Developmental Programming of Bone Marrow Failure in a Murine Model of Fanconi Anemia

Abstract 1334 Bone marrow failure is the most common cause of morbidity and mortality from Fanconi anemia (FA), a recessively inherited disorder resulting from mutations in one of 15 known genes that cooperate in a DNA repair pathway. The underlying etiology is thought to reflect an accelerated postnatal exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool. However, laboratory evidence of compromised hematopoietic function in patients generally precedes symptoms of cytopenia, and several other mesodermal-derived organ systems show defects with prenatal onset, including the skeletal system, heart, kidneys, and others. Further, recent experimental evidence in human embryonic stem cell lines suggested that RNA interference-mediated knock-down of FANCD2 and FANCA impairs development of hematopoietic cells. The fetal liver provides a unique microenvironment for development of definitive hematopoietic function and serves as a site of massive HSPC expansion. However, neither the potential developmental onset of bone marrow failure or non-stem cell-autonomous contributions in FA have been systematically clarified to-date. We relied on a murine model of FA with a transgenic disruption of Fancc to test the hypothesis that hematopoietic failure for this disease may have developmental origins. Although spontaneous bone marrow failure does not occur in this FA mouse model, animals recapitulate impaired repopulating ability, characteristic cell cycle abnormalities, and impaired cytokine responses. To determine whether number and function of fetal liver (FL) HSPCs affect postnatal hematopoietic function in FA mice, we plated unfractionated cells from 14.5 days post coitum (dpc) FL in methylcellulose and undertook a chronologic assessment of postnatal bone marrow progenitor clonogenicity. These studies showed that, compared with wild-type (wt) littermates, Fancc −/− animals demonstrate a progressive deficiency in progenitor number and function that increases with age, suggesting that HSPC attrition is developmentally programmed. Fancc −/− fetal mice revealed a 10% reduction in body mass and 33% lower total liver cell count compared with wt littermates. Cytogenetic analysis shows Fancc −/− FL cells exhibit mitomycin-c hypersensitivity characteristic of FA, with increased chromosomal breakage and radial formation. Livers of 14.5±.5 dpc Fancc −/− fetuses contain approximately 43% fewer c-Kit + Sca-1 + progenitor-enriched cells, compared with wt littermates. Cell cycle status of fetal livers revealed a characteristically increased proportion of Fancc −/− fetal liver progenitor-enriched (c-Kit + Sca-1 + ) cells in G2-M phase of cell cycle, compared to wt littermate liver. When plated in methylcellulose assays, Fancc −/− FL showed an approximately 20% reduction in progenitor frequency, compared to wt littermates, and plating in mitomycin-c resulted in outgrowth of fewer colonies. Further, studies to determine the relative in vivo repopulating cell frequency were performed using CD45-isotype mismatched, submyeloablatively irradiated (750 cGy) animals. Recipients receiving unfractionated 14.5±.5 dpc Fancc −/− liver cells showed a slight, but consistent reduction in peripheral blood chimerism at serial timepoints (1–5 months) and bone marrow chimerism at sacrifice. We also found a 21% reduction in total Fancc −/− clonogenic bone marrow progenitor frequency by methylcellulose assay in primary recipients, compared to wt-transplanted controls. In sum, these studies suggest a developmental origin of hematopoietic failure in FA, whereby the prenatal onset potentially contributes to disease progression. Results contrast with a conventional model of postnatal stem cell attrition and may impact the development of preemptive therapies for FA patients. Disclosures: No relevant conflicts of interest to declare.