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Chronic mucocutaneous candidiasis (CMC) is a disease that is characterized by susceptibility to chronic or recurrent infections with Candida spp. due to mutations affecting mainly the IL-17 signaling of T-Cells. The most common etiologies of CMC are gain-of-function (GOF) mutations in the STAT1 gene. In this paper we report the generation of a hiPSC line from a patient suffering from CMC due to a heterozygous GOF STAT1 p.R274Q mutation which can be used for disease modeling purposes.

Summary Interleukin-3 (IL-3) is capable of supporting the proliferation of a broad range of hematopoietic cell types, whereas granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) represent critical cytokines in myeloid differentiation. When this was investigated in a pluripotent-stem-cell-based hematopoietic differentiation model, IL-3/G-CSF or IL-3/M-CSF exposure resulted in the continuous generation of myeloid cells from an intermediate myeloid-cell-forming complex containing CD34+ clonogenic progenitor cells for more than 2 months. Whereas IL-3/G-CSF directed differentiation toward CD45+CD11b+CD15+CD16+CD66b+ granulocytic cells of various differentiation stages up to a segmented morphology displaying the capacity of cytokine-directed migration, respiratory burst response, and neutrophil-extracellular-trap formation, exposure to IL-3/M-CSF resulted in CD45+CD11b+CD14+CD163+CD68+ monocyte/macrophage-type cells capable of phagocytosis and cytokine secretion. Hence, we show here that myeloid specification of human pluripotent stem cells by IL-3/G-CSF or IL-3/M-CSF allows for prolonged and large-scale production of myeloid cells, and thus is suited for cell-fate and disease-modeling studies as well as gene- and cell-therapy applications., Graphical Abstract, Highlights • Myeloid specification of human PSCs by IL-3-/M-CSF, G-CSF, or GM-CSF • Large-scale and continuous generation of M2-MΦ or granulocytes by M-CSF or G-CSF • Functional iPSC-derived macrophages or granulocytes similar to in-vivo-derived cells, In this article, Moritz and colleagues show that IL-3 in combination with M-CSF/G-CSF or GM-CSF is able to instruct human pluripotent stem cells toward a myeloid lineage. The use of IL-3/G-CSF or IL-3/M-CSF allows for prolonged and large-scale production of human granulocytes or macrophages, respectively, that demonstrate similar functionalities compared with their in-vivo-derived counterparts.

Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood.To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages.Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays.Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA.These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific mAb, inhibits the release of human immunodeficiency virus (HIV-1), and canine distemper virus (CDV)-, but not measles virus (MV)-induced cell-cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop (LEL) of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas. High resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins β1-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the fusion machinery to cell contact areas.

Hematopoietic in-vitro-differentiation of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) holds great promise for disease modeling, drug testing, as well as cell- and gene-therapy strategies. Although hematopoietic differentiation of PSC has been shown to be feasible, the majority of current protocols apply a large number of different cytokines to direct differentiation. In this line, priming the differentiation process by a multitude of cytokines may alter the endogenous hematopoietic differentiation program of PSCs, thus hampering the usefulness of such protocols to gain insight into physiologic human hematopoietic development. To overcome this problem we have investigated the hematopoietic differentiation potential of human PSC, based on minimal cytokine application. Given the emerging role of IL3 as a critical factor in adult hematopoiesis and the pivotal role of M-CSF and G-CSF for terminal myeloid differentiation, we here employed IL3 in combination with either M-CSF or G-CSF on hematopoietic development. To prove our concept, human CD34+ cell-derived iPSC clones were subjected to an embryoid body (EB)-based myeloid differentiation protocol employing cytokines from day 5 onwards and yielding so-called “myeloid cell forming complexes” (MCFCs) within 7-10 days. Analysis of MCFC within 10 days of differentiation revealed expression of MIXL1, KDR1, GATA2, and RUNX1, as well as an early CD34+/CD45- population undergoing transition to a CD34+/CD45+ and thereafter CD34-/CD45+ phenotype. The hypothesis of a primitive hematopoietic cell arising from a population with dual (hematopoietic and vascular epithelial) potential was supported by co-staining of these populations with VE-cadherin (CD144). Here primarily the CD34+/CD45+/CD144- cells were capable of colony formation in vitro. Differentiation of PSC for more than 15-days resulted in the continuous shedding of hematopoietic cells from MCFCs and further differentiation along the IL3/M-CSF let to the generation of >99% pure monocytes/ macrophages (iPSC-MΦ), while IL3/G-CSF promoted granulopoiesis (iPSC-gra, purity >95%). Of note, hardly any CD34+ cells were detected among MCFC-shedded cells for the IL3/M-CSF as well as the IL3/G-CSF combination. In contrast, differentiation in IL3 only resulted in 10% MCFC-derived CD34+ cells, an observation further confirmed by a 10-times increased clonogenicity for cells shedded from MCFC exposed to IL3 only when compared to IL3/G-CSF or IL3/M-CSF cultures. Furthermore, cells cultured in IL3 maintained the capacity of subsequent M-CSF-driven terminal differentiation, whereas no suspension cells were observed following differentiation of PSC with G-CSF alone. Most strikingly, IL3/M-CSF or IL3/G-CSF cultures generated iPSC-MΦ or iPSC-gra from day 14-15 onwards over a period of 3-5 months at a quantity of 0.4-2.0 x 106 cells/week (cumulative 0.8-4.0 x 107 cells) per 3.5 cm well. For IL3/M-CSF cultures detailed characterization of mature myeloid cells demonstrated a typical MΦ-morphology of iPSC-MΦ by cytospins and a surface-marker profile of CD45, CD11b, CD14, CD163, and CD68. In addition, iPSC-MΦ had the ability to phagocytose latex-coated beads similar to peripheral blood (PB)-MΦ polarized to M2 and upon LPS stimulation secreted MCP1, IL6, IL8, and IL10, whereas IFNy, IL1b, IL4, IL5, and IL12 were absent. iPSC-gra showed surface expression of CD45, CD11b, CD16, CD15, CD66b and a differential count containing pro-myelocyte (3%), myelocyte (5%), meta-myelocyte (30%), bands (22%), eosinophils (2%), basophils (1%), and segmented-neutrophils (37%) . Moreover, iPSC-gra were able to migrate towards an IL8 or fMLP gradient, formed neutrophil extracellular traps, and up-regulated NADPH activity and ROS production upon PMA stimulation to a similar degree as PB granulocytes. In summary, we here present an in vitro differentiation protocols for human iPSC requiring minimal cytokine stimulation, which appears highly suited to model human hematopoietic development or generate cells for gene and cell-replacement strategies. We further provide evidence that IL3 constitutes a key cytokine driving the early hematopoietic specification of human PSC, whereas M-CSF and G-CSF function primarily as downstream “supporter” cytokines regulating the terminal differentiation towards macrophages and granulocytes, respectively. Disclosures No relevant conflicts of interest to declare.