Abstract
Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee autotetraploids and allotetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or autotetraploid iPSCs. Analysis of gene expression patterns in interspecific allotetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allotetraploid cells. We successfully generated derivative cells with nested deletions or interspecific recombination on the X chromosome. These studies confirm an important role for the X chromosome in trans regulation of expression differences between species and illustrate the potential of this system for more detailed cis and trans mapping of the molecular basis of human and chimpanzee evolution.
Highlights
Humans have had a long-standing interest in the features that distinguish our species from other animals [1, 2]
We generated two human autotetraploid lines (“H1H1” lines, from human induced pluripotent stem cells (iPSCs) line H23555 [H1]); five chimpanzee autotetraploid lines (“C1C1” lines from chimpanzee iPSC line C3649 [C1]); and 22 human–chimpanzee allotetraploid lines from different fusion events including 12 “H1C1” lines derived from H1 and C1 and 10 “H2C2” lines derived from human iPSC line H20961 (H2) and chimpanzee iPSC line C8861 (C2) (Dataset S1)
Tetraploid iPSCs were larger than diploid cells but had normal morphology and could be routinely propagated under the same conditions as diploid iPSCs (SI Appendix, Fig. S1)
Summary
Humans have had a long-standing interest in the features that distinguish our species from other animals [1, 2]. We develop a genetic approach in fused stem cell lines that makes it possible to map human–chimpanzee molecular and cellular differences to specific regions of the genome. We illustrate this approach by mapping chromosome regions responsible for species-specific gene expression differences in fused tetraploid cells. This approach is general, and could be used in the future to map the genomic changes that control many other human– chimpanzee differences in various cell types or organoids in vitro
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