Specific cell states underlie complex tissue regeneration in spiny mice

SUMMARY STATEMENTComparing regenerative vs. fibrotic healing, we identify injury-induced cell states associated with persistent cell cycle progression and complex tissue regeneration in mammals. Cell proliferation is an elemental feature of epimorphic regeneration in vertebrate taxa. We previously reported that in contrast to fibrotic repair observed in laboratory mouse (Mus) strains, highly regenerative spiny mice (Acomys spp.) exhibit cell cycle progression and cell proliferation to faithfully replace missing tissue. However, little is known about proliferation dynamics, and specific cell types and states that may contribute to complex tissue regeneration in mammals. Using temporal pulse-chase experiments, we show that stromal cells in Acomys dimidiatus rapidly re-enter the cell cycle in response to injury and maintain tight spatiotemporal control of cell cycle progression to restrict the proliferative population to a distal area relative to the injury. Conversely, Mus stromal cells incorporate thymidine analogs without cell division supporting an S-phase arrest after D10. Deploying immunostaining and scRNA-seq, we identify several key cell types (CRABP1+, SMA+) differentially associated with regenerating versus scar tissue. Importantly, our single cell data revealed distinct gene expression profiles for cross-species stromal cell types, identifying cell states specific for regenerative or fibrotic healing. While CRABP1+ fibroblasts are enriched in Acomys ears before and after injury, similar fibroblasts enriched in young, postnatal Mus ears remain unable to promote regeneration. Our data underscore the finely regulated dynamics of proliferating cells during regeneration and emphasize that regeneration depends on multiple factors including the presence of specific cell types and the ability of cells to acquire specific states. Key Conclusions-Differentiated cells in Acomys, Mus and Danio re-enter the cell cycle in response to injury, while homeostatic cycling cells contribute to blastema formation in Ambystoma -Pulse-chase thymidine analog labeling shows tight spatiotemporal control of proliferating stromal cells during regeneration in Acomys. -Following injury, CRABP1 and SMA are expressed in distinct stromal cell populations in Acomys but are co-expressed in Mus stromal cell populations. -Species-specific cell states underlie regenerative and fibrotic repair -CRABP1+ cells are lost during embryonic development in Mus ear pinna but are retained in Acomys to adulthood. -Young neonatal Mus with abundant CRABP1+ cells still fail to execute regenerative healing