Identification and comparison of orthologous cell types from primate embryoid bodies shows limits of marker gene transferability

The foundation of many human-specific traits is likely established during early embryonic development. To understand these molecular processes, we compared gene expression dynamics in embryoid bodies (EBs) differentiated from iPSCs of four primate species: human, orangutan, cynomolgus, and rhesus macaque. We performed scRNA-seq on ~ 85,000 cells after 8 and 16 days of EB differentiation, identifying 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. 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 using primates. Overall design: We generated embryoid bodies (EBs) from iPSCs of two human individuals, two orangutan clones, three cynomolgus clones from two individuals, and three rhesus clones. iPSCs were dissociated to clumps and kept in floating culture for 8 days to form EBs, followed by 8 days of attached culture in gelatin-coated wells. At day 8 and day 16 of the differentiation, we dissociated EBs into single-cells and equally pooled cells from all 4 species, before generating sequencing libraries. We performed the experiment in three independent replicates, generating a total of 4x lanes and 6x lanes of 10x Genomics scRNA-seq at day 8 and day 16, respectively.