Induction of digit regeneration by Wnt active nail epithelium

Wnt活性指甲上皮诱导手指再生

• 批准号: 9542711
• 负责人: Mayumi Ito
• 金额: $ 37.29万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9542711
• 关键词: Address; Amputation; Amputees; Area; Bone Regeneration; Cell Lineage; Cell Therapy; Cells; Cues; Defect; Digit structure; Disease; Distal; Epithelial; Epithelial Cells; Epithelium; FGF2 gene; FGFR1 gene; Family member; Fibroblast Growth Factor; Foundations; Genetic; Genetic Transcription; Goals; Growth; In Vitro; Injury; Leg; Limb structure; Mediating; Mediator of activation protein; Mesenchymal; Molecular; Molecular Profiling; Nail plate; Natural regeneration; Organ; Organ Model; Paracrine Communication; Pathway interactions; Play; Regenerative Medicine; Regenerative response; Role; Signal Transduction; Skin; Stem cells; Testing; Transgenic Mice; Transgenic Organisms; Transplantation; Undifferentiated; WNT Signaling Pathway; arm; base; beta catenin; blastema; bone; digit regeneration; experimental study; in vivo; innovation; keratinocyte; limb regeneration; mouse model; nerve supply; novel strategies; organ regeneration; overexpression; progenitor; public health relevance; regenerative; stem cell niche; transcription factor; wound; wound epidermis

DESCRIPTION (provided by applicant): Currently, there is no way to regenerate limbs that are lost to injuries or diseases. Our long term goal is to understand the role of the nail epithelim during digit tip regeneration and propose a novel strategy to promote regeneration of the digit/limb. The mammalian digit tip including its bone can regenerate upon amputation. Following amputation of the digit tip, the nail epithelium regrows together with the underlying undifferentiated mesenchymal blastema cells that regenerates the digit bone. Regeneration occurs only in areas associated with the nail and it is unknown why this regenerative limitation exists. We recently identified and discovered nail epithelial stem cells (NSCs) undergo Wnt-dependent nail differentiation. Remarkably, Wnt activation in the nail epithelium is required not only for nail regeneration but also for mesenchymal blastema growth that leads to digit bone regeneration upon digit tip amputation. When the digit is amputated proximal to the Wnt-active nail progenitors, Wnt activation does not occur in epithelial cells in the wound area and the nail/digit fails to regenerate. Nevertheless, forced Wnt activation in epithelial cells including NSCs can overcome this limitation. We hypothesize that Wnt-active nail epithelium holds a lineage specific function to promote digit regeneration by emitting paracrine signals. Aim1: We found that FGF2 is expressed in nail epithelial cells in a Wnt-dependent manner after amputation and that FGF2 promotes blastema proliferation through FGFR1 signaling in vitro. We will test whether FGFR1 signaling is essential for blastema growth in vivo, by genetically deleting fgfr1 in blastema cells. We will also test if overexpression of fgf2 in epithelial cells cn overcome the proximal limitation in regeneration. These experiments will be the first to dissect the role of a molecular pathway in digit tip regeneration by separately targeting the epithelial an mesenchymal cells. Aim2: [While the essential role of Wnt-active nail epithelial cells in inducing digit regeneration has been demonstrated, it remains unclear whether Wnt-active NSC-derived epithelial cells hold a unique lineage-dependent function to promote regeneration. We will thus test whether Wnt-activation in non-NSC-derived epithelial cells (i.e. epithelial cells proximal to nail epithelium) can similarly function to promote regeneration as those derived from the NSC lineage. Non-NSC-derived epithelial cells with stabilized ß-catenin lack expression of TCF/LEF1 family members, transcription factors essential for Wnt induced transcription, in contrast to nail epithelial cells that display both ß-catenin stabilization and express TCF1. We plan to induce Wnt signal activation in non- NSC lineage by genetically expressing TCF1 in combination with stabilized ß-catenin following amputations proximal to the NSC niche and examine whether this is sufficient to induce digit regeneration. Additionally, we will transplant NSCs and normal skin keratinocytes that constitutively activate Wnt signaling underneath the wound epidermis. We will test if this cell therapy can induce digit regeneration following proximal amputation.] These experiments will create a foundation to exploit NSCs in regenerative medicine for treating amputees.

描述（申请人提供）：目前，还没有办法使因受伤或疾病而失去的肢体再生。我们的长期目标是了解指甲上皮在指尖再生过程中的作用，并提出一种促进手指/肢体再生的新策略。哺乳动物的指尖（包括骨头）可以在截肢后再生。指尖截除后，指甲上皮与下面的未分化间充质胚细胞一起重新生长，从而再生指骨。再生仅发生在与指甲相关的区域，并且尚不清楚为什么存在这种再生限制。我们最近鉴定并发现指甲上皮干细胞（NSC）经历 Wnt 依赖性指甲分化。值得注意的是，指甲上皮中的 Wnt 激活不仅是指甲再生所必需的，也是间充质胚基生长所必需的，从而在指尖截肢时导致指骨再生。当手指在 Wnt 活性指甲祖细胞附近被截肢时，伤口区域的上皮细胞不会发生 Wnt 激活，指甲/手指无法再生。然而，包括 NSC 在内的上皮细胞中的强制 Wnt 激活可以克服这一限制。我们假设 Wnt 活性指甲上皮具有谱系特异性功能，可通过发射旁分泌信号来促进手指再生。目的1：我们发现FGF2在截肢后以Wnt依赖性方式在指甲上皮细胞中表达，并且FGF2在体外通过FGFR1信号传导促进胚基增殖。我们将通过基因删除胚基细胞中的 fgfr1 来测试 FGFR1 信号传导对于体内胚基生长是否至关重要。我们还将测试上皮细胞中 fgf2 的过度表达是否能够克服再生的近端限制。这些实验将首次通过分别针对上皮细胞和间充质细胞来剖析分子途径在指尖再生中的作用。目标 2：[虽然已证明 Wnt 活性指甲上皮细胞在诱导手指再生中的重要作用，但仍不清楚 Wnt 活性 NSC 衍生的上皮细胞是否具有独特的谱系依赖性促进再生功能。因此，我们将测试非 NSC 来源的上皮细胞（即邻近指甲上皮的上皮细胞）中的 Wnt 激活是否可以与 NSC 谱系来源的细胞类似地发挥促进再生的作用。具有稳定的 β-连环蛋白的非 NSC 来源的上皮细胞缺乏 TCF/LEF1 家族成员的表达，TCF/LEF1 家族成员是 Wnt 诱导转录所必需的转录因子，而指甲上皮细胞则表现出 β-连环蛋白的稳定性并表达 TCF1。我们计划在接近 NSC 生态位的截肢后通过基因表达 TCF1 与稳定的 ß-连环蛋白相结合来诱导非 NSC 谱系中的 Wnt 信号激活，并检查这是否足以诱导数字再生。此外，我们将移植 NSC 和正常皮肤角质形成细胞，这些细胞在伤口表皮下持续激活 Wnt 信号传导。我们将测试这种细胞疗法是否可以在近端截肢后诱导手指再生。]这些实验将为利用再生医学中的 NSC 来治疗截肢者奠定基础。