Dedifferentiation induced by newt extract

蝾螈是一种两栖动物，可以再生附属物、眼睛结构和许多重要器官。在再生过程中，损伤部位的组织从分化状态恢复到胚胎样状态。这种去分化过程产生增殖祖细胞群，这些祖细胞可以再分化成再生结构的多个组织。更好地理解去分化过程将对我们理解蝾螈和高等脊椎动物的再生潜力产生巨大的影响。蝾螈和哺乳动物的去分化能力已经在体外使用肌肉来源的细胞系进行了广泛的研究。对蝾螈A1肌细胞系的研究表明，成熟分化的A1细胞在胎牛血清（FBS）处理后可以重新进入细胞周期。哺乳动物分化的肌肉细胞不具备这种能力，仍然处于有丝分裂后。然而，小鼠C2C12肌管在接受再生蝾螈肢体提取物治疗后可以重新进入细胞周期，这表明哺乳动物细胞仍然具有去分化能力，但可能没有适当的触发因素来启动反应。我们最近的研究表明，原代小鼠肌管在提取物处理后不会重新进入细胞周期。由此可见，C2C12细胞的去分化能力低于蝾螈细胞，但高于原代肌细胞。本研究的目的是研究蝾螈和哺乳动物肌肉细胞的去分化过程，确定控制它们去分化能力的遗传差异，最终改善哺乳动物细胞的去分化过程。我们已经分离出另一种蝾螈肌肉细胞系（称为A2）。与A1细胞不同，A2肌管对FBS治疗没有反应，无法重新进入细胞周期。在Aim 1中，我们将描述蝾螈A1和A2细胞的去分化过程。通过比较A1和A2细胞的转录组，我们将确定哪些突变允许细胞周期重新进入。我们还将进一步表征去分化过程，并确定是否涉及细胞死亡（caspase激活）和p53激活。***在Aim 2中，我们将描述哺乳动物C2C12和原代肌细胞的去分化过程。由于C2C12细胞对提取物的反应是片段化和重新进入细胞周期，而原代肌细胞只是片段化，我们将确定两种细胞类型之间的差异，并评估细胞周期基因和凋亡基因是否参与细胞对提取物的反应。***最终，更好地了解蝾螈细胞和哺乳动物细胞去分化能力的差异可能有助于我们更好地了解蝾螈的再生能力，并提高哺乳动物细胞的再生能力。**

The newt, Notophthalmus viridescens, is an amphibian that can regenerate appendages, eye structures and many vital organs. During the regeneration process, tissues at the injury site revert from their differentiated form into an embryonic-like state. This dedifferentiation process generates a population of proliferating progenitor cells that can then redifferentiate into the multiple tissues of the regenerated structure. A better understanding of the dedifferentiation process would have tremendous implications for our understanding of regenerative potential in both newts and higher vertebrates. ***Dedifferentiation capabilities in both newts and mammals have been extensively studied in vitro using muscle-derived cell lines. Studies with the newt A1 muscle cell line have shown that mature differentiated A1 cells can re-enter the cell cycle following treatment with foetal bovine serum (FBS). Mammalian differentiated muscle cells do not possess this capability and remain post-mitotic. However, mouse C2C12 myotubes can re-enter the cell cycle following treatment with extract derived from a regenerating newt limb, suggesting that mammalian cells still possess dedifferentiation capabilities but may not have the appropriate triggers to initiate the response. We have recently shown that primary mouse myotubes do not re-enter the cell cycle following extract treatment. Thus it appears that C2C12 cells possess less dedifferentiation capability than newt cells but more than primary muscle cells. The aim of this proposal is to study the dedifferentiation process in newt and mammalian muscle cells, to determine the genetic differences that govern their abilities to dedifferentiate, and to ultimately improve the dedifferentiation process in mammalian cells. ***We have isolated another newt muscle cell line (called A2). Unlike A1 cells, A2 myotubes do not respond to FBS treatment by re-entering the cell cycle. In Aim 1, we will characterize the dedifferentiation process in newt A1 and A2 cells. By comparing the transcriptomes of A1 and A2 cells, we will determine which mutations allow cell cycle re-entry. We will also further characterize the dedifferentiation process and determine if cell death (caspase activation) and p53 activation are involved. ***In Aim 2, we will characterize the dedifferentiation process in mammalian C2C12 and primary muscle cells. Since C2C12 cells respond to extracts by fragmenting and re-entering the cell cycle, whereas primary muscle cells only fragment, we will determine the differences between the two cell types and assess whether cell cycle genes and apoptotic genes are involved in the responses of the cells to extract. ***Ultimately, a better understanding of the differences in dedifferentiation capability between newt cells and mammalian cells may help us to better understand the regenerative capability of newts and to improve regenerative ability in mammalian cells.**