绍: The Canadian Journal of Veterinary Research, published by the Canadian Veterinary Medical Association, is Canada''s only veterinary research publication. This quarterly peer-reviewed online-only journal has earned a wide international readership through the publishing of high quality scientific papers in the field of veterinary medicine. The Journal publishes the results of original research in veterinary and comparative medicine.
期刊最新文献
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