Modeling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency.To study IRF8 in human hematopoiesis I generated human IRF8–/– iPS cells and IRF8–/– embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. I differentiated iPS and ES cells into primitive hematopoietic progenitors with colony forming and short-term engraftment potential. Upon induction of hematopoietic differentiation, I demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. I differentiated iPS and ES cell derived progenitors into the classical DC (cDC) subsets cDC1 and cDC2 and plasmacytoid DC (pDC). These subsets presented similar surface marker profiles than their in vivo equivalents. I found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. However, in an unrestricted differentiation regimen, IRF8–/– iPS and ES cells demonstrated disturbed cDC2 and monocyte development, while exhibiting a clear bias toward neutrophils and eosinophils. Moreover, IRF8–/– DC showed reduced MHC class II expression and were impaired in cytokine responses, chemotactic migration and antigen presentation. Taken together, I engineered a human IRF8 knockout model that suggests differences between mice and men regarding the role of IRF8 during cDC2 and eosinophil development. Yet, this model recapitulates the clinical phenotype of individuals carrying IRF8 loss-of-function mutations. Therefore, the iPS and ES cell based IRF8 knockout model represents a valid tool to study molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC independent of donor variation or availability