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”，和特定的挑战主题11-GM-101，“建立再生能力”。与幼年哺乳动物相比，成年哺乳动物的肌肉干细胞(MuSCs)增殖较少，新生的肌肉较少，纤维化程度较高。这些效应至少部分归因于调节MSC及其后代的可溶性分子(康博伊，2005)。这些分子调节剂的浓度在再生过程中呈现时间变化，这种时间依赖性对于成功激活MUSC和预防纤维化非常重要(Brack，2007)。我们的目标是为这些调节剂提供适当的时间分布，以建立成年小鼠MSC及其后代的再生能力。我们的建议解决了这样一个事实，即目前没有技术在没有基因工程的情况下以生理方式在小鼠身上改变调节分子的浓度和时间，这是很难同时使用多个调节分子的，也需要世代繁殖，因此速度很慢，或者通过外部系绳机械地将小鼠连接到泵上，这有几个缺点：系绳阻止群体居住，并且与在小鼠身上执行的许多行为和成像分析不兼容。为了迎接这一挑战，我们发明了一种可植入鼠标的遥控微泵。我们已经成功地使用该泵将荧光素输送给携带表达萤火虫荧光素酶的MUSCs的小鼠，使MUSCs以剂量依赖和时间依赖的方式发出光(生物发光)。我们在背部皮肤下植入泵，并在皮肤下和肌肉筋膜下放置一根导管，携带表达荧光素酶的MuSCs。我们假设我们的泵可以利用生理时间剂量谱在再生过程中长期将干细胞调节分子输送到MUSCs。这些配置文件将自动与包括锻炼和睡眠在内的鼠标行为同步，如果候选的MUSC调节器与这些行为自然同步，如IGF-1。为此，我们在运行轮和跟踪植入泵中的红外光发射器的摄像头中建立了定制的笼子，使我们能够自动确定在任何给定时间哪个鼠标正在运动，并向该鼠标中的泵发送无线电信号，指示该泵提供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正在工作的泵原型转化为两家当地公司可以大量生产的版本，以实现统计意义(也支持当地就业)，使用具有已知时间分布(IGF-1)的再生调节器测试我们的泵和定制笼子，然后确定其他5种MUSCs及其后代的可溶性调节因子(WNT7a、睾酮、HGF、WNT3a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：将非荧光素酶的小鼠移植到表达荧光素酶的MUSCs。在MSC有时间植入后，我们将使用蛇毒肌毒素Notex in来损伤肌肉，这是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身上更容易测量。具体地说，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、凋亡和产生生物发光信号的细胞的表型；以及3)功能分析以测量动物步态和活动度以及肌肉收缩力量。我们将为每个调节器尝试两个时间配置文件。前两个时间分布将包括基于文献估计的范围。第三个时间概况将由我们对前两个时间概况的经验进一步了解。鉴于在原位建立成体细胞的再生能力以促进伤口愈合和减少瘢痕形成的浓厚兴趣，利用生理时间剂量谱在原位刺激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一个有价值的工具，并最终影响人类组织的再生。经济影响：我们的提案将对经济产生积极影响，直接在斯坦福医学公司、斯坦福工程公司以及当地公司EoPlex(加州山景城)和Bestek(加州圣何塞)创造或保留8个工作岗位(见支持信)。此外，根据加州生物医学行业2009年的报告，斯坦福医药公司直接雇用的每个人都会产生乘数效应，提供商品和服务的公司会另外雇用3到5个人。成人成功的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，没有办法以一种与标准群体住房以及标准行为和成像分析兼容的方式向小鼠提供时变模式的多种药物，这大大推迟了用于人类的再生医学疗法的到来。我们的可植入鼠标的远程控制微泵技术克服了这一限制；能够更快速、高效和经济地发现和提供再生疗法。

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.