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 we generated human IRF82/2 iPS cells and IRF82/2 embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we 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. We differentiated iPS cell and ES cell derived progenitors into CD1411 cross-presenting cDC1 and CD1c1 classical cDC2 and CD3031 plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF82/2 iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF82/2 DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. STEM CELLS 2017;35:898-908 SIGNIFICANCE STATEMENTPluripotent stem cells and CRISPR/Cas9n technology are particularly well suited for engineering cells to study the impact of specific factors on cell development, including antigen presenting dendritic cells (DC). So far, DC research was limited to primary cell samples obtained for example, from mice or men. In the mouse system, genetically modified DC are readily obtained by using transgenic, knockout, and knockin mice. In the human system studies with mutated DC relied on patients harboring specific mutations and there was a paucity of techniques for genetic engineering directly in human cells. Induced pluripotent stem (iPS) cell and CRISPR/ Cas9n technology now allow to overcome these limitations. Here, we generated interferon regulatory factor 8 (IRF8) knockout human iPS cells and ES cells, and IRF82/2 DC derived thereof. We show that IRF82/2 cells recapitulate the phenotype of individuals with an IRF8 loss of function mutation. Our IRF82/2 iPS cells and ES cells provide a platform to study IRF8 deficient DC subset specification and DC function independent of donor variation or availability. In summary, our IRF82/2 iPS cells and ES cells represent a valid and powerful model to elucidate mechanisms of human DC development and functional diversity.

Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells;Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells and dendritic cells (DC) derived thereof. This makes iPS cells particularly well suited for studying molecular mechanisms of diseases, drug discovery and regenerative therapy (Grskovic et al., 2011;Bellin et al., 2012;Robinton and Daley, 2012).DC are the major antigen presenting cells of the immune system and thus they are key players in modulating and directing immune responses (Merad et al., 2013). DC patrol peripheral and interface tissues (e.g., lung, intestine and skin) to detect invading pathogens, and upon activation they migrate to lymph nodes to activate and prime lymphocytes.DC comprise a phenotypically heterogeneous family with functionally specialized subsets (Schlitzer and Ginhoux, 2014). Generally, classical DC (cDC) and plasmacytoid DC (pDC) are distinguished, exhibiting a classical and plasma cell-like DC morphology, respectively. cDC recognize a multitude of pathogens and secrete proinflammatory cytokines upon activation, while pDC are specialized to detect intracellular pathogens and secrete type I interferons (Merad et al., 2013;Schlitzer and Ginhoux, 2014). cDC are further divided into cross-presenting cDC1 and conventional cDC2, in the human system referred to as CD141 + Clec9a + cDC1 and CD1c + CD14 -cDC2. Human pDC are characterized as CD303 +