Reprogramming Cells to Enable Limb Regeneration

重新编程细胞以实现肢体再生

• 批准号: 8131057
• 负责人: Jonathan M. Slack
• 金额: $ 29.6万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8131057
• 关键词: Affect; Amputation; Animal Model; Antibiotic Resistance; Antibiotics; Biological Assay; Cell Culture Techniques; Cells; Characteristics; Chromatin; Competence; DNA; Development; Fibroblasts; Gene Targeting; Genes; Genetic Markers; Healed; Health; Hindlimb; In Vitro; Leg; Limb Bud; Limb structure; Methods; Natural regeneration; Pattern; Phenotype; Process; Public Health; Rana; Research; Staging; Subfamily lentivirinae; Tadpoles; Techniques; Testing; Time; Xenopus; Xenopus laevis; gain of function; healing; innovation; limb regeneration; promoter; regenerative; stem cell technology; tissue culture; transcription factor

DESCRIPTION (provided by applicant): The project will apply induced pluripotential stem cell (iPS) technology to understand the process of limb regeneration in a model organism. Tadpoles of the frog Xenopus laevis are normally able to regenerate their limbs following amputation at early developmental stages but not at later stages. This situation provides a potential gain-of-function assay for factors promoting limb regeneration. My central hypothesis is that regeneration of Xenopus froglet or late stage tadpole limbs will occur if the limb fibroblasts can be respecified to an early tadpole limb bud phenotype. This will be achieved by introducing specific transcription factors into froglet limb cells cultured in vitro. Fibroblasts will be cultured from limbs of froglets that carry an antibiotic resistance gene driven by a limb bud- specific promoter: prx1. Transcription factors that encode limb bud status will then be introduced using inducible lentivirus, in the presence of treatments or other genes that will open chromatin and enable the transcription factors to locate their target genes in the DNA. Cells that are respecified to the early limb bud state will activate the prx1 promoter and will then be isolated by antibiotic selection. The cells will be characterized by expression analysis for other limb bud genes. A new assay for regeneration-competence will be refined. This is done by injecting limb bud cells subcutaneously into froglet hindlimbs, allowing to heal, amputating through the graft region, and then assessing the degree of regeneration achieved. This method will be refined using regeneration-competent tadpole limb cells for the graft, and will then used to test the regenerative capacity of the cells modified in culture. The use of intrinsic genetic markers in the donor cells will enable us to detect whether the regenerate has arisen from donor cells, host cells, or a mixture of the two, and whether there are positional information type interactions between graft and host affecting the pattern of the regenerated limbs. This project is innovative in several ways. Firstly it is the first time that iPS technology has been applied to a problem of regeneration. Secondly, it applies tissue culture to a regeneration problem, which has seldom been done before. Thirdly it makes use of selective techniques to obtain the desired cells, again a technique that has not previously been used in regeneration research. The progressive loss of Xenopus tadpole limbs to regenerate is correlated with an inability to re-activate expression of early limb bud genes. This is also characteristic of mammalian limbs. Once developed, the proposed method is therefore expected also to be capable of stimulating regeneration in mammalian limbs. PUBLIC HEALTH RELEVANCE: Early tadpole legs will regenerate after they are cut off, later ones will not. This innovative proposal will use modern stem cell technology to test a hypothesis about why this is so. If the hypothesis is confirmed, the methods developed should assist in facilitating the regeneration of mammalian limbs.

描述（由申请人提供）：该项目将应用诱导多能干细胞（iPS）技术来了解模型生物体的肢体再生过程。非洲爪蟾的蝌蚪通常能够在发育早期截肢后再生四肢，但在发育后期则不能。这种情况为促进肢体再生的因素提供了潜在的功能获得测定。我的中心假设是，如果肢体成纤维细胞可以被重新指定为早期蝌蚪肢芽表型，则爪蟾幼蛙或晚期蝌蚪肢体将会再生。这将通过将特定的转录因子引入体外培养的蛙肢细胞中来实现。将从携带由肢芽特异性启动子 prx1 驱动的抗生素抗性基因的青蛙四肢中培养成纤维细胞。然后，在治疗或其他基因存在的情况下，使用诱导型慢病毒引入编码肢芽状态的转录因子，这些基因将打开染色质并使转录因子能够在 DNA 中定位其目标基因。重新指定为早期肢芽状态的细胞将激活 prx1 启动子，然后通过抗生素选择进行分离。这些细胞将通过其他肢芽基因的表达分析来表征。将完善一种新的再生能力测定方法。这是通过将肢芽细胞皮下注射到青蛙后肢中，使其愈合，通过移植区域截肢，然后评估所实现的再生程度来完成的。该方法将使用具有再生能力的蝌蚪肢体细胞进行移植，然后用于测试培养中修饰的细胞的再生能力。在供体细胞中使用内在遗传标记将使我们能够检测再生是否是由供体细胞、宿主细胞或两者的混合物产生的，以及移植物和宿主之间是否存在影响再生模式的位置信息类型相互作用。再生的肢体。该项目在多个方面具有创新性。首先，iPS技术首次应用于再生问题。其次，它将组织培养应用于再生问题，这在以前很少有人做过。第三，它利用选择性技术来获得所需的细胞，这也是一种以前从未用于再生研究的技术。非洲爪蟾蝌蚪肢体再生的逐渐丧失与无法重新激活早期肢芽基因的表达有关。这也是哺乳动物四肢的特征。一旦开发出来，所提出的方法预计也能够刺激哺乳动物四肢的再生。公众健康相关性：早期的蝌蚪腿被切断后会再生，后来的蝌蚪则不会。这项创新提案将利用现代干细胞技术来检验这一假设。如果这一假设得到证实，所开发的方法应有助于促进哺乳动物肢体的再生。