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

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”，以及具体的挑战主题11- gm -101，“建立再生能力”。与年轻哺乳动物相比，成年哺乳动物的肌肉干细胞（MuSCs）增殖更少，产生的新肌肉更少，纤维化更多。这些影响至少部分是由于调节musc及其后代的可溶性分子（Conboy, 2005）。这些分子调节因子的浓度在再生过程中表现出时间上的变化，这种时间依赖性对于MuSC的成功激活和防止纤维化是重要的（Brack, 2007）。我们的目标是提供这些具有适当时间谱的调节因子，以在成年小鼠中建立musc及其后代的再生能力。我们的建议解决了这样一个事实，即目前没有技术可以在没有基因工程的情况下以生理方式改变老鼠体内调节分子的浓度和时间，而基因工程很难同时处理多个调节分子，而且还需要世代繁殖，因此速度很慢，或者通过外部系绳将老鼠机械地连接到泵上，这有几个缺点：系绳防止群居，与许多对小鼠进行的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老鼠体内的遥控微泵。我们已经成功地使用泵将荧光素输送到携带表达萤火虫酶荧光素酶的musc的小鼠体内，使musc以剂量依赖和时间依赖的方式发光（生物发光）。我们将泵植入背部皮肤下，在皮肤下和肌肉筋膜下放置导管，携带表达荧光素酶的musc。我们假设我们的泵可以在再生过程中使用生理时间剂量谱将干细胞调节分子长期输送到musc。在候选MuSC调节因子（如IGF-1）与这些行为自然同步的情况下，这些特征将自动与小鼠的行为同步，包括运动和睡眠。为此，我们在跑步轮上安装了传感器，并在植入的泵中安装了摄像头，摄像头可以跟踪红外光发射器，这样我们就可以自动确定哪只老鼠在任何给定的时间运动，并向老鼠体内的泵发送无线电信号，指示泵输送MuSC调节器。我们的泵目前是手工制作的，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批量生产的版本，两家当地公司可以生产足够数量的产品，以达到统计显著性（也支持当地就业），使用具有已知时间特征的再生调节因子（IGF-1）测试我们的泵和定制的保持架，然后确定其他5种可溶性musc调节因子及其后代(Wnt7a，睾酮，HGF，Wnt3a抑制剂和MGF)。我们的模型将与我们的荧光素泵试验相同：非荧光素酶小鼠移植表达荧光素酶的musc。在musc有时间植入后，我们将使用蛇毒肌毒素，诺特辛来损伤肌肉，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小鼠作为对照，因为我们旨在改善的再生缺陷在老年小鼠中更容易测量。具体来说，我们的读数将是：1)生物发光来量化增殖反应；2)组织学定量分析产生生物发光信号的细胞的纤维化、凋亡和表型；3)功能分析，测量动物的步态、运动和肌肉收缩力。我们将为每个调节器尝试两个时间概况。前两个时间剖面将包含基于文献估计的范围。我们对前两个时间剖面的经验将进一步说明第三个时间剖面。考虑到建立原位成体细胞的再生能力以改善伤口愈合和减少疤痕的强烈兴趣，使用生理时间剂量曲线刺激原位干细胞功能的方法应该是有吸引力的，为再生医学领域提供了有价值的工具，并最终影响人体组织的再生。经济影响：我们的提案将通过在斯坦福医学、斯坦福工程以及当地公司EoPlex （Mountain View， CA）和BesTek （San Jose， CA）直接创造或保留8个工作岗位，对经济产生积极影响（见支持信）。此外，根据《2009年加州生物医学产业报告》，斯坦福医学院每雇用一个人，就会产生乘数效应，另外还有三到五个人受雇于提供商品和服务的公司。成功的成人组织再生可能需要多种药物以特定的时间（时变）剂量模式输送到特定的组织。目前还没有办法将多种药物的时变模式以一种与标准群体住房和标准行为和成像分析相兼容的方式传递给小鼠，这大大推迟了人类再生医学疗法的到来。我们的鼠标植入遥控微泵技术克服了这一限制；使再生疗法的发现和提供更加快速、有效和经济。

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.