Injectable Hydrogels to Improve the Efficacy of iPSC-derived Therapies

 描述：可注射干细胞疗法是治疗多种损伤和退行性疾病的一种有前途的微创策略。目前基于干细胞的临床试验用于治疗心血管疾病，如外周动脉疾病(PAD)，通常显示疗效有限，部分原因是细胞存活率较低。我们以前已经使用PAD的动物模型证明，人诱导的多能干细胞来源的内皮细胞(hiPSC-ECs)在注射到缺血组织后存活率迅速下降，导致血液灌流恢复仅有轻微改善。为了解决细胞存活的这一限制，我们以前设计了水凝胶，可以联合注射，以保护细胞在注射器针头注射期间免受机械性膜损伤。然而，这种水凝胶非常顺应性(G‘~10Pa.)，不适合于许多生物医学应用。因此，我们建议开发聚乙二醇聚N-异丙基丙烯酰胺共混改性双组分水凝胶(Mitch-PEG-PNIPAM)。我们的目标是设计水凝胶，提供可调节的机械硬度和持续输送促生存因子，以抑制缺氧诱导的细胞凋亡，同时在注射过程中仍提供显著的膜保护。因此，在特定的目标1中，我们将评估这样的假设，即调节TH水凝胶的刚性和促进生存因子的释放动力学将显著提高暴露于注射流和低氧下的干细胞的活性。人IPSC-ECs将被包裹在不同硬度(G‘=10-100pa)和促生存因子(Rho相关激酶抑制剂Y-27632和胰岛素样生长因子-1)的工程水凝胶中。单元格 将受到体外注射模型的影响，并对膜损伤、线粒体活性、代谢活性和凋亡标记物进行急性检测。在注射后7、14和28天，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)培养条件下进行量化，以模拟体内缺血。根据这些检测的结果，我们将选择最大限度地提高细胞存活率的水凝胶硬度和促存活因子。在具体目标2中，我们将通过评估水凝胶在提高细胞存活率和治疗效果方面的效果，验证在NOD-SCID小鼠诱导的后肢缺血(PAD的实验模型)中的体外结果。在注射之前，细胞将被包裹在最佳水凝胶和促存活因子中 进入缺血肢体。对照包括注射生理盐水(有和没有细胞，有和没有细胞 细胞存活和血液灌流将分别通过生物发光成像和激光多谱勒光谱进行无创跟踪。组织学外植体将分析坏死、炎症、新生血管、组织再生和移植细胞的存在。建议的研究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效果。

 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.