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

Abstract

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.

Progress and Future Prospectives in Skin-on-Chip Development with Emphasis on the use of Different Cell Types and Technical Challenges

“…Langerhans cells) is less clear. However during the last years it is shown that iPSC can be differentiated into several types of immune cells; T-lymphocytes, macrophages, granulocytes, erythrocytes and dendritic cells [59–62]. More research is needed to look into the differentiation of iPSC towards skin specific Langerhans cells.…”

Induced pluripotent stem cells reprogrammed from primary dendritic cells provide an abundant source of immunostimulatory dendritic cells for use in immunotherapy

“…The importance of DCs in the initiation of immune responses among antigen-naïve T cells has fueled interest in iPSCs as a renewable and scalable source of rare DC subsets with desirable properties not normally vested in moDCs, 3,4 the population most frequently used in clinical trials. 2 ( Figure 1B,D), a phenotype equally evident among ipDCs of mouse 37 and human origin.…”

“…2 Although such trials have demonstrated only limited efficacy, induced pluripotent stem cells (iPSCs) may provide an unlimited source of previously inaccessible subsets of DCs, such as plasmacytoid DCs 3 and CD141 + DCs displaying superior capacity for the cross-presentation of TAAs. 4 Unlike moDCs, such a novel source is amenable to scale-up, quality control and genome editing, 3,5 making it attractive for downstream therapeutic applications. 6 Nevertheless, iPSC-derived DCs (ipDCs) appear arrested at a stage of development more reminiscent of a fetal rather than an adult phenotype, a finding common to many cell types differentiated from a pluripotent source, including hepatocytes, 7 cardiomyocytes, 8 and forebrain neurons.…”

Nintedanib Targets KIT D816V Neoplastic Cells Derived from Induced Pluripotent Stem cells of Systemic Mastocytosis

“…[25][26][27] Additionally, iPSCs and embryonic stem cells (ESCs) are readily subjected to genome engineering by CRISPR/Cas9 to precisely introduce or repair oncogenic mutations. 26,28,29 We report here on the generation of KIT D816V iPSCs from SM patients. SM-derived KIT D816V iPSCs differentiated into hematopoietic progenitor cells (HPCs) and MCs with patient-specific features.…”