Nanoflare-based isolation of cardiac progenitor cells derived from murine and human pluripotent stem cells

目前，在多能干细胞体外分化过程中衍生的心脏祖细胞的分离受到缺乏合适的细胞表面标记物的限制。心脏祖细胞的分离是心脏再生策略、心脏发育研究以及使用基于iPS的体外疾病模型的不可或缺的要求。目前，心脏祖细胞群体由它们的不同转录因子的特异性表达谱来定义，如Tbx5和Nkx2.5对于第一心野（PHF）的平行表达和Isl1对于第二心野（SHF）的平行表达。在拟议的项目中，我们将使用nanoflare技术来特异性分离多能干细胞体外分化过程中衍生的PHF和SHF祖细胞。nanoflare技术是一种检测内源性活mRNA的新方法。Nanoflares由一个中心的金纳米颗粒与共轭荧光标记的DNA探针组成，用于细胞特异性mRNA检测。mRNA与纳米耀斑探针的结合导致荧光信号的释放。然后，可以使用FACS分选通过该特异性和瞬时荧光信号分离靶细胞。使用nanoflare技术分离特定心脏祖细胞的拟议项目包括以下里程碑：1）通过纳米片在体外分化小鼠和人多能干细胞期间进行有效的细胞标记2）使用针对Tbx 5的特异性纳米片检测小鼠心脏祖细胞，合适的转基因多能干细胞系体外分化过程中的Nkx2.5和Isl 1 3）基于nanoflare的分离的鼠心脏祖细胞的详细表征4）在人多能干细胞的体外分化过程中，通过纳米片分离的人心脏祖细胞的检测和表征总之，所提出的项目将建立一种特异性的、高效的、简单的和生理特性保持的技术用于从多能干细胞衍生的心脏祖细胞的分离是不可或缺的。除此之外，所提出的技术可以很容易地转移到不同的研究领域，这些领域使用来自多能干细胞的细胞衍生物，因此也为科学界带来了巨大的普遍潜力。

Today, the isolation of cardiac progenitor cells derived during in-vitro differentiation of pluripotent stem cells is limited by the lack of appropriate cell surface marker. The isolation of cardiac progenitor cells is an indispensible requirement for cardiac regenerative strategies, research in cardiac development as well as the use of iPS based in vitro disease models. Currently, cardiac progenitor cell populations are defined by their specific expression profile of different transcription factors like the parallel expression of Tbx5 and Nkx2.5 for the primary heart field (PHF) and Isl1 for the second heart field (SHF). In the proposed project we will use the nanoflare technology for a specific isolation of PHF and SHF progenitor cells derived during in vitro differentiation of pluripotent stem cells. The nanoflare technology is a new approach for endogenous live mRNA detection. Nanoflares consists of a central gold nanoparticle with conjugated fluorescent-labeled DNA probes for cell specific mRNA detection. The binding of the mRNA to the nanoflare probe leads to the release of the fluorescence signal. Afterwards the target cells can be isolated by this specific and transient fluorescence signal using FACS sorting. The proposed project for specific cardiac progenitor cell isolation using the nanoflare technology consists of the following milestones: 1) Efficient cell labeling during in vitro differentiation of muirne and human pluripotent stem cells by nanoflares 2) Detection of murine cardiac progenitor cells using specific nanoflares for Tbx5, Nkx2.5 and Isl1 during in vitro differentiation of appropriate transgenic pluripotent stem cell lines 3) Detailed characterization of nanoflare based isolated murine cardiac progenitor cells 4) Detection and characterization of human cardiac progenitor cells isolated by nanoflares during in vitro differentiation of human pluripotent stem cells In summary, the proposed project will build the indispensible requirement for a specific, efficient, easy and the physiological properties preserving technology for the isolation of cardiac progenitor cells derived from pluripotent stem cells. Beside this, the proposed technology could be easily transferred to different research areas, which use cell derivates derived from pluripotent stem cells and therefore harbors also a great general potential for the scientific community.