Dedifferentiation induced by newt extract

蝾螈，Notophthalmus viridescens，是一种两栖动物，可以再生附肢，眼睛结构和许多重要器官。在再生过程中，损伤部位的组织从其分化形式恢复为胚胎样状态。该去分化过程产生增殖祖细胞群，其然后可以再分化成再生结构的多种组织。 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源性细胞系进行了广泛的研究。对蝾螈A1肌肉细胞系的研究表明，成熟分化的A1细胞在用胎牛血清（FBS）处理后可以重新进入细胞周期。 哺乳动物分化的肌细胞不具备这种能力，并保持有丝分裂后。 然而，小鼠C2C12肌管可以重新进入细胞周期后，从再生蝾螈肢体提取物的治疗，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器启动响应。我们最近发现，原代小鼠肌管在提取物处理后不会重新进入细胞周期。因此，C2C12细胞似乎具有比蝾螈细胞更低的去分化能力，但比原代肌细胞更高。该提案的目的是研究蝾螈和哺乳动物肌肉细胞的去分化过程，以确定控制其去分化能力的遗传差异，并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处理不响应，不重新进入细胞周期。 在目的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（半胱天冬酶激活）和p53激活。* 在目标2中，我们将表征哺乳动物C2C12和原代肌细胞中的去分化过程。 由于C2C12细胞通过片段化和重新进入细胞周期来响应提取物，而原代肌细胞仅片段化，因此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响应。* 最终，更好地了解蝾螈细胞和哺乳动物细胞之间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.**