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

左心发育不全综合征是多基因和异型的，但尚未完全了解。关于细胞周期、表观遗传因素、信号通路和基本心脏基因突变的几种失调已经被描述过。为了了解这些，各种人类iPS模型已经被开发出来。患者产生的HIPS(人类诱导多能干细胞)与健康对照的比较为识别心脏发育的差异和揭示可能导致HLHS的机制提供了可能性。这一领域的发现可能为HLHS的治疗提供新的治疗靶点。在我部，几个国家和国际合作取得了显着的成果。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使用完整的分析方法在异质细胞群上产生的。然而，在iPS培养中，心肌细胞(CM)的最大比例约为90%，即使在CM中，这些细胞在功能和分布(房室、结节)上也有显著差异。由于RNA-Seq谱，单细胞RNA-Seq分析可以用于区分室性、房室和结节CM。因此，我的目标是在单细胞水平上建立更详细的转录组图像。到目前为止，我们已经使用了3个HLHS系(根据它们的基因选择)。手术治疗成功后心脏功能受损的患者的进一步样本已被送往肖恩·吴的实验室。尤其是“遗传负担”的HLHS iPS与其他HLHS iPS系之间的差异可能会导致特定的治疗策略。在我的研究项目中，HLHS患者CM的单细胞RNA-Seq分析将使用Chromium Controler®进行，重点关注CM亚型(心室、心房和结节)。实验将包括HLHS患者和健康对照组产生的CM的比较，以及个别HLHS系之间的比较。由于HLHS是一种多因素疾病，不同HLHS患者的CMS之间的细胞内差异也是可以预期的。与斯坦福大学(过去的哈佛大学)心血管研究所的吴教授的实验室已经有了长期的合作。吴教授和他的团队在培养和建立人类iPS细胞方面拥有丰富的专业知识。此次停留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。

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.