Efficient differentiation of human Fanconi anemia hematopoietic progenitor cells by inducible complementation reveals a role for p53 in regulation of self-renewal and differentiation

Fanconi anemia (FA) is a genetic disorder of DNA repair that manifests as bone marrow dysfunction typically occurring in childhood. The lack of human model systems has impeded progress in identifying effective therapies. To address this, efforts have focused on using directed differentiation of patient-derived human induced pluripotent stem cells (IPSCs) to hematopoietic stem and progenitor cells (HSPCs). However, the requirement for an intact FA DNA repair pathway for efficient reprogramming of patient cells to pluripotency requires genetic complementation of FA pathway defects to efficiently generate IPSCs, precluding derivation of FA pathway-deficient human HSPCs for study. To overcome this barrier, we employed a system of doxycycline-inducible complementation of FANCA deficient patient-derived IPS cells. Doxycycline exposure allowed for the robust maintenance of undifferentiated IPS cells in culture. Removal or retention of doxycycline exposure during directed differentiation of HSPCs allowed for the production of isogenic FANCA-deficient and FANCA-complemented human HSPCs. IPS-derived, FANCA-deficient HSPCs showed impaired response to genotoxic stress, proliferation, and survival compared to complemented HSPCs. Using these cells, we found that FANCA-deficient HSPCs showed expected impaired clonogenicity in methylcellulose culture and unexpected accelerated erythroid differentiation relative to complemented cells. Activation of p53-mediated p21 transactivation in FANCA-deficient HSPCs serves to drive accelerated erythropoiesis as the expense of clonogenicity. This study demonstrates the feasibility of inducible complementation in IPSCs as a method to generate isogenic FA-deficient and FA-complemented human HPSCs for disease modeling and uncovers a novel mechanism by which the FA pathway regulates the balance between stem cell maintenance and terminal differentiation.