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

描述（由申请人提供）：心肌梗死（MI）后产生的纤维化疤痕是坚硬的细胞外基质（ECM），由于组织变薄和坏死时胶原蛋白的分泌增强。先前改善心肌梗死后心肌功能的方法，如心脏贴片和细胞注射，并不能充分模拟基质的固有特性，如刚度（表示为E）。此外，它们通常使用成体干细胞，这些干细胞尚未显示出具有显着的重塑能力，而是对异常基质条件更敏感；因此，已经观察到细胞不正确地分化为成骨细胞样细胞，或在3倍僵硬的梗死心肌中完全不能分化，即梗死b>。我们最近开发了一种动态的，巯基修饰的透明质酸（HA-S）为基础的水凝胶，随着时间的推移，通过时间依赖性交联显示出发育适当的硬度。我们还表明，这可以改善成熟心肌细胞的心脏祖细胞分化，比不重塑的软基质提高近一个数量级。在这个建议中，我们将首先将我们的发现扩展到胚胎干细胞（ESCs），它应该比以前的干细胞类型在基质重塑方面更有效。首先在ESCs中监测2D和3D HA-S水凝胶中心脏特异性基因的表达，以确定HA-S是否可以诱导心肌形成，相对于心脏祖细胞和年龄匹配的对照动物，如果不能，至少确保它能增强HA-S水凝胶的分化，而HA-S水凝胶已经通过碘乙酰胺处理去除了它们的时间依赖性交联。还将测量基质分泌、组装和重塑（通过透明质酸酶降解），并与心脏祖细胞进行比较，以确保ESCs确实可以有效地重塑基质，并且细胞可以在材料中充分迁移。细胞和HA-S水凝胶随后将用于皮下大鼠模型，以确保生物相容性并监测水凝胶在体内的特性，例如时间依赖性硬化。最后，在心肌梗死大鼠模型中，ESCs和/或心脏祖细胞将与HA-S结合使用，以确定我们的HA-S水凝胶与常规治疗（如细胞注射）相比，在多大程度上可以改善心肌梗死后的心肌功能。

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.