Unresolved s-phase delays in fa hsc lead to deficits in fetal liver hematopoietic stem cell expansion in fanconi ko cells

Fanconi Anemia (FA) is a heritable multisystem disorder and most patients present with hematopoietic failure in early school age. Mutations in genes encoding FA proteins lead to deficits in DNA interstrand crosslink (ICL)-repair. More broadly, FA proteins play a role in guarding the integrity of the hematopoietic stem cell (HSC) pool from cellular stress. The physiologic role of FA proteins during HSC development, when the HSC pool is first formed, however, remains unclear. In mice, the critical phase of HSC pool expansion begins with HSC migration to the fetal liver (FL) and we previously demonstrated the reduction of HSC numbers during midgestation by embryonic day (E)14.5 in both Fancc-/- and Fancd2-/- mouse models (Yoon et al., Stem Cell Reports 2016). To begin to understand how FA-deficiency constrains fetal HSC development, we tracked HSC subpopulations in Fancd2-/- mice throughout ontogeny and found that a proportionally intact HSC pool at E12.5 gave way to significant quantitative deficits by E14.5. By contrast, the subsequent migration of cells to the fetal bone marrow and the canonical molecular switch toward a more quiescent adult HSC phenotype involving Lin28 and HMGA2 were intact. In focusing on E14.5 HSC, we next determined that the deficits were not reflective of apoptotic loss, but rather a lack of HSC and progenitor cell expansion. Fancd2-/- HSC were less quiescent based on Ki67 staining, and in vivo cell cycle measurements using sequential EdU /BrdU uptake instead revealed a significant delay in S-phase entry with subsequent accumulation in G2/M. We propose that FA proteins are required to resolve S-phase replication stress during FL-HSC expansion, and the complementary molecular and functional profiling of the HSC population during S-phase transit is currently underway. Understanding fetal deficits in FA will help resolve the disease pathophysiology and may yield insight into FA-specific obstacles to expansion and differentiation of reprogrammed pluripotent cells for regenerative therapies.