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获得的心脏祖细胞的分离受到缺乏适当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、心脏发育研究以及基于体外疾病模型的iPS使用的必要条件。目前，心脏祖细胞群是通过不同转录因子的特定表达谱来定义的，如Tbx5和Nkx2.5在初级心脏野（PHF）和Isl1在第二心脏野（SHF）的平行表达。在该项目中，我们将使用纳米耀斑技术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。纳米耀斑技术是一种检测内源性活mRNA的新方法。纳米耀斑由中心金纳米粒子和共轭荧光标记的DNA探针组成，用于细胞特异性mRNA检测。mRNA与纳米火炬探针的结合导致荧光信号的释放。然后用流式细胞仪（FACS）分选，利用特异的瞬时荧光信号分离靶细胞。使用纳米耀斑技术分离特定心脏祖细胞的拟议项目包括以下里程碑：1)利用纳米耀斑对小鼠和人多能干细胞体外分化过程中的高效细胞标记2)利用Tbx5特异性纳米耀斑检测小鼠心脏祖细胞3)基于纳米耀斑的小鼠离体心脏祖细胞的详细表征4)在人多能干细胞离体分化过程中利用纳米耀斑分离的人心脏祖细胞的检测和表征。多能干细胞衍生心脏祖细胞的分离及其生理特性保存技术。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，这些研究领域使用来自多能干细胞的细胞衍生物，因此对科学界来说也具有巨大的普遍潜力。

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.