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CMS被认为是替代受损CMS的替代细胞来源。各种类型的干细胞正在研究它们在CM分化方面的潜力。骨骼肌源性干细胞(MDSCs)是从骨骼肌标本中获得的一组体细胞干细胞。研究表明，MDSCs可沿多个谱系分化，包括骨骼肌、骨、肌腱、神经、内皮细胞、造血细胞和平滑肌细胞。MDSCs易于体外扩增，可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MDSCs具有心脏修复的潜力，并可以分化为CMS。最近，我们利用MDSC-聚集球形成和MDSC-SPHERE-三维心脏凝胶生物反应器(MDSC-SPHERE-3DGB)相结合的策略，成功地从培养的大鼠MDSCs诱导出具有功能的CM。MDSC-SPHERE-3DGB是可伸缩的，MDSC来源的CMS很容易通过酶消化的方式提取出来。MDSC-SPHERE-3DGB还允许直接评估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一种组织培养方法，以获得从人MDSCs分化而来的具有功能的CMS。具体目的：确定人MDSC-SPHERE-3DGB诱导CM的最佳条件。我们将检验4个假设，1)。使用MDSC-SPHERE-3DGB系统将人类MDSC分化为功能正常的CMS；2)在MDSC-SPHERE-3DGB中添加维甲酸处理进一步增加了功能性CM的分化；3)周期性机械拉伸刺激增加人MDSC来源的CM的增殖和收缩特性，类似于未成熟的胎儿型CMS；4)循环电刺激可促进人骨髓间充质干细胞来源的CM成熟，并提高对低氧条件的耐受性。意义：本提案的目标是建立一个使用MDSC-SPHERE-3DGB系统的范例，以定义功能正常的人类MDSC来源的CMS的区别。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：我们最近成功地使用三维组织培养系统(生物反应器)从大鼠骨骼肌源性干细胞中培养出可工作的心肌细胞。从心脏病患者的骨骼肌活检中相对容易地获得骨骼肌源性干细胞，并且从骨骼肌干细胞中诱导的心肌细胞可以移植到相同的患者身上，以治愈受损的心脏。本研究旨在探讨人骨骼肌源性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.