Generating and characterizing primate iPSCs for evolutionary analyses

Geuder, Johanna (2022): Generating and characterizing primate iPSCs for evolutionary analyses. Dissertation, LMU München: Fakultät für Biologie

Abstract

The similarities and differences between us and our closest relatives, the primates, have fascinated researchers for decades and evoked various approaches to better understand the underlying genotype-phenotype relationship. Starting with early comparisons of protein sequences between humans and chimpanzees, substantial technological advances in genomics have led to a deeper understanding of the complexities in this relationship, ranging from cataloging genetic differences to modeling genetic differences in cellular and animal systems. Furthermore, the lack of genetic differences - sequence conservation - is crucial to annotate the human genome and interpret biomedically relevant variants within humans. Charting differences and similarities in molecular and cellular properties can take such a comparative approach to the next phenotypic level. In particular, similar to the information obtained from DNA conservation, expression conservation could help annotating and interpreting human gene expression patterns and thus also provide biomedically relevant information. However, the major limiting factor in this venture is the availability of comparable samples of different primates, mainly due to ethical constraints. Induced pluripotent stem cells (iPSCs) are used in humans to overcome such limitations, as they can be propagated indefinitely and differentiated to many different cell types. Thus, they can provide a valuable and unique resource for functional primate genomics. In this context, I established a method to generate iPSCs from primates. One of the major challenges in generating iPSCs from non-model organisms is the acquisition of the somatic cells for reprogramming. Therefore, I focused on urine as a non-invasive cell source and could show that cells can be isolated from very small amounts of primate urine samples, which were collected in an unsterile manner. These cells can be efficiently reprogrammed into iPSCs using the footprint-free Sendai Virus reprogramming method. Utilizing this approach, we generated four iPSC lines from two orangutans, three iPSC lines from one gorilla and nine lines from five humans. We validated the pluripotecy of these lines using immunocytochemistry, differentiation assays and also classified the cells as pluripotent using bulk RNA-sequencing. We further showed that expression differences among clones are comparable to those among individuals and considerably larger than technical sources of variation, suggesting that these cells are a suitable resource for functional primate genomics. As RNA-sequncing (RNA-seq) is a decisive assay to classify cells and to study gene expression in a comparative context, a robust and affordable method to quantify RNA expression levels is indispensable. I contributed to develop prime-seq, a sensitive bulk RNA-seq protocol that we showed to perform equivalently to standard bulk RNA-seq methods, but at a fourfold higher efficiency due to almost 50-fold cheaper library costs. This is highly useful to e.g. classify generated iPSCs as described above. However, to compare heterogenous cell populations, as they arise for example during the differentiation of iPSCs, RNA-seq with single-cell resolution (scRNA-seq) is crucial. I contributed to develop mcSCRB-seq, a sensitive, powerful and efficient single cell RNA-seq method, that is plate-based and hence, can be used for scRNA-seq on sorted single cells. Finally, I utilized mcSCRB-seq to compare gene expression trajectories during differentiation of our primate iPSCs towards neural precursor cells (NPCs). We sampled single cells of nine different clones from three species at six different time points during early neural differentiation and thus generated a comprehensive dataset to study this process in a comparable manner. We identify genes with a conserved constant up-regulation throughout the trajectory and find that these genes have a higher probability of being mutation intolerant and a higher probability to be associated with neurodevelopmental disorders. This strengthens the hypothesis that identifying conserved expression patterns in primate iPSCs could carry unique functional information to annotate and interpret the human genome.\par In summary, within my thesis I describe the basis for comparative research settings, by providing a non-invasive and footprint-free method to generate iPSCs from various primates. Additionally, I contributed to efficient methods to characterize these cells and showcase in an encompassing study how expression conservation can help to better understand the human genome.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 14. September 2022
• 1. Bericht­erstatter:in: Enard, Wolfgang