Remote-control mouse-implantable micropumps for establishment of regenerative cap

用于建立再生帽的遥控小鼠植入式微泵

• 批准号: 7827111
• 负责人: Helen M Blau
• 金额: $ 48.95万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7827111
• 关键词: Address; Adult; Animals; Apoptosis; Area; Back; Behavior; Behavioral; Biological Assay; Bioluminescence; Breeding; California; Catheters; Cell Count; Cell Proliferation; Cell physiology; Cells; Cicatrix; Code; Collaborations; Computers; Contracts; Custom; Data; Degenerative Disorder; Dependence; Disadvantaged; Dose; Drug Delivery Systems; Employment; Engineering; Enzymes; Equipment; Exercise; Exhibits; Fascia; Feedback; Fibrosis; Fireflies; Gait; Generations; Genetic Engineering; Goals; Hand; Histology; Hour; Housing; Human; Image; Implant; In Situ; Individual; Industry; Injury; Insulin-Like Growth Factor I; Laboratories; Letters; Light; Literature; Luciferases; Mammals; Measures; Medicine; Modeling; Molecular; Movement; Mus; Muscle; Muscle Fibers; Muscle function; Muscle satellite cell; Natural regeneration; Occupations; Pattern; Pharmaceutical Preparations; Phenotype; Physiological; Procedures; Process; Production; Proliferating; Protocols documentation; Publications; Pump; Quality Control; Radio; Regenerative Medicine; Reporting; Research; Rotation; Running; Saline; Services; Signal Pathway; Signal Transduction; Skin; Sleep; Snake Venoms; Speed; Stem cells; System; Technology; Testing; Testosterone; Time; Tissues; Translating; Transplantation; Trauma; Variant; Weight; Work; Wound Healing; base; disability; dosage; economic impact; experience; improved; inhibitor/antagonist; injured; interest; luciferin; meetings; muscle regeneration; notexin; prevent; prototype; regenerative; regenerative therapy; research study; response; satellite cell; sensor; success; tissue regeneration; tool

DESCRIPTION (provided by applicant): Our application addresses broad Challenge Area 11, "Regenerative Medicine", and specific Challenge Topic 11-GM-101, "Establishment of regenerative capabilities". In adult mammals, muscle stem cells (MuSCs) proliferate less, there is less new muscle produced, and there is more fibrosis, than in young mammals. These effects are at least partly due to soluble molecules that regulate MuSCs and their progeny (Conboy, 2005). The concentrations of these molecular regulators exhibit temporal variation during regeneration, and this time-dependence is important for successful MuSC activation and preventing fibrosis (Brack, 2007). Our goal here is to deliver these regulators with appropriate temporal profiles to establish regenerative capabilities of MuSCs and their progeny in adult mice. Our proposal addresses the fact that there is currently no technology to vary the concentrations and timing of regulator molecules in a physiologic manner in mice without either genetic engineering, which is difficult to do with more than one regulator simultaneously and also requires breeding for generations and is thus slow, or by mechanically connecting the mouse to a pump via an external tether, which has several disadvantages: tethers prevent group housing and are incompatible with many behavioral and imaging assays performed on mice. To meet this challenge we invented a mouse-implantable remote-control micropump. We have successfully used the pump to deliver luciferin to mice carrying MuSCs expressing the firefly enzyme luciferase, causing the MuSCs to emit light (bioluminescence) in a dose-dependent and time-dependent manner. We implant the pump under the skin of the back and run a catheter under the skin and under the fascia of the muscle carrying the luciferase-expressing MuSCs. We hypothesize that our pump can deliver stem cell regulator molecules to the MuSCs on a long-term basis during regeneration using physiologic temporal dosage profiles. These profiles will be automatically synchronized with mouse behaviors including exercise and sleep in the case of candidate MuSC regulators that are naturally synchronized with these behaviors, such as IGF-1. To this end we have built custom cages with sensors in running wheels and cameras which track infrared light emitters in the implanted pumps, allowing us to determine automatically which mouse is exercising at any given time and send a radio signal to the pump in that mouse to instruct the pump to deliver the MuSC regulator. Our pump is currently made by hand, a long and tedious process. Our specific objectives are to translate our current working pump prototype into a mass-producible version that two local companies can manufacture in sufficient quantities to achieve statistical significance (also supporting local employment), to test our pumps and custom cages using a regulator of regeneration with a known temporal profile (IGF-1), and then to identify effective temporal profiles for 5 other soluble regulators of MuSCs and their progeny (Wnt7a, testosterone, HGF, Wnt3a inhibitor, and MGF). Our model will be the same as for our luciferin pump test: non-luciferase mice transplanted with luciferase-expressing MuSCs. After the MuSCs have had time to engraft we will injure the muscle using a snake venom myotoxin, notexin, an injury model with which we have experience. We will use old mice and young controls, because the regenerative deficits we aim to ameliorate are easier to measure in old mice. Specifically, our readouts will be: 1) bioluminescence to quantify the proliferative response; 2) histology to quantify fibrosis, apoptosis, and the phenotypes of the cells producing the bioluminescence signal; and, 3) functional assays to measure animal gait and mobility and muscle contractile force. We will try two temporal profiles for each regulator. The first two temporal profiles will bracket a range estimated based on the literature. The third temporal profile will be further informed by our experience with the first two temporal profiles. Given the strong interest in establishing regenerative capabilities in adult cells in situ to improve wound healing and reduce scarring, a means of stimulating stem cell function in situ using physiologic temporal dosage profiles should be attractive, provide a valuable tool for the regenerative medicine field, and ultimately impact regeneration of tissues in humans. Economic impact: Our proposal would positively impact the economy by directly creating or retaining 8 jobs at Stanford Medicine, Stanford Engineering, and at local companies EoPlex (Mountain View, CA) and BesTek (San Jose, CA) (see supporting letters). In addition, according to the California Biomedical Industry 2009 report, for every individual directly employed by Stanford Medicine there is a multiplier effect, with another three to five people employed in firms that offer goods and services. Successful tissue regeneration in adults will likely require multiple drugs delivered to specific tissues with specific temporal (time-varying) dosage patterns. Currently there is no way to deliver time-varying patterns of multiple drugs to mice in a manner compatible with standard group housing and standard behavioral and imaging assays, significantly delaying the arrival of regenerative medicine therapies for humans. Our mouse- implantable remote-control micropump technology overcomes this limitation; enabling more rapid, efficient, and economical discovery and delivery of regenerative therapies.

描述（由申请人提供）：我们的申请涉及广泛的挑战领域11，“再生医学”和特定的挑战主题11-GM-101，“再生能力的建立”。在成年哺乳动物中，肌肉干细胞（MuSC）增殖较少，产生的新肌肉较少，并且比年轻哺乳动物中的纤维化更多。这些作用至少部分是由于调节MuSC及其后代的可溶性分子（Conboy，2005）。这些分子调节剂的浓度在再生期间表现出时间变化，并且这种时间依赖性对于成功的MuSC活化和预防纤维化是重要的（Brack，2007）。我们的目标是提供这些具有适当时间分布的调节剂，以建立成年小鼠中MuSC及其后代的再生能力。我们的建议解决了以下事实：目前没有技术在没有基因工程的情况下以生理方式改变小鼠中调节剂分子的浓度和时间，基因工程难以同时使用一种以上的调节剂，并且还需要繁殖几代，因此缓慢，或者通过外部系链将小鼠机械连接到泵，这具有几个缺点：系链阻止了群体圈养，并且与在小鼠上进行的许多行为和成像分析不相容。为了应对这一挑战，我们发明了一种可植入小鼠体内的遥控微型泵。我们已经成功地使用该泵向携带表达萤火虫酶荧光素酶的MuSC的小鼠递送了Escherin，使MuSC以剂量依赖性和时间依赖性的方式发光（生物发光）。我们将泵植入背部皮肤下，并在皮肤下和肌肉筋膜下运行导管，携带表达端粒酶的MuSC。我们假设我们的泵可以在再生期间使用生理时间剂量曲线长期将干细胞调节分子递送到MuSC。这些配置文件将自动与小鼠行为同步，包括运动和睡眠，在候选MuSC调节剂的情况下，这些调节剂与这些行为自然同步，例如IGF-1。为此，我们已经建立了定制的笼子，在转轮上安装了传感器，在植入的泵中安装了摄像头，跟踪红外光发射器，使我们能够在任何给定时间自动确定哪只小鼠正在锻炼，并向该小鼠的泵发送无线电信号，以指示泵输送MuSC调节器。我们的泵目前是手工制造的，这是一个漫长而繁琐的过程。我们的具体目标是将我们目前的工作泵原型转化为可大规模生产的版本，两家当地公司可以生产足够的数量，以达到统计学意义（也支持当地就业），使用具有已知时间分布的再生调节剂（IGF-1）测试我们的泵和定制笼，然后鉴定MuSC及其后代的5种其他可溶性调节剂（Wnt 7a、睾酮、HGF、Wnt 3a抑制剂和MGF）的有效时间分布。我们的模型将与我们的荧光素泵测试相同：移植有表达荧光素酶的MuSC的非荧光素酶小鼠。在MuSC有时间移植后，我们将使用蛇毒肌毒素notexin损伤肌肉，这是一种我们有经验的损伤模型。我们将使用老年小鼠和年轻对照组，因为我们旨在改善的再生缺陷在老年小鼠中更容易测量。具体而言，我们的读数将是：1）生物发光，以量化增殖反应; 2）组织学，以量化纤维化，细胞凋亡和产生生物发光信号的细胞的表型;和，3）功能测定，以测量动物步态和运动性和肌肉收缩力。我们将为每个调节器尝试两个时间曲线。前两个时间曲线将包括根据文献估计的范围。第三个时间分布图将进一步借鉴我们在前两个时间分布图方面的经验。鉴于在原位建立成体细胞的再生能力以改善伤口愈合和减少瘢痕形成的强烈兴趣，使用生理时间剂量分布原位刺激干细胞功能的手段应该是有吸引力的，为再生医学领域提供有价值的工具，并最终影响人类组织的再生。经济影响：我们的提议将直接在斯坦福大学医学、斯坦福大学工程以及当地公司EoTech（山景，加利福尼亚州）和BeTek（圣何塞，加利福尼亚州）创造或保留8个工作岗位，从而对经济产生积极影响（见支持信）。此外，根据《加州生物医学产业2009年报告》，斯坦福大学医学院每直接雇用一个人，就会产生乘数效应，另外三到五个人受雇于提供商品和服务的公司。成人的成功组织再生可能需要多种药物以特定的时间（随时间变化）剂量模式递送到特定组织。目前，没有办法以与标准群体饲养和标准行为和成像测定兼容的方式向小鼠提供多种药物的时变模式，这显著延迟了人类再生医学疗法的到来。我们的小鼠植入式远程控制微泵技术克服了这一限制;能够更快速，有效和经济地发现和提供再生疗法。