Gonçalves N J N, Bressan F F, Roballo K C S, Meirelles F V, Xavier P L P, Fukumasu H, Williams C, Breen M, Koh S, Sper R, Piedrahita J, Ambrósio C E
Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, FZEA/USP, São Paulo, Brazil.
Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, FZEA/USP, São Paulo, Brazil; Department of Veterinary Surgery, Sector Anatomy, Faculty of Veterinary Medicine and Animal Science, FMVZ/USP, São Paulo, Brazil.
Theriogenology. 2017 Apr 1;92:75-82. doi: 10.1016/j.theriogenology.2017.01.013. Epub 2017 Jan 8.
Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.
高桥和山中建立了第一种技术，即将与多能性相关的转录因子整合到体细胞基因组中，以使这些细胞能够重编程。这些转录因子的表达使分化的体细胞能够将其表型逆转到胚胎状态，从而产生诱导多能干细胞（iPSC）。通过慢病毒多顺反子人和小鼠载体（hOSKM/mOSKM）从犬胎儿成纤维细胞中产生iPSC，旨在在该动物模型中获得具有与胚胎干细胞（ESC）相似特征的多能干细胞。本研究中获得的细胞系不依赖于白血病抑制因子（LIF）或任何其他补充抑制剂，对酶处理（TrypLE Express酶）具有抗性，并且依赖于碱性成纤维细胞生长因子（bFGF）。从略有不同的方案中获得克隆系，最大重编程效率为0.001%。所有集落碱性磷酸酶、胚状体形成和自发分化均呈阳性，并高表达内源性八聚体结合转录因子4（OCT4）和性别决定区Y框蛋白2（SOX2）。犬iPSC在体内注射后120天形成肿瘤。通过荧光原位杂交（FISH）进行初步染色体评估，未发现染色体异常。据我们所知，本报告首次描述了通过慢病毒载体对犬体细胞进行重编程的能力，无需补充且对酶作用具有抗性，从而证明了这些细胞系的多能性。
