Reprogramming Cells to Enable Limb Regeneration

重新编程细胞以实现肢体再生

• 批准号: 7912844
• 负责人: Jonathan M. Slack
• 金额: $ 29.9万
• 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/7912844
• 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; 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; public health relevance; 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）：该项目将采用引起的多能干细胞（IPS）技术来了解模型生物体中肢体再生的过程。青蛙爪蟾laevis的t通常能够在早期发育阶段进行截肢后的肢体再生，但在较晚的阶段不可再生其四肢。这种情况为促进肢体再生的因素提供了潜在的功能收益测定。我的中心假设是，如果可以将肢体成纤维细胞重新定义为早期的t骨四肢芽表型，则爪蟾果冻或晚期t的再生将发生。这将通过将特定的转录因子引入体外培养的Froglet肢体细胞中来实现。成纤维细胞将从froglets的肢体中培养，这些蛙的四肢携带由肢体芽的启动子驱动的抗生素耐药性基因：PRX1。然后，将使用可诱导的慢病毒或其他将打开染色质的基因并使转录因子在DNA中定位其靶基因的情况下，使用诱导式慢病毒来引入编码肢体状态的转录因子。被解释到早期肢体状态的细胞将激活PRX1启动子，然后通过抗生素选择分离。细胞将以其他肢体芽基因的表达分析来表征。将完善用于再生能力的新测定法。这是通过将肢体芽细胞皮下注射到蛙的后肢中来完成的，从而可以愈合，通过移植区截肢，然后评估达到的再生程度。该方法将使用竞争性的the脚肢体细胞进行完善，然后用来测试在培养中修饰的细胞的再生能力。供体细胞中固有的遗传标志物的使用将使我们能够检测到再生是由供体细胞，宿主细胞或两者的混合物引起的，以及是否存在关节和宿主之间的位置信息类型相互作用，并影响了再生肢体的模式。该项目在几种方面具有创新性。首先，这是IPS技术首次应用于再生问题。其次，它将组织培养物应用于再生问题，以前很少进行。第三，它利用选择性技术来获取所需的细胞，这再次是一种以前在再生研究中尚未使用的技术。爪蟾t肢的逐渐损失与再生的逐渐丧失与无法重新激活早期肢体芽基因的表达相关。这也是哺乳动物四肢的特征。因此，一旦开发，提出的方法也有望刺激哺乳动物肢体的再生。公共卫生相关性：早期的t腿在切断后将再生，后来不会再生。这项创新的建议将使用现代干细胞技术来检验关于为什么这样的假设。如果确认该假设，则开发的方法应有助于促进哺乳动物的肢体再生。