Allelic bias and genetic mapping of differentiation potential revealed by single-cell transcriptomics in recombinant hybrid mouse embryonic stem cells

Abstract

Self-renewal and pluripotency are twin hallmarks of embryonic stem cells. Every cell in an early embryo must balance between differentiation and proliferation, often involving irreversible fate decisions. However, there are very few studies that directly investigate how these gene pathways evolve between species, because crosses between species usually fail. Here, we combine in vitro recombination (IVR) and single-cell transcriptomics in F1 hybrid embryonic stem (ES) cells to determine the genes and loci influencing the decision between renewal vs. differentiation. Through RNAi suppression of Blm helicase, we effectively produced recombinant ES cells between the laboratory C57BL/6N mouse and Mus spretus. We then induced differentiation and obtained single-cell transcriptomes on ~20,000 IVR cells. Using reference scRNAseq datasets from naïve and differentiated non-recombinant cells, we show that in vitro recombination boosted genotypic and expression diversity. We found that the genetic reshuffling under IVR produced cells that can sustain robust Nanog stemness marker expression despite differentiating treatments. Moreover, we observed that allele-specific expression increased with differentiation, possibly from recombinant cells. We also found that SPRET alleles were enriched in highly differentiated cells. Our work shows that by combining in vitro recombination and single-cell assays, it is now possible to directly map genes controlling otherwise inaccessible cellular phenotypes across species