Injectable Hydrogels to Improve the Efficacy of iPSC-derived Therapies

 描述：注射干细胞疗法是一种有前途的微创策略，可治疗各种损伤和退行性疾病。目前基于干细胞的治疗心血管疾病（如外周动脉疾病（PAD））的临床试验通常显示出有限的疗效，部分原因是细胞存活率低。我们之前已经使用PAD动物模型证明，人诱导多能干细胞衍生的内皮细胞（hiPSC-EC）的存活率在注射到缺血组织后迅速下降，导致血液灌注恢复仅略有改善。为了解决细胞存活的这种限制，我们先前已经设计了可以共注射的水凝胶，以保护细胞在注射针注射期间免受机械膜损伤。然而，这样的水凝胶是非常柔顺的（G' ~10 Pa），并且不适合于许多生物医学应用。因此，我们建议开发混合诱导的双组分水凝胶改性聚乙二醇和聚（N-异丙基丙烯酰胺）（MITCH-PEG-PNIPAM）。我们的目标是设计水凝胶，提供可调的机械刚度和持续递送促生存因子，以抑制缺氧诱导的细胞凋亡，同时在注射过程中仍然提供显着的膜保护。因此，在具体目标1中，我们将评估以下假设：水凝胶刚性的调节和促存活因子的释放动力学将显著改善暴露于注射流和缺氧的干细胞的活力。将人iPSC-EC包封在不同刚度（G '= 10-100 Pa）和促存活因子（Rho相关激酶抑制剂Y-27632;和胰岛素样生长因子-1）的工程化水凝胶内。细胞 将进行体外注射模型，并对膜损伤、线粒体活性、代谢活性和凋亡标记物进行急性测定。在注射后第7、14和28天，将检测细胞增殖率、代谢活性、凋亡标志物和EC表型。 在常氧（20%O2）和低氧（1%O2）培养条件下定量以模拟体内缺血。基于这些测定的结果，我们将选择使细胞存活最大化的水凝胶刚度和促存活因子。在具体目标2中，我们将通过评价水凝胶在增强细胞存活和细胞治疗功效方面的功效来验证具有诱导后肢缺血的NOD-SCID小鼠（PAD的实验模型）中的体外结果。细胞将在注射前被封装在最佳水凝胶和促存活因子中 注入缺血肢体对照包括盐水注射（有和没有细胞，有和没有细胞）。 促存活因子）和水凝胶注射（无细胞，有和无促存活因子）分别通过生物发光成像和激光多普勒光谱法非侵入性地跟踪细胞存活和血液灌注。将分析组织学外植体的坏死、炎症、新血管形成、组织再生和移植细胞的存在。拟议研究的结果将导致一种新的生物材料方法，以提高干细胞治疗的临床应用的有效性。

 DESCRIPTION: Injectable stem cell therapy is a promising, minimally invasive strategy to treat a wide range of injuries and degenerative diseases. Current stem cell-based clinical trials to treat cardiovascular diseases such as peripheral arterial disease (PAD) have generally shown limited efficacy, in part due to poor cell survival. We have previously demonstrated using animal models of PAD that the survival of human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) declines rapidly after injection into ischemic tissue, leading to only a modest improvement in blood perfusion recovery. To address this limitation of cell survival, we have previously engineered hydrogels that can be co-injected to protect cells from mechanical membrane damage during syringe-needle injection. However, such hydrogels are very compliant (G' ~10 Pa), and not suitable for many biomedical applications. Therefore, we propose to develop Mixing-Induced Two-Component Hydrogels modified with polyethylene glycol and poly(N-isopropylacrylamide) (MITCH-PEG-PNIPAM). Our goal is to engineer hydrogels that provide tunable mechanical stiffness and sustained delivery of pro-survival factors to inhibit hypoxia-induced apoptosis while still providing significant membrane protection during injection. Accordingly, in Specific Aim 1, we will evaluate the hypothesis that tuning of th hydrogel rigidity and the release kinetics of pro-survival factors will significantly improve the viability of stem cells exposed to injection flow and hypoxia. Human iPSC-ECs will be encapsulated within the engineered hydrogels of varying stiffness (G'= 10-100 Pa) and pro-survival factors (Rho-associated kinase inhibitor Y-27632; and insulin-like growth factor-1). Cells will be subjected to an in vitro model of injection and acutely assayed for membrane damage, mitochondrial activity, metabolic activity, and apoptotic markers. At 7, 14, and 28 days post-injection, cell proliferation rate, metabolic activity, apoptotic markers, and EC phenotype will be quantified in both normoxic (20% O2) and hypoxic (1% O2) culture conditions to mimic in vivo ischemia. Based on the results of these assays, we will choose the hydrogel stiffness and pro-survival factor that maximizes cell survival. In Specific Aim 2 we will validate the in vitro resuls in NOD-SCID mice with induced hindlimb ischemia, an experimental model of PAD, by evaluating the efficacy of the hydrogel in enhancing cell survival and therapeutic efficacy of the cells. Cells will be encapsulated in the optimal hydrogel and pro-survival factors before injection into the ischemic limb. Controls include saline injection (with and without cells, with and without pro-survival factors) and hydrogel injection (without cells, with and without pro-survival factors) Cell survival and blood perfusion will be tracked noninvasively by bioluminescence imaging and laser Doppler spectroscopy, respectively. Histological explants will be analyzed for necrosis, inflammation, neovascularization, tissue regeneration, and presence of transplanted cells. The results of the proposed studies will lead to a new biomaterials approach to enhance the efficacy of stem cell therapy for clinical applications.