Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, Calgary, AB T2N 4N1, Canada.
Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
Stem Cell Reports. 2023 Dec 12;18(12):2328-2343. doi: 10.1016/j.stemcr.2023.10.009. Epub 2023 Nov 9.
Sus scrofa domesticus (pig) has served as a superb large mammalian model for biomedical studies because of its comparable physiology and organ size to humans. The derivation of transgene-free porcine induced pluripotent stem cells (PiPSCs) will, therefore, benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. PiPSCs can be differentiated into derivatives representing the primary germ layers in vitro and can form teratomas in immunocompromised mice. Furthermore, the transgene-free PiPSCs preserve intrinsic species-specific developmental timing in culture, known as developmental allochrony. This is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ∼3.7 h, a timescale recapitulating in vivo porcine somitogenesis. We conclude that the transgene-free PiPSCs can serve as a powerful tool for modeling development and disease and developing transplantation strategies. We also anticipate that they will provide insights into conserved and unique features on the regulations of mammalian pluripotency and developmental timing mechanisms.
家猪（猪）因其与人类相当的生理学和器官大小，一直是生物医学研究的优秀大型哺乳动物模型。因此，无转基因猪诱导多能干细胞（PiPSCs）的衍生将有益于再生生物学及其医学应用中猪特异性模型的发展。过去，由于缺乏对稳定体外猪多能状态的信号环境的了解，这一努力受到了阻碍。在这里，我们报告说，通过使用针对该物种优化的方案，无转基因 PiPSCs 可以通过附加体载体和 microRNA-302/367 从猪成纤维细胞中高效衍生。PiPSCs 可以在体外分化为代表初级胚层的衍生物，并可以在免疫缺陷小鼠中形成畸胎瘤。此外，无转基因 PiPSCs 在培养中保留了内在的物种特异性发育时间，称为发育异时性。这是通过建立一个猪体外分段时钟模型来证明的，该模型首次显示出约 3.7 h 的特定周期性，这一时间尺度再现了体内猪体节发生。我们得出结论，无转基因 PiPSCs 可以作为建模发育和疾病以及开发移植策略的强大工具。我们还预计，它们将为哺乳动物多能性和发育时间机制的调控提供关于保守和独特特征的见解。
