Yamazaki Atsushi, Shiozawa Seiji, Koushige Yuko, Kondo Hirotaka, Shibuya Hisashi, Edamura Kazuya
Laboratory of Veterinary Surgery, Department of Veterinary Medicine, College of Bioresource and Sciences, Nihon University, Fujisawa, Kanagawa, Japan.
Institute for Disease Modeling, Kurume University School of Medicine, Kurume, Fukuoka, Japan.
PLoS One. 2025 Jun 18;20(6):e0315161. doi: 10.1371/journal.pone.0315161. eCollection 2025.
In our previous study, canine induced pluripotent stem cells (iPSCs) were successfully generated from skin-derived fibroblasts, without the use of viral vectors. However, for clinical application of canine iPSCs in veterinary regenerative medicine, iPSCs generated from less invasive cell sources would be desirable. Therefore, the purpose of this study was to generate iPSCs from canine umbilical cords discarded at fetal birth. Canine umbilical cords were cut into small pieces and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Episomal vectors carrying 10 reprogramming gene sets were introduced into fibroblasts obtained from the umbilical cord using electroporation. When putative iPSCs colonies emerged, constitutive cell characterization was performed to evaluate cell morphology, proliferative potential, alkaline phosphatase staining, expression of stem cell markers, and the ability to differentiate into a trilineage following embryoid body and teratoma formation. Multiple putative iPSC colonies formed when reprogramming gene sets were introduced into fibroblasts obtained from the canine umbilical cord. The resulting colony cells stained positive for alkaline phosphatase, and showed expression of OCT4, SOX2, NANOG, SSEA1, and SSEA3 on fluorescent immunostaining for stem cell markers. Furthermore, the mRNA expression of canine endogenous OCT4, SOX2, and NANOG significantly increased, confirming the multi-differentiation potential of cells after embryoid and teratoma formation. In this study, iPSCs were successfully generated from canine umbilical cord without the use of a viral vector. Furthermore, canine umbilical cord-derived iPSCs were successfully cultured in a feeder-free manner. This study contributes to the development of veterinary regenerative medicine by using canine iPSCs.
在我们之前的研究中,成功地从皮肤来源的成纤维细胞中生成了犬诱导多能干细胞(iPSCs),且未使用病毒载体。然而,对于犬iPSCs在兽医再生医学中的临床应用而言,从侵入性较小的细胞来源生成iPSCs将是理想的。因此,本研究的目的是从胎儿出生时丢弃的犬脐带中生成iPSCs。将犬脐带切成小块,在补充有10%胎牛血清的杜氏改良 Eagle 培养基中培养。使用电穿孔法将携带10个重编程基因集的游离型载体导入从脐带获得的成纤维细胞中。当假定的iPSCs集落出现时,进行组成型细胞表征,以评估细胞形态、增殖潜力、碱性磷酸酶染色、干细胞标志物的表达以及在形成胚状体和畸胎瘤后分化为三系的能力。当将重编程基因集导入从犬脐带获得的成纤维细胞时,形成了多个假定的iPSC集落。所得集落细胞碱性磷酸酶染色呈阳性,并且在干细胞标志物的荧光免疫染色中显示出OCT4、SOX2、NANOG、SSEA1和SSEA3的表达。此外,犬内源性OCT4、SOX2和NANOG的mRNA表达显著增加,证实了胚状体和畸胎瘤形成后细胞的多分化潜力。在本研究中,未使用病毒载体成功地从犬脐带中生成了iPSCs。此外,犬脐带来源的iPSCs成功地以无饲养层方式培养。本研究通过使用犬iPSCs为兽医再生医学的发展做出了贡献。