Artificial Stem Cells for Vascular Tissue Engineering

摘要 目前用于小直径动脉搭桥术或用于腹膜透析的房室通路的血管移植物 理想且有显著的故障率。组织工程血管移植物(TEVGs) 间充质干细胞(MSCs)前景看好，但有两个主要局限性可能会阻碍其临床应用 翻译。首先，心血管疾病的高危患者--如老年人和糖尿病患者-- 功能障碍的MSCs，可能无法产生可行的TEVG。其次，使用任何类型的细胞 需要长期的培养扩展-包括MSCs-打开了细胞污染风险的大门，或者 在制造TEVG之前，还面临着成本高、等待时间长的问题。这个 这项拟议工作的总体目标是开发一种新颖的、临床可行的、快速制造的无细胞TEVG。这个 这一提议的首要假设是，可以从人类间充质干细胞中获得分泌因子， 由“人造间充质干细胞”(ArtMSCs)包装和运送，然后可以取代 我们TEVG中的MSCs。三个具体目标将解决我们的假设： 目标1：发展由可降解和可调节微球家族组成的“人造干细胞” 人骨髓间充质干细胞的条件培养液。ArtMSCs的目标是取代我们体内对细胞的需求 TEVG，但保持其关键的秘密因素。我们期望我们可以封装和时间释放 利用PLGA微球从具有生物活性的人间充质干细胞中提取可溶性因子。我们将使用以下方法进行验证 释放的推动血管细胞迁移和增殖的因子。 目的2：对artMSCs进行调节，以最优替代MSC分泌活动的时间进程，并 在植入的TEVG内居住。这一目标将包括两个部分：a)体内时间进程评估 大鼠模型中基于MSC的TEVGs，监测移植的MSCs的存在和宿主SMC和 内皮细胞的募集和重塑。B)利用该定时信息来制造离散的 ArtMSCs将在不同的时间释放他们的货物。我们预计ART MSC家庭的净影响将 与重塑的TEVG内主动分泌的MSC的旁分泌活性大致相同。 目的3：测试由我们的artMSCs组成的TEVG的体内疗效。为此，我们将为这些家庭提供种子 将artMSCs转化为可生物降解的支架，并在大鼠模型中评估其作为TEVG的作用。我们预计会有一个 微球负载的无细胞支架将至少与TEVG和负载MSCs的支架一样有效。 TEVG将根据成功的衡量标准进行评估，包括通畅度和动脉样成分。 一种基于标准化人类MSC细胞系(即来自 健康患者)将提供从患者到患者的统一的治疗策略，而不是固有变量 基于自体细胞的策略。我们方法的无细胞本质更容易移植到临床上， 而且，从单个患者身上采集细胞的成本和时间将被消除。

SUMMARY Vascular grafts that are currently used in small-diameter arterial bypass or in AV access for dialysis are not ideal and have significant failure rates. Tissue-engineered vascular grafts (TEVGs) using autologous mesenchymal stem cells (MSCs) show promise, but have two main limitations that may prevent their clinical translation. First, patients at high risk for cardiovascular disease - such as the elderly and diabetics - have dysfunctional MSCs which may not be able to yield a viable TEVG. Second, the use of any cell type that requires extended culture expansion – including MSCs – opens the door to the risk of cellular contamination or transformation, as well as high costs and a substantial waiting time before a TEVG can be fabricated. The overall goal of the proposed work is to develop a novel, clinically-viable, rapidly-fabricated, cell-free TEVG. The overarching hypothesis of this proposal is that secreted factors from human MSCs can be harvested, packaged, and delivered by “artificial MSCs” (artMSCs) that can then replace the paracrine activity of the MSCs in our TEVG. Three specific aims will address our hypotheses: Aim 1: Develop “artificial stem cells” comprised of families of degradable and tunable microspheres loaded with conditioned media from human MSCs. The goal of the artMSCs is to replace the need for cells in our TEVG, but maintain their critical secreted factors. We expect that we can encapsulate and time-release the soluble factors from biologically active human MSC using PLGA microspheres. We will validate this by using the released factors to drive vascular cell migration and proliferation. Aim 2: Tune the artMSCs in order to most optimally replace the time course of MSC secretory activity and residency within an implanted TEVG. This aim will have two parts: A) An in-vivo time course evaluation of MSC-based TEVGs in a rat model, monitoring presence of implanted MSCs and timing of host SMC and endothelial cell recruitment and remodeling. B) Utilize this timing information to fabricate discrete families of artMSCs that will each release their cargo at different times. We expect the net effect of our artMSC families to approximate the paracrine activity of actively secreting MSC present within the remodeling TEVG. Aim 3: Test the in vivo efficacy of a TEVG comprised of our artMSCs. For this aim, we will seed the families of artMSCs into biodegradable scaffolds and evaluate them as a TEVG in a rat model. We expect that a microsphere-loaded, acellular scaffold will be at least as effective as a TEVG as a scaffold loaded with MSCs. The TEVGs will be assessed by metrics of success including patency and an artery-like composition. An innovative therapy based on secreted factors from standardized human MSC cell lines (i.e., from healthy patients) would offer a uniform treatment strategy from patient to patient than an inherently variable autologous cell-based strategy. The cell-free nature of our approach is more easily translatable to the clinic, and the cost and time spent harvesting cells from individual patients would be eliminated.