Abstract
The evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human (Homo sapiens) and six chimpanzee (Pan troglodytes) induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.
Highlights
Primates display a range of skeletal morphologies and susceptibilities to skeletal diseases, but the molecular basis of these phenotypic differences is unclear
To study primate skeletal gene expression patterns, we differentiated previously characterized and validated induced pluripotent stem cell lines (iPSCs) from six humans and six chimpanzees [34–39] through an intermediary mesenchymal stem cell (MSC) state, and subsequently into osteogenic cells, which are the primary cells in bone (Figs 1, S1, and S2, Methods, S1–S8 Tables, and S1 Text)
Genes previously identified in iPSCs as differentially expressed (DE) between species [36,48] are enriched among genes we identified as interspecific DE in pluripotent cells but not among the interspecific DE genes we identified in mesenchymal or osteogenic cells
Summary
The skeleton is a biologically and evolutionarily important organ system that consists of several tissues, including bone and cartilage. The skeletal system serves a variety of functions, most notably supporting body weight and facilitating locomotion. While these broad functions are conserved across vertebrates, different species have developed distinct skeletal morphologies, which enable differential use of skeletal elements. Clarifying the mechanisms that contribute to such complex traits will improve our understanding of skeletal evolution and development. As with all complex traits, skeletal traits are affected by both genetic [11–15] and environmental factors [16–20], and these effects may be mediated, at least in part, through gene expression changes. While the contribution of environmental factors to skeletal differences has been widely studied in the fields of comparative anatomy, forensics, and paleoanthropology, molecular variation in skeletal tissues is not well characterized, especially among primates
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