NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes.

Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.

A 3D iPSC-differentiation model identifies interleukin-3 as a regulator of early human hematopoietic specification.

Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional, organoid-like differentiation system (hemanoid) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show, that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43-/CD45- hemato-endothelial progenitor cells (HEPs) forming organized, vasculature-like structures and giving rise to CD34low/CD144-/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEPs is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signalling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of hemato-endothelial progenitor cells and highlight the potential of a hemanoid-based model to study human hematopoietic development.

Ex vivo Generation of Genetically Modified Macrophages from Human Induced Pluripotent Stem Cells.

BACKGROUND: Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), have the capacity to differentiate towards all three germ layers and have been highlighted as an attractive cell source for the field of regenerative medicine. Thus, stable expression of therapeutic transgenes in iPSCs, as well as thereof derived progeny of hematopoietic lineage, may lay the foundation for innovative cell replacement therapies. METHODS: We have utilized human iPSC lines genetically modified by lentiviral vector technology or targeted integration of reporter genes to evaluate transgene expression during hematopoietic specification and differentiation towards macrophages. RESULTS: Use of lentiviral vectors equipped with an ubiquitous chromatin opening element (CBX3-UCOE) as well as zinc finger nuclease-mediated targeting of an expression cassette into the human adeno-associated virus integration site 1 (AAVS1) safe harbor resulted in stable transgene expression in iPSCs. When iPSCs were differentiated along the myeloid pathway into macrophages, both strategies yielded sustained transgene expression during the hematopoietic specification process including mature CD14+ and CD11b+ macrophages. CONCLUSION: Combination of human iPSC technology with either lentiviral vector technology or designer nuclease-based genome editing allows for the generation of transgenic iPSC-derived macrophages with stable transgene expression which may be useful for novel cell and gene replacement therapies.

Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

Gene correction of human induced pluripotent stem cells repairs the cellular phenotype in pulmonary alveolar proteinosis.

RATIONALE: 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. OBJECTIVES: 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. METHODS: 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. MEASUREMENTS AND MAIN RESULTS: 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. CONCLUSIONS: 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.

An Improved Protocol for Targeted Differentiation of Primed Human Induced Pluripotent Stem Cells into HLA-G-Expressing Trophoblasts to Enable the Modeling of Placenta-Related Disorders.

Abnormalities at any stage of trophoblast development may result in pregnancy-related complications. Many of these adverse outcomes are discovered later in pregnancy, but the underlying pathomechanisms are constituted during the first trimester. Acquiring developmentally relevant material to elucidate the disease mechanisms is difficult. Human pluripotent stem cell (hPSC) technology can provide a renewable source of relevant cells. BMP4, A83-01, and PD173074 (BAP) treatment drives trophoblast commitment of hPSCs toward syncytiotrophoblast (STB), but lacks extravillous trophoblast (EVT) cells. EVTs mediate key functions during placentation, remodeling of uterine spiral arteries, and maintenance of immunological tolerance. We optimized the protocol for a more efficient generation of HLA-Gpos EVT-like trophoblasts from primed hiPSCs. Increasing the concentrations of A83-01 and PD173074, while decreasing bulk cell density resulted in an increase in HLA-G of up to 71%. Gene expression profiling supports the advancements of our treatment regarding the generation of trophoblast cells. The reported differentiation protocol will allow for an on-demand access to human trophoblast cells enriched for HLA-Gpos EVT-like cells, allowing for the elucidation of placenta-related disorders and investigating the immunological tolerance toward the fetus, overcoming the difficulties in obtaining primary EVTs without the need for a complex differentiation pathway via naïve pluripotent or trophoblast stem cells.

Generation of two human NRF2 knockout iPSC clones using CRISPR/Cas9 editing.

The nuclear factor erythroid 2-related factor 2 (NFE2L2, known as NRF2) regulates the expression of antioxidative and anti-inflammatory proteins. In order to investigate its impact during viral infections and testing of antiviral compounds, we applied CRISPR/Cas9 editing to eliminate NRF2 in the human iPS cell line MHHi001-A and generated two NRF2 knockout iPSC clones MHHi001-A-6 and MHHi001-A-7. After differentiation into epithelia or endothelial cells, these cells are useful tools to examine the antiviral effects of activators of the NRF2 signaling pathway.

Generation of human induced pluripotent stem cell lines encoding for genetically encoded calcium indicators RCaMP1h and GCaMP6f.

Calcium plays a key role in cardiomyocytes (CMs) for the translation of the electrical impulse of an action potential into contraction forces. A rapid, not-invasive fluorescence imaging technology allows for the monitoring of calcium transients in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) to investigate the cardiac electrophysiology in vitro and after cell transplantation in vivo. The genetically encoded calcium indicators (GECIs) GCaMP6f or RCaMP1h were successfully transfected in the previously established hiPSC line MHHi001-A, together with a cardiac specific antibiotic selection cassette facilitating the monitoring of the calcium handling in highly pure populations of hiPSC-CMs.

Generation of two TMEM16A knockout iPSC clones each from a healthy human iPSC line, from a Cystic Fibrosis patient specific line with p.Phe508del mutation and from the gene corrected iPSC line.

The Transmembrane member 16A (TMEM16A), also known as anoctamin-1 (ANO1), is a calcium-activated chloride channel present in the airway epithelium. It is known to be involved in the apical chloride secretion indicating that TMEM16A could be addressed for the treatment of chloride secretion defects like in Cystic- Fibrosis (CF). In this paper we generated knockout cell lines using CRISPR/Cas9-mediated ablation in a healthy human iPSC line (MHHi001-A), in a CF patient iPSC line (MHHi002-A) and in its corrected counterpart (MHHi002-A-1). These lines can be used for gaining information about the role of TMEM16A for mucus secretion and/or production and evaluating its therapeutic potential.

Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies.

Drug-inducible suicide systems may help to minimize risks of human induced pluripotent stem cell (hiPSC) therapies. Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed. We have introduced a drug-inducible Caspase 9 suicide system (iCASP9) into the AAVS1 safe-harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. After transplantation into mice, drug treatment generally led to rapid elimination of teratomas, but single animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ∼ 3 × 10-8. Besides transgene elimination, presumably via loss of heterozygosity (LoH), silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, no escapees from biallelic iCASP9 cells were observed after treatment of up to 0.8 billion hiPSCs. The highly increased safety level provided by biallelic integration of the iCASP9 system may substantially contribute to the safety level of iPSC-based therapies.

ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives.

BACKGROUND: Congenital ISG15 deficiency is a rare autoinflammatory disorder that is driven by chronically elevated systemic interferon levels and predominantly affects central nervous system and skin. METHODS AND RESULTS: We have developed induced pluripotent stem cell-derived macrophages and endothelial cells as a model to study the cellular phenotype of ISG15 deficiency and identify novel treatments. ISG15-/- macrophages exhibited the expected hyperinflammatory responses, but normal phagocytic function. In addition, they displayed a multifaceted pathological phenotype featuring increased apoptosis/pyroptosis, oxidative stress, glycolysis, and acylcarnitine levels, but decreased glutamine uptake, BCAT1 expression, branched chain amino acid catabolism, oxidative phosphorylation, ß-oxidation, and NAD(P)H-dependent oxidoreductase activity. Furthermore, expression of genes involved in mitochondrial biogenesis and respiratory chain complexes II-V was diminished in ISG15-/- cells. Defective mitochondrial respiration was restored by transduction with wild-type ISG15, but only partially by a conjugation-deficient variant, suggesting that some ISG15 functions in mitochondrial respiration require ISGylation to cellular targets. Treatment with itaconate, dimethyl-itaconate, 4-octyl-itaconate, and the JAK1/2 inhibitor ruxolitinib ameliorated increased inflammation, propensity for cell death, and oxidative stress. Furthermore, the treatments greatly improved mitochondria-related gene expression, BCAT1 levels, redox balance, and intracellular and extracellular ATP levels. However, efficacy differed among the compounds according to read-out and cell type, suggesting that their effects on cellular targets are not identical. Indeed, only itaconates increased expression of anti-oxidant genes NFE2L2, HMOX1, and GPX7, and dimethyl-itaconate improved redox balance the most. Even though itaconate treatments normalized the elevated expression of interferon-stimulated genes, ISG15-/- macrophages maintained their reduced susceptibility to influenza virus infection. CONCLUSIONS: These findings expand the cellular phenotype of human ISG15 deficiency and reveal the importance of ISG15 for regulating oxidative stress, branched chain amino acid metabolism, and mitochondrial function in humans. The results validate ruxolitinib as treatment for ISG15 deficiency and suggest itaconate-based medications as additional therapeutics for this rare disorder.

Congenital deficiency reveals critical role of ISG15 in skin homeostasis.

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-ß1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.

Generation of pulmonary arterial hypertension patient-specific induced pluripotent stem cell lines from three unrelated patients with a heterozygous missense mutation in exon 12, a heterozygous in-frame deletion in exon 3 and a missense mutation in exon 11 of the BMPR2 gene.

Loss-of-function mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene are common in heritable or idiopathic pulmonary arterial hypertension (PAH), and can result in functional impairment of both endothelial and vascular smooth muscle cells. Here, we report 3 PAH patient-specific induced pluripotent stem cells (iPSC) lines from 3 unrelated patients harbouring different mutations in the BMPR2 gene: a heterozygous missense mutation in exon 12, a heterozygous frame shift deletion in exon 3, and a heterozygous missense mutation in exon 11. These cell lines will serve as a valuable resource to model PAH in vitro.

Generation of an induced pluripotent stem cell line (MHHi018-A) from a patient with Cystic Fibrosis carrying p.Asn1303Lys (N1303K) mutation.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene which encodes for a chloride ion channel regulating the balance of salt and water across secretory epithelia. Here we generated an iPSC line from a CF patient homozygous for the p.Asn1303Lys mutation, a Class II folding defect mutation. This iPSC line provides a useful resource for disease modeling and to investigate the pharmacological response to CFTR modulators in iPSC derived epithelia.

Human STAT1 gain-of-function iPSC line from a patient suffering from chronic mucocutaneous candidiasis.

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.

Generation of a NKX2.1 - p63 double transgenic knock-in reporter cell line from human induced pluripotent stem cells (MHHi006-A-4).

Tumor protein p63 (p63) encodes for a transcription factor of the p53 family and is a marker for respiratory basal cells. Based on a NKX2.1 knock-in reporter cell line from human induced pluripotent stem cells (hiPSCs) (MHHi06-A-2) we established a NKX2.1/p63 double transgenic knock-in reporter cell line using TALEN technology. The reporter enables the optimization and monitoring of hiPSC differentiation towards NKX2.1/p63 double positive cells as well as enrichment for single or double positive cells.

Generation of three induced pluripotent stem cell lines (MHHi012-A, MHHi013-A, MHHi014-A) from a family with Loeys-Dietz syndrome carrying a heterozygous p.M253I (c.759G>A) mutation in the TGFBR1 gene.

Loeys-Dietz syndrome (LDS) is a rare connective tissue disorder characterized by a genetic predisposition for thoracic aortic aneurysm and dissection. Despite heterozygous loss-of-function mutations in genes for ligand, receptor, or downstream mediators of the transforming growth factor ß (TGFß) pathway, LDS is associated with a signature of high TGFß signaling. We generated induced pluripotent stem cell (iPSC) lines from three adult LDS-patients (two male, one female) of a family with a heterozygous point mutation in exon 4 of the TGFß-receptor1 (TGFBR1) gene (p.M253I; c.759G>A). The lines offer a valuable resource for modeling the pathophysiology of genetically mediated aortic disease.

Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions.

Most monogenic disorders have a primary clinical presentation. Inherited ISG15 deficiency, however, has manifested with two distinct presentations to date: susceptibility to mycobacterial disease and intracranial calcifications from hypomorphic interferon-II (IFN-II) production and excessive IFN-I response, respectively. Accordingly, these patients were managed for their infectious and neurologic complications. Herein, we describe five new patients with six novel ISG15 mutations presenting with skin lesions who were managed for dermatologic disease. Cellularly, we denote striking specificity to the IFN-I response, which was previously assumed to be universal. In peripheral blood, myeloid cells display the most robust IFN-I signatures. In the affected skin, IFN-I signaling is observed in the keratinocytes of the epidermis, endothelia, and the monocytes and macrophages of the dermis. These findings define the specific cells causing circulating and dermatologic inflammation and expand the clinical spectrum of ISG15 deficiency to dermatologic presentations as a third phenotype co-dominant to the infectious and neurologic manifestations.

Generation of a NKX2.1 knock-in reporter cell line from human induced pluripotent stem cells (MHHi006-A-2).

NK homeobox 1 (NKX2.1; also known as thyroid transcription factor 1, TTF-1) encodes for a transcription factor involved in the development of thyroid, lung and brain. Here, we established a NKX2.1 knock-in reporter cell line from human induced pluripotent stem cells (iPSCs) using TALEN technology. The reporter enables the optimization and monitoring of the differentiation of pluripotent stem cells (PSCs) into NKX2.1 expressing cells on a single cell level, as well as the enrichment of NKX2.1 positive cells.