Differential Single Cell Responses of Embryonic Stem Cells Versus Embryoid Bodies to Gravity Mechanostimulation

The forces generated by gravity have shaped life on Earth and impact gene expression and morphogenesis during early development. Conversely, disuse on Earth or during spaceflight, reduces normal mechanical loading of organisms, resulting in altered cell and tissue function. Although gravity mechanical loading in adult mammals is known to promote increased cell proliferation and differentiation, little is known about how distinct cell types respond to gravity mechanostimulation during early development. In this study we sought to understand, with single cell RNA-sequencing resolution, how a 60-min pulse of 50 g hypergravity (HG)/5 kPa hydrostatic pressure, influences transcriptomic regulation of developmental processes in the embryoid body (EB) model. Our study included both day-9 EBs and progenitor mouse embryonic stem cells (ESCs) with or without the HG pulse. Single cell t-distributed stochastic neighbor mapping shows limited transcriptome shifts in response to the HG pulse in either ESCs or EBs; this pulse however, induces greater positional shifts in EB mapping compared to ESCs, indicating the influence of mechanotransduction is more pronounced in later states of cell commitment within the developmental program. More specifically, HG resulted in upregulation of self-renewal and angiogenesis genes in ESCs, while in EBs, HG loading was associated with upregulation of Gene Ontology-pathways for multicellular development, mechanical signal transduction, and DNA damage repair. Cluster transcriptome analysis of the EBs show HG promotes maintenance of transitory cell phenotypes in early development; including EB cluster co-expression of markers for progenitor, post-implant epiblast, and primitive endoderm phenotypes with HG pulse but expression exclusivity in the non-pulsed clusters. Pseudotime analysis identified three branching cell types susceptible to HG induction of cell fate decisions. In totality, this study provides novel evidence that ESC maintenance and EB development can be regulated by gravity mechanostimulation and that stem cells committed to a differentiation program are more sensitive to gravity-induced changes to their transcriptome.