Artificial Stem Cells for Vascular Tissue Engineering

总结 目前用于小直径动脉旁路术或AV通路透析的血管移植物不适用于 理想，并具有显著的失败率。使用自体移植物的组织工程化血管移植物（TEVG） 间充质干细胞（MSC）显示出希望，但有两个主要的限制，可能会阻止其临床应用。 翻译.首先，患有心血管疾病的高风险患者-如老年人和糖尿病患者- 功能失调的MSC可能不能产生有活力的TEVG。第二，使用任何细胞类型， 需要延长培养扩增-包括MSC-打开了细胞污染风险的大门， 在制造TEVG之前，需要高成本和大量的等待时间。的 所提出的工作的总体目标是开发一种新的、临床上可行的、快速制造的、无细胞的TEVG。的 该提议的首要假设是可以收获来自人MSC的分泌因子， 包装，并通过“人工MSC”（artMSC）递送，然后可以替代细胞的旁分泌活性。 我们的TEVG中的MSC。三个具体目标将解决我们的假设： 目的1：开发由可降解和可调微球组成的“人工干细胞” 用来自人MSC的条件培养基。artMSCs的目标是取代我们的细胞需求， TEVG，但保持其关键的分泌因子。我们希望我们可以封装和时间释放 使用PLGA微球从生物活性人MSC中分离可溶性因子。我们将通过使用 释放的因子驱动血管细胞迁移和增殖。 目的2：调节artMSC以最佳地替代MSC分泌活性的时间过程， 在植入的TEVG内驻留。该目的将有两个部分：A）体内时间过程评估， 在大鼠模型中基于MSC的TEVG，监测植入MSC的存在和宿主SMC的时间， 内皮细胞募集和重塑。B）利用该定时信息来制造离散的族 artMSC，其各自将在不同时间释放其货物。我们希望我们的artMSC家族的净效应 近似于重塑TEVG内存在的主动分泌MSC的旁分泌活性。 目的3：测试由我们的artMSC组成的TEVG的体内功效。为了这个目标，我们将播种家庭 将artMSCs植入可生物降解的支架中，并在大鼠模型中将其作为TEVG进行评估。我们预计， 因此，微球负载的无细胞支架将至少与TEVG一样有效，如负载MSC的支架。 TEVG将通过成功指标进行评估，包括通畅性和动脉样成分。 一种基于来自标准化人MSC细胞系的分泌因子的创新疗法（即，从 健康的患者）将为患者之间提供统一的治疗策略，而不是固有的变量 基于自体细胞的策略。我们的方法的无细胞性质更容易转化为临床， 并且将消除从个体患者收集细胞所花费的成本和时间。

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.