Abstract：
Cellular reprogramming is a process by which adult differentiated cells lose their identity and are converted into pluripotent stem cells, known as induced pluripotent stem (iPS) cells. This process can be achieved in vitro and in vivo and is relevant for many fields including regenerative medicine and cancer. Cellular reprogramming is commonly induced by the ectopic expression of a transcription factor cocktail composed by Oct4, Sox2, Klf4, and Myc (abbreviated as OSKM), and its efficiency and kinetics are strongly dependent on the presence of Myc. Here, we describe a versatile method to study reprogramming in vivo based on the use of adeno-associated viral (AAV) vectors, which allows the targeting of specific organs and cell types. This method can be used to test Myc mutations or genes that may replace Myc, or be combined with different Myc regulators. In vivo reprogramming can be scored by the presence of teratomas and the isolation of in vivo iPS, thereby providing a simple surrogate for the function of Myc in dedifferentiation and stemness. Our protocol can be divided into five steps: (1) intravenous inoculation of AAV vectors; (2) monitoring the animals until the appearance of teratomas; (3) analysis of teratomas; (4) histopathological analysis of mouse organs; and (5) isolation of in vivo-generated iPS cells from teratomas, blood, and bone marrow. The information obtained by this in vivo testing platform may provide relevant information on the role of Myc in tissue regeneration, stemness, and cancer.
摘要：
细胞重编程是成年分化细胞失去其身份并转化为多能干细胞的过程，被称为诱导多能干细胞（iPS）。该过程可以在体外和体内实现，并且与包括再生医学和癌症在内的许多领域有关。细胞重编程通常由Oct4，Sox2，Klf4和Myc（缩写为OSKM）组成的转录因子混合物的异位表达诱导，其效率和动力学强烈依赖于Myc的存在。这里,我们描述了一种基于使用腺相关病毒（AAV）载体的体内研究重编程的通用方法，它允许靶向特定的器官和细胞类型。此方法可用于测试Myc突变或可能替代Myc的基因，或与不同的Myc调节器结合使用。体内重编程可以通过畸胎瘤的存在和体内iPS的分离来评分，从而为Myc在去分化和干性中的功能提供了简单的替代。我们的方案可分为五个步骤：（1）静脉内接种AAV载体；（2）监测动物直至畸胎瘤出现；（3）畸胎瘤分析；（4）小鼠器官的组织病理学分析；（5）从畸胎瘤中分离体内产生的iPS细胞，血,还有骨髓.该体内测试平台获得的信息可能提供有关Myc在组织再生中的作用的相关信息，stemness,和癌症。
