In-vitro generation of cardiomyocytes from MAPC

描述（由申请人提供）：干细胞和组织工程方法的发展和建立具有针对许多最重要的心血管疾病的潜力，并对美国医疗保健系统中主要死亡原因之一的治疗产生巨大影响。目前的提案是Athersys公司和明尼苏达大学干细胞研究所在其细胞治疗项目中的合作成果。该项目的主要重点是开发多能成体祖细胞（MAPC）技术，作为体外生成心肌细胞的新平台，用于心脏病的同种异体细胞治疗。该提案的总体目标是协同Athersys的小分子化合物高通量筛选（HTS）能力与MAPC平台。工作假设是，这提供了一个独特的机会来确定可以启动MAPCs向心肌细胞或祖细胞特异性和受控分化的因素和途径。第一阶段的工作将侧重于利用转基因小鼠模型获得的MAPCs，其中绿色荧光蛋白（GFP）报告基因通过心肌特异性启动子序列的活性在心肌中特异性表达。MAPCs将从这些转基因小鼠中分离出来，并扩增用于HTS试验，该试验测量GFP荧光的诱导，作为MAPCs向心肌细胞谱系分化的定量读数。第一阶段的两个具体目标是；1)从心肌特异性表达GFP的转基因小鼠中分离扩增MAPC；2)建立一种基于GFP荧光定量测定MAPC向心肌细胞谱系分化的灵敏特异性方法。该提案的最终目标是在II期结束时建立小鼠和人类MAPCs离体生产心肌细胞祖细胞的条件。这将为临床和商业上相关的心肌细胞提供新的来源，作为近期产品的体外毒理学研究或心脏病同种异体细胞疗法的进一步发展对心肌细胞有很大的需求。

DESCRIPTION (provided by applicant): The development and institution of stem cell and tissue engineering methodologies hold the potential to target many of the most significant cardiovascular diseases and have a dramatic impact on the treatment of one of the leading causes of death in the United States healthcare system. The current proposal is a collaborative effort between Athersys, Inc. and the University of Minnesota Stem Cell Institute within their cell therapy program. This project has a main focus on exploiting the Multipotent Adult Progenitor Cell (MAPC) technology as a novel platform for the in-vitro generation of cardiomyocytes for potential application in allogeneic cell therapy in heart disease. The overall objective of this proposal is to synergize the small molecule compound high-throughput screening (HTS) capability at Athersys with the MAPC platform. The working hypothesis is that this provides a unique opportunity to identify factors and pathways that can initiate specific and controlled differentiation of MAPCs into cardiomyocytes or progenitors. The Phase I effort will focus on the use of MAPCs acquired from transgenic mouse models in which the green fluorescent protein (GFP) reporter is specifically expressed in cardiac muscle through the activity of cardiac muscle specific promoter sequences. MAPCs will be isolated from these transgenic mice and expanded for use in HTS assays that measure the induction of GFP fluorescence as a quantitative read-out for differentiation of MAPCs into the cardiomyocyte lineage. The two specific aims in Phase I are; 1) to isolate and expand MAPC from transgenic mice with cardiac muscle specific expression of GFP, and; 2) to establish a sensitive and specific assay for quantification of MAPC differentiation into the cardiomyocyte lineage based on GFP fluorescence. The ultimate ambition of this proposal is to establish the conditions for ex vivo production of cardiomyocyte progenitors from mouse as well as human MAPCs by the end of Phase II. This will provide clinically and commercially relevant new sources for cardiac muscle cells, which are in great demand for in vitro toxicology studies as a near term product, or for the further development of allogeneic cellular therapies for heart disease.