Supplemental Digital Content is Available in the Text. Human sodium channel Na V 1.7 in induced pluripotent stem cell–derived sensory neurons sets the action potential threshold but does not support subthreshold depolarizations.

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 (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs – this is of relevance for standardization and automation of cell culture procedures.

The KIT D816V mutation is found in more than 80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. KIT D816V therefore represents a prime therapeutic target for SM. Here we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM (ASM) and mast cell leukemia (MCL) to develop a patient-specific SM disease model for mechanistic and drug discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, an FDA approved angiokinase inhibitor that targets VEGFR, PDGFR and FGFR, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the ATP binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V targeted therapy of advanced SM.

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Graphical AbstractPatient-specific iPS cells with JAK2 V617F mutation differentiate into DC3 dendritic cells, megakaryocytes and red blood cells.

Abstract:The aim of the current study was to estimate total intake and dietary sources of eicosapentaenoic acid (EPA), docosapentanoic (DPA), and docosahexaenoic acid (DHA) and compare DHA intakes with the recommended intakes in a cohort of pregnant and lactating women. Twenty-four-hour dietary recalls and supplement intake questionnaires were collected from 600 women in the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort at each trimester of pregnancy and 3 months postpartum. Dietary intake was estimated in 2 ways: by using a commercial software program and by using a database created for APrON. Only 27% of women during pregnancy and 25% at 3 months postpartum met the current European Union (EU) consensus recommendation for DHA. Seafood, fish, and seaweed products contributed to 79% of overall n-3 long-chain polyunsaturated fatty acids intake from foods, with the majority from salmon. The estimated intake of DHA and EPA was similar between databases, but the estimated DPA intake was 20%-30% higher using the comprehensive database built for this study. Women who took a supplement containing DHA were 10.6 and 11.1 times more likely to meet the current EU consensus recommendation for pregnancy (95% confidence interval (CI): 6.952-16.07; P < 0.001) and postpartum (95% CI: 6.803-18.14; P < 0.001), respectively. Our results suggest that the majority of women in the cohort were not meeting the EU recommendation for DHA during pregnancy and lactation, but taking a supplement significantly improved the likelihood that they would meet recommendations.

BackgroundDifferentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modeling, drug screening, and cellular therapy. Various differentiation protocols have been established to generate iPSC-derived HPCs (iHPCs) that resemble their primary counterparts in morphology and immunophenotype, whereas a systematic epigenetic comparison was yet elusive.ResultsIn this study, we compared genome-wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. After 20 days of in vitro differentiation, cells revealed typical hematopoietic morphology, CD45 expression, and colony-forming unit (CFU) potential. DNAm changes were particularly observed in genes that are associated with hematopoietic differentiation. On the other hand, the epigenetic profiles of iHPCs remained overall distinct from natural HPCs. Furthermore, we analyzed if additional co-culture for 2 weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward the hematopoietic lineage. Proliferation of iHPCs and maintenance of CFU potential was enhanced upon co-culture. However, DNAm profiles support the notion that additional culture expansion with stromal support did not increase epigenetic maturation of iHPCs toward natural HPCs.ConclusionDifferentiation of iPSCs toward the hematopoietic lineage remains epigenetically incomplete. These results substantiate the need to elaborate advanced differentiation regimen while DNAm profiles provide a suitable measure to track this process.Electronic supplementary materialThe online version of this article (10.1186/s13148-019-0617-1) contains supplementary material, which is available to authorized users.