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

• Applicant: Dr. Nazan Puluca
• Subject Area: Human Genetics ; General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2018 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 414228177

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.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Sean Wu