Three-Dimensional Cardiac Gel Bioreactor for Functioning Cardiomyocyte Induction

描述（由申请人提供）：受损心肌重新激活心肌细胞（CM）增殖的能力有限，是心肌再生的主要障碍。干细胞来源的CM被认为是替代受损CM的替代细胞来源。各种类型的干细胞正在研究其CM分化的潜力。骨骼肌源性干细胞（MDSC）是从骨骼肌标本中获得的体干细胞群。已显示MDSC沿沿着多个谱系分化，包括骨骼肌、骨、腱、神经、内皮、造血和平滑肌细胞。MDSC易于在体外扩增，并且可以作为自体移植物移植。以前的研究，包括我们自己的工作，已经表明MDSC具有心脏修复的潜力，并且可以分化为CM。最近，我们成功地诱导功能CM从培养的大鼠MDSC使用的策略相结合的MDSC聚集球形成和MDSC球三维心脏凝胶生物反应器（MDSC-sphere-3DGB）。MDSC-sphere-3DGB是可扩展的，并且通过构建体的酶消化容易地提取MDSC衍生的CM。MDSC-sphere-3DGB还允许直接评价MDSC衍生的CM作为工程化心脏组织的收缩特性。因此，该提案的目的是优化组织培养方法以获得从人MDSC分化的功能性CM。具体目的：确定人MDSC-sphere-3DGB用于功能性CM诱导的最佳条件。我们将测试4个假设，1）。使用MDSC-球体-3DGB系统，人MDSC分化成功能性CM; 2）. MDSC-球体-3DGB中的添加剂视黄酸处理进一步增加功能性CM分化; 3）。循环机械拉伸刺激增加人MDSC衍生的CM增殖和收缩特性，类似于未成熟的胎儿型CM; 4）。周期性电刺激增加人MDSC衍生的CM成熟并提高对缺氧条件的耐受性。重要性：本提案的目标是使用MDSC-球体-3DGB系统建立一个范例，以定义功能性人MDSC衍生CM的分化。这些研究支持我们实施自体细胞心肌成形术以修复先天性和获得性心脏病的长期目标。 公共卫生相关性：我们最近成功地产生了可工作的心肌细胞从大鼠骨骼肌来源的干细胞使用三维组织培养系统（生物反应器）。骨骼肌来源的干细胞相对容易地从心脏病患者的骨骼肌活检获得，并且来自骨骼肌干细胞的诱导的心肌细胞可以移植到相同的患者以治愈受损的心脏。本研究旨在探索人骨骼肌干细胞来源的心肌细胞治疗先天性和获得性心脏病的最佳条件。

DESCRIPTION (provided by applicant): The limited capacity of the injured myocardium to reactivate cardiomyocyte (CM) proliferation is a major barrier to myocardial regeneration. Stem cell derived CMs are considered as an alternative cell source to replace damaged CMs. Various types of stem cells are under investigation of their potential for CM differentiation. Skeletal muscle derived stem cells (MDSCs) are a somatic stem cell population obtained from skeletal muscle specimens. MDSCs have been shown to differentiate along multiple lineages including skeletal muscle, bone, tendon, nerve, endothelial, hematopoietic, and smooth muscle cells. MDSCs are easily expanded in vitro and can be transplanted as autologous grafts. Previous studies, including our own work, have shown that MDSCs have potential for cardiac repair and can differentiate into CMs. We have recently succeeded in the induction of functioning CM from cultured rat MDSCs using the strategy of combined MDSC-aggregate sphere formation and MDSC-sphere-3-dimensional cardiac gel bioreactor (MDSC-sphere-3DGB). MDSC-sphere-3DGB is scalable and the MDSC-derived CMs are easily extracted by enzymatic digestion of the construct. MDSC-sphere-3DGB also allows the direct evaluation of MDSC-derived CM contractile properties as an engineered cardiac tissue. Thus, the aim of the proposal is to optimize a tissue culture method to obtain functioning CMs differentiated from human MDSCs. Specific Aim: Define the optimal condition of human MDSC-sphere-3DGB for functioning CM induction. We will test 4 hypotheses, 1). Human MDSCs differentiate into functioning CMs using a MDSC-sphere-3DGB system; 2). Additive retinoic acid treatment in MDSC-sphere-3DGB further increases functioning CM differentiation; 3). Cyclic mechanical stretch stimulation increases human MDSC-derived CM proliferation and contractile properties similar to the immature fetal type CMs; 4). Cyclic electric stimulation increases human MDSC-derived CM maturation and improves tolerance to the hypoxic conditions. Significance: The goal of the present proposal is to establish a paradigm using a MDSC-sphere-3DGB system to define the differentiation of the functioning human MDSC-derived CMs. These studies support our long term goal of implementing autologous cellular cardiomyoplasty to repair congenital and acquired heart diseases. PUBLIC HEALTH RELEVANCE: We have recently succeeded in generating workable heart muscle cell from rat skeletal muscle derived stem cells using a 3-dimensional tissue culture system (bioreactor). The skeletal muscle derived stem cell is relatively easily obtained from a skeletal muscle biopsy of heart disease patients and the induced heart muscle cells from the skeletal muscle stem cells can be transplanted to the same patients to cure the damaged heart. This proposal is to investigate the optimal conditions of HUMAN skeletal muscle derived stem cell-derived heart muscle cells to cure congenital and acquired heart diseases.