Stem Cells and Dynamic Materials Improve Cardiac Function Post-mycardial Infarcti

描述(申请人提供)：心肌梗死(MI)后导致的纤维性瘢痕是僵硬的细胞外基质(ECM)，原因是随着组织变薄和坏死，胶原蛋白的分泌增加。以往改善心肌梗死后心肌功能的方法，如心脏补片和细胞注射，不能充分模拟基质的固有属性，如硬度(表示为E)。此外，他们经常使用成年干细胞，这些干细胞尚未被证明具有显著的重塑能力，相反，它们对异常的基质条件更敏感；因此，人们观察到，在过于僵硬3倍的梗塞心肌中，细胞不正确地分化为成骨样细胞，或者完全无法分化，即EInfarct&gt；eCardio。我们最近开发了一种动态的硫醇修饰的透明质酸(HA-S)水凝胶，它通过时间依赖的交联会随着时间的推移表现出发育适当的硬度。我们还表明，这可以改善成熟心肌细胞的心脏前体细胞分化，比不重塑的软基质高出近一个数量级。在这项提案中，我们将首先将我们的发现扩展到胚胎干细胞(ESCs)，它在基质重塑方面应该比以前的干细胞类型更有效。首先将在胚胎干细胞中监测2D和3D HA-S水凝胶中心脏特异基因的表达，以确定HA-S是否能够相对于心脏祖细胞和年龄匹配的对照组动物诱导心肌生成，如果不能，至少确保它促进了与HA-S水凝胶的分化，后者已经通过碘乙酰胺处理消除了其时间依赖的交联。还将测量基质分泌、组装和重塑(通过透明质酸酶降解)，并与心脏前体细胞进行比较，以确保ESCs确实可以有效地重塑基质，并确保细胞能够在材料中充分迁移。细胞和HA-S水凝胶随后将用于大鼠皮下模型，以确保生物相容性和监测体内的水凝胶特性，例如时间依赖的僵硬。最后，在心肌梗死大鼠模型中，胚胎干细胞和/或心脏祖细胞将与HA-S联合使用，以确定与常规治疗(如细胞注射)相比，我们的HA-S水凝胶在多大程度上可以改善心肌梗死后的心肌功能。 公共卫生相关性：作为美国主要的死亡原因，心肌梗死(MI)后充血性心力衰竭(CHF)促使人们需要开发新的技术来防止形成僵硬、伤痕累累的肌壁，从而损害心脏功能。在过去的二十年里，使用干细胞贴片或注射的新策略一直未能充分重塑组织并恢复其功能，部分原因可能是它们无法解决疾病利基的特定设计标准，例如僵硬(表示为E)；通常将成年干细胞添加到这个利基导致干细胞对环境做出反应，而不是重塑环境，因此，干细胞行为异常发生，因为利基相对于健康的肌肉eCardio太硬了3-4倍。也有人提出了与细胞联合治疗心肌梗死的材料，但它们往往不能充分模拟天然心肌设计要求，也不能使用具有足够能力的细胞来重塑利基。对此，我们设计了一种基于透明质酸(HA)的材料，它具有随时间变化的交联性，专门模拟心肌如何从柔软的胚胎干细胞(EESC)到更坚硬的心壁eCardio；随时间变化的硬化诱导心脏祖细胞表达比静态培养更成熟的心脏标志物3倍。它还使85%的祖细胞形成成熟的收缩肌节，而静态培养只有15%。当与胚胎干细胞结合时，这种基于HA和ESC的方法可能能够在胚胎干细胞发育时充分保护它们，迁移到伤痕累累的宿主心肌，并随后对其进行重塑，试图恢复某种程度的收缩功能。在这项提案中，我们将首先研究工程HA材料在多大程度上促进ESCs的心脏分化和基质分泌、组装和重塑。在了解了这种相互作用并得到更好的了解后，该组合将被引入大鼠模型皮下以确定生物相容性，然后用于大鼠梗死模型以评估其改善心脏结局的能力。

DESCRIPTION (provided by applicant): The fibrotic scar that results after a myocardial infarction (MI) is stiff extracellular matrix (ECM), owing to the enhanced secretion of collagen as the tissue thins and undergoes necrosis. Pervious methods to improve myocardial function post-MI, e.g. cardiac patches and cell injections, do not sufficiently mimic the intrinsic properties of the matrix, such as stiffness (denoted E). Moreover, they often employ adult stem cells which have not been shown to have significant remodeling capacity and instead are more responsive to aberrant matrix conditions; thus cells have been observed to improperly differentiate into osteoblast-like cells or to fail to differentiate altogether in infarcted myocardium that is 3-fold too stiff, i.e. EInfarct >> ECARDIO. We have recently developed a dynamic, thiol-modified hyaluronic acid (HA-S)-based hydrogel that displays developmentally appropriate stiffness over time via time-dependent crosslinking. We have also shown that this can improve cardiac progenitor differentiation in mature cardiomyocytes by nearly an order of magnitude over soft matrix that does not remodel. In this proposal, we will first extend our findings to embryonic stem cells (ESCs), which should be even more effective at matrix remodeling than previous stem cell types. Expression of cardiac-specific genes in 2D and 3D HA-S hydrogels will first be monitored in ESCs to determine if HA-S can induce cardiomyogenesis relative to cardiac progenitor cells and age-matched control animals, and if not, at least ensure that it enhances differentiation over HA-S hydrogels that have had their time-dependent crosslinking removed by treatment with iodacetamide. Matrix secretion, assembly, and remodeling (via degradation by hyaluronidase) will also be measured and compared to cardiac progenitor cells to ensure that ESCs can indeed remodel matrix effectively and that cells can migrate sufficiently in the material. Cells and HA-S hydrogels will subsequently be used in a subcutaneous rat model to ensure biocompatibility and monitor hydrogel properties in vivo, e.g. time-dependent stiffening. Finally in a rat model of MI, ESCs and/or cardiac progenitor will be used in conjunction with the HA-S to determine to what degree our HA-S hydrogel can improve myocardial function post-MI versus convention treatments, e.g. cell injection. PUBLIC HEALTH RELEVANCE: As a leading cause of death in the United States, congestive heart failure (CHF) post-myocardial infarction (MI) has incited the need to develop novel techniques that prevents the formation of a stiff, scarred muscle wall which impairs heart function. Over the past two decades, novel strategies using stem cell patches or injections have not been able to sufficiently remodel the tissue and restore its function, which may be in part due to their inability to address specific design criteria of the diseased niche, e.g. stiffness (denoted E); often the addition of adult stem cells into this niche results in stem cells responding to the environment rather than remodeling it, and as such, aberrant stem cell behavior occurs given that the niche is 3- to 4-fold too stiff, EInfarct, relative to healthy muscle, ECARDIO. Materials have also been proposed in conjunction with cells to treat MI, but they often do not adequately mimic native myocardial design requirements or use cells with sufficient capacity to remodel the niche. In response, we have engineering a hyaluronic acid (HA)-based material that has time-dependent crosslinking to specifically mimic how the myocardium stiffens from soft embryonic stem cells (ESCs), EESC, to the stiffer heart wall, ECARDIO; time-dependent stiffening induces cardiac progenitors to express 3-fold more mature cardiac markers versus static cultures. It also causes 85% of progenitors to form mature, contractile sarcomeres versus only 15% for static cultures. When combined with ESCs that are uniquely tuned to remodel and shape their niche as they develop, this HA- and ESC-based approach may be able to sufficiently protect ESCs as they develop, migrate into the scarred host myocardium, and subsequently remodel it to attempt to restore some level of contractile function. In this proposal, we will first study to what extent the engineered HA material enhances cardiac differentiation and matrix secretion, assembly, and remodeling in ESCs. After understanding this interaction and better, the combination will be introduced subcutaneously in a rat model to determine biocompatibility and then in used in a rat infarct model to assess its ability to improve cardiac outcome.