Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit^® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.

中文翻译：

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犬外周血单核细胞有效重编程为诱导多能干细胞

转录因子Oct3/4、Klf4、Sox2和c-Myc 的强制共表达将体细胞重编程为多能干细胞 (PSC)。这种诱导的 PSC (iPSC) 可以生成成人身体的任何细胞类型或无限增殖而不会失去其潜力。因此，iPSCs 可以作为无限的细胞来源，用于开发各种疾病模型和动物和人类的再生疗法。虽然犬外周血单个核细胞 (PBMC) 很容易获得，但它们的 iPSC 重编程效率非常低。在这项研究中，我们确定了犬 PBMC 在几种条件下的重编程效率，涉及三种类型的培养基，辅以小分子化合物。我们发现使用 N2B27 培养基补充白血病抑制因子 (LIF)、碱性成纤维细胞生长因子 (bFGF)、l-抗坏血酸）。我们生成了五个 ciPSC 系，它们可以在补充了 LIF 的StemFit ^®培养基中维持。SeVdp(KOSM)302L 载体在四个 ciPSC 系中被适当地沉默。在所表征的两个品系中，两个品系的碱性磷酸酶活性均呈阳性并表达多能性标志物，包括Oct3/4、Sox2和Nanog转录物，以及八聚体结合转录因子 (OCT) 3/4 和 NANOG 蛋白，以及 SSEA-1 碳水化合物抗原。ciPSCs 可以形成胚状体并分化为三个胚层，如标记基因和蛋白质表达所示。此外，一个 ciPSC 系形成了畸胎瘤，包括来自每个胚层的几个组织。即使经过多次传代，我们的 ciPSC 系仍保持正常的核型。此外，我们的新重编程方法能够从多个供体 PBMC 生成 ciPSC。总之，我们开发了一种简单有效的策略，用于从 PBMC 生成无足迹 ciPSC。我们相信该策略可用于兽医领域的疾病建模和再生医学。