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Single cell RNA Sequencing in Hypoplastic Left Heart Syndrome (HLHS) derived from human induced pluripotent stem cells

来自人类诱导多能干细胞的左心发育不全综合征 (HLHS) 的单细胞 RNA 测序

基本信息

批准号：
414228177
负责人：
Dr. Nazan Puluca
金额：
--
依托单位：
Klinik für Herz- und Gefäßchirurgie
依托单位国家：
德国
项目类别：
Research Fellowships
财政年份：
2018
资助国家：
德国
起止时间：
2017-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/414228177?language=en
关键词：
Single cell RNA Sequencing Hypoplastic

项目摘要

The hypoplastic left heart syndrome is multigenic and heteregenous but not completely understood. Several dysregulations regarding the cell cycle, epigenetic factors, signaling paths and mutations in essential cardiac genes were described before. To understand these, various human ips models have been developed. The comparison of patient-generated hiPS (human induced pluripotent stem cells) and healthy controls provides the possibilty to identify differences in cardiac development und to declare mechanisms which may lead to HLHS.Findings in this field may lead to new therapeutic targets for the treatment of HLHS. In our department several national and international cooperations showed remarkable findings. During my research project covering 18 months at the Cardiovascular Institute, Stanford University (head: Prof.Sean Wu) I aim to analyze dysregulations in HLHS patients on a single cell RNA level. All known data and results for now were generated on a heterogenous cell population using a complete assay. However, the maximum achievable proportion of cardiomyocytes (CM) in iPS cultures is about 90% and even within the CM these are significantly different in their function and distribution (atrial, ventricular, nodal).Due to the RNA-Seq profile, single-cell RNA-Seq analyzes can be used to distinguish between ventricular, atrial and nodal CM.Therefore, I aim to create a more detailed picture of the transcriptome on a single cell level.So far we have used 3 HLHS lines (selected by their genotype) for all experiments. Further samples from patients who have an impaired ventriclular function after successful surgical therapy have already been sent to Sean Wu's laboratory.In particular, the difference between "genetically burdened" HLHS iPS and the other HLHS iPS lines could lead to specific therapeutic strategies.During my research project, single cell RNA-Seq analyzes of CM of HLHS patients will be performed using Chromium Controller® paying particular attention on the CM subtypes (ventricular, atrial and nodal).The experiments will include the comparison of CM generated from HLHS patients and healthy controls, as well as between individual HLHS lines. Since HLHS is a multifactorial disease, intracellular differences between the individual CMs of the different HLHS patients are also expected.There is already a long-standing cooperation with the laboratory of Prof. S. Wu at the Cardiovascular Institute of Stanford University (Harvard University in the past). Prof. Wu and his group have great expertise in the cultivation and establishment of human iPS cells. Another goal of this stay is to integrate those new skills and techniques in new projects at our research center in Munich.

左心发育不全综合征是多基因和异质性的，但尚未完全了解。关于细胞周期、表观遗传因素、信号通路和心脏必需基因突变的几种失调在之前已经描述过。为了理解这些，已经开发了各种人类ips模型。比较患者产生的人诱导多能干细胞（hiPS）和健康对照提供了识别心脏发育差异和宣布可能导致HLHS的机制的可能性。这一领域的发现可能会为HLHS的治疗带来新的治疗靶点。在我们部门，几项国内和国际合作取得了显著成果。在斯坦福大学心血管研究所（主任：sean Wu教授）为期18个月的研究项目中，我的目标是在单细胞RNA水平上分析HLHS患者的失调。目前所有已知的数据和结果都是在异质细胞群上使用完整的测定法产生的。然而，iPS培养中心肌细胞（CM）的最大可实现比例约为90%，即使在CM内，它们的功能和分布（心房、心室、结）也有显著差异。由于RNA-Seq谱，单细胞RNA-Seq分析可用于区分室性、心房性和结性CM。因此，我的目标是在单细胞水平上创建一个更详细的转录组图。到目前为止，我们在所有实验中使用了3个HLHS系（按其基因型选择）。手术治疗成功后脑室功能受损患者的进一步样本已被送往吴尚恩的实验室。特别是，“遗传负担”的HLHS iPS与其他HLHS iPS系之间的差异可能导致特定的治疗策略。在我的研究项目中，我将使用Chromium Controller®对HLHS患者的CM进行单细胞RNA-Seq分析，特别关注CM亚型（心室、心房和结）。实验将包括比较HLHS患者和健康对照产生的CM，以及个别HLHS系之间的CM。由于HLHS是一种多因素疾病，因此不同HLHS患者个体CMs之间的细胞内差异也是预期的。与美国斯坦福大学心血管研究所（曾为哈佛大学）吴教授实验室有长期合作关系。吴教授和他的团队在人类iPS细胞的培养和建立方面具有丰富的专业知识。这段时间的另一个目标是将这些新技能和技术整合到我们慕尼黑研究中心的新项目中。