Generation and characterization of primate iPSCs for cross-species comparisons

Jocher, Jessica (2025): Generation and characterization of primate iPSCs for cross-species comparisons. Dissertation, LMU München: Fakultät für Biologie

Abstract

For centuries, scientists have been intrigued by the phenotypic similarities and differences between humans and our closest living relatives, the primates. This has led to the development of various approaches aiming to understand the underlying genotype-phenotype relationship. Starting with investigating DNA sequence divergence between humans and chimpanzees, increased sequencing and advances in computational methods have enabled researchers to identify variants that are associated with human-specific phenotypes. In contrast, sequence conservation across multiple species with a close phylogenetic relationship can help to identify and interpret functional genetic variants. However, as changes in protein-coding sequences alone cannot account for the striking differences in phenotypes between species, gene expression regulation is considered as a pivotal contributor. Thus, the study of gene expression in humans and other primates can reveal unique insights into the evolution of gene regulation and can be used to identify genes and pathways that are associated with human-specific adaptations. However, a major obstacle to comparative studies, especially those involving primates, is the availability of comparable samples. This is mainly due to ethical and practical reasons that complicate the acquisition of cells, particularly during developmental stages. To overcome these limitations, induced pluripotent stem cells (iPSCs) can be used, as they can be grown indefinitely in culture while maintaining their pluripotency. Thus, iPSCs can be differentiated into almost any cell type of the adult body, thereby allowing the study of rare and otherwise inaccessible cell types. Nevertheless, one of the major challenges in generating iPSCs from primates is obtaining the primary cells to be reprogrammed. To this end, I contributed to establishing a method that uses urine as a completely non-invasive cell source for deriving primate iPSCs. In the study, we demonstrate that urine-derived stem cells (UDSCs) can be isolated even from small volumes of primate urine and that the addition of a broad-spectrum antibacterial agent prevents contamination due to the unsterile collection from the zoo floor. Using this method, we were able to isolate and efficiently reprogram UDSCs from human, gorilla, and orangutan, thereby contributing to expanding the number of available cell lines from great apes for cross-species comparisons. To facilitate the use of this method by other researchers, I created a detailed protocol outlining the most crucial steps. These include the isolation and expansion of UDSCs from urine samples, as well as their reprogramming using a commercially available Sendai Virus (SeV) kit. In addition to the protocol, the manuscript also includes a video demonstrating how the most critical steps are performed in the lab. Moreover, we provide some best practices and troubleshooting guides that will enable a broader community to apply our method to their species of choice. In recent years, not only great apes but also Old world monkeys have been extensively studied for the generation of iPSCs. Although iPSC lines exist for most of these primates, the number of different individuals and clones per species is still rather limited. However, given the high variability in gene expression among individuals and clones of the same individual, it is crucial for comparative primate genomics to increase the sample size to as many individuals or clones as possible. In this context, I generated new iPSC lines from one rhesus macaque, one baboon, and two vervet monkey individuals. To do this, I reprogrammed skin fibroblasts on feeder cells using a footprint-free SeV reprogramming method and later gradually transitioned the iPSCs to feeder-free culture conditions. We further characterized all iPSC lines using human iPSC characterization standards to prove their pluripotency and validate their undifferentiated state. As all of these cell lines can be cultured under feeder-free conditions in commercially available medium, this enhances their value for cross-species comparisons. The invention of single-cell RNA-sequencing (scRNA-seq) technologies to measure gene expression, together with the possibility to generate iPSCs from primates that can be differentiated to nearly any desired cell type, have enabled the implementation of huge evolutionary studies. One example for such a study are scRNA-seq CRISPR inference (CRISPRi) screens to analyze molecular phenotypes across species. To this end, I contributed to generating human, gorilla and cynomolgus iPSCs that carry a doxycycline-inducible KRAB-dCas9 construct. The cell lines exhibited comparable down-regulation of target genes and comparable phenotypic effects in a scRNA-seq CRISPRi screen. Thereby, I helped to provide a valuable resource for performing CRISPRi in various primates, which can offer unique insights into human biology and evolution. In addition, comparative primate genomic studies can help to elucidated the evolutionary forces that drive the conservation or divergence of gene expression levels between species. In this regard, I used scRNA-seq to quantify gene expression during differentiation of our primate iPSCs towards embryoid bodies (EBs), a model for early development. I sampled single cells from ten different clones from four species at two time points, corresponding to differentiating and terminal cell types. This resulted in a comprehensive dataset comprising 85,000 cells, which we used to identify diverse cell types across all three germ layers. To accurately compare cell types across species, we developed a semi-automated computational pipeline combining classification and label transfer across clusters to identify orthologous cell types. This approach allowed us to investigate cross-species reproducibility of marker genes, revealing that human markers were less effective in macaques and vice versa. Furthermore, we found transcription factors to be the most conserved markers, highlighting their potential for cross-species studies. Overall, our study enhances the understanding of conserved and diverged molecular features in early primate development and we provide a well curated cell type reference for future in vitro studies. In summary, within my thesis I contributed to the field of comparative primate genomics, by assisting in the development of a method to generate iPSCs from urine as a non-invasive cell source, and by providing a detailed protocol on how to apply this method. Moreover, I generated iPSC lines from fibroblasts of rhesus, vervet and baboon and contributed to the implementation of CRISPRi screens from primates. Additionally, I demonstrated, through a comparative differentiation approach to EBs, how orthologous cell types can be identified between species, thereby establishing a foundation for the identification of human-specific adaptations in gene regulation.

• 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: 8. Mai 2025
• 1. Bericht­erstatter:in: Enard, Wolfgang