Unbiased mapping of non-myocyte cell (NMC) populations in the human heart – Cellular composition & single cell transcriptome analysis considering age, anatomical location and cardiac pathologies

心脏非心肌细胞(NMC)群体对心脏发育、内环境稳定和各种心脏疾病有重要作用。因此，NMC是创新再生战略的有趣目标。然而，心脏中的NMC在数量和体积方面的比例以及它们各自的基因表达谱仍然存在争议，因为它们的分析受到细胞鉴定和分离的困难。在应用项目中，首次打算利用能够绕过上述问题的最先进的单细胞技术，以公正的方式分析出生后“人类”心脏的NMC组成和NMC转录本。我们试图分析来自不同解剖位置的不同心脏诊断的新生儿、婴儿和成人患者的心脏活检的单细胞悬液。我们的总体目标是(I)鉴定人类心脏内NMC的细胞组成，(Ii)创建hNMC群体的分子基因表达图谱。这些目标将在四个工作包(WP)中阐述。(Wp1)利用新鲜采集的人心脏组织建立单细胞RNA测序(sc-RNAseq)技术。(WP2)对30个特定部位、年龄类别和心脏疾病的人类心脏组织样本进行SC-RNAseq。(WP3)根据(A)心脏内的定位，(B)出生后发育年龄和(C)基本诊断，确定hNMC组成和基因表达谱的差异。(WP4)关于hNMC内心脏成纤维细胞亚群的定位、年龄和疾病的定义。我们假设，通过使用最先进的sc-RNAseq，首次可以定义人类心脏内不同NMC群体的可重复性和明显区分的分子表型。因此，该项目将产生受上述变量影响的hNMC人群的详细图谱，这是开发新疗法和更好地了解某些心脏病的病理生理学的不可或缺的先决条件。

Cardiac non-myocyte cell (NMC) populations significantly contribute to cardiac development, homeostasis and various cardiac diseases. Thus, NMCs are interesting targets for innovative regenerative strategies. The proportion of NMCs in the heart, however, with respect to number and volume, and their respective gene expression profiles still remain controversial since their analysis has been hindered by difficulties in cell identification and isolation. In the applied project it is intended, for the first time, to analyse NMC composition and NMC transcriptomes of the postnatal “human” heart in an unbiased fashion by state-of-the-art single cell technology which allows circumventing the above mentioned problems. We seek to analyse single cell suspensions from cardiac biopsies of different anatomical locations of neonatal, infant and adult patients with different cardiac diagnosis. Our overall aim is (I) to identify the cellular composition of NMCs within the human heart and, (II) to create a molecular gene expression map for hNMC populations. These objectives will be addressed in four work packages (WP). (WP1) Establishment of the single cell RNA sequencing (sc-RNAseq) technology using freshly harvested human cardiac tissue. (WP2) Sc-RNAseq of 30 human cardiac tissue samples of defined localizations, age classes and cardiac diseases. (WP3) Identification of differences in the hNMC composition and gene expression profiles depending on (a) localization within the heart, (b) postnatal developmental age and (c) underlying diagnosis. (WP4) Definition of cardiac fibroblast subpopulations within the hNMCs with respect to localization, age and disease.We hypothesize that by using state-of-the-art sc-RNAseq, for the first time, a reproducible and clearly distinguished molecular phenotype of different NMC populations within the human heart could be defined. This project will therefore result in a detailed mapping of hNMC populations influenced by the above mentioned variables which is an indispensable prerequisite for the development of new therapies and a better pathophysiologic understanding of certain cardiac diseases.