28 results on '"Stephanie Sontag"'
Search Results
2. Influence of Pennation Angle and Muscle Thickness on Mechanomyographic Amplitude–Torque Relationships and Sex-Related Differences in the Vastus Lateralis
- Author
-
Michael Trevino, Sergio Perez, Stephanie Sontag, Alex Olmos, Sunggun Jeon, and Lyric Richardson
- Subjects
females ,log transform ,males ,mechanomyography ,muscle thickness ,pennation angle ,Diseases of the musculoskeletal system ,RC925-935 - Abstract
This study examined potential sex-related differences and correlations among the pennation angle (PA), muscle thickness (MT), and mechanomyographic amplitude (MMGRMS)–torque relationships of the vastus lateralis (VL) in 11 healthy males and 12 healthy females. The PA and MT of the VL were quantified with ultrasound. Participants performed an isometric muscle action of the knee extensors that linearly increased to 70% of maximal strength followed by a 12 s plateau. MMG was recorded from the VL. Linear regression models were fit to the log-transformed MMGRMS–torque relationships to calculate b terms (slopes) for the linearly increasing segment. MMGRMS was averaged during the plateau. Males exhibited greater PA (p < 0.001), MT (p = 0.027), b terms (p = 0.005), and MMGRMS (p = 0.016). The b terms were strongly (p < 0.001, r = 0.772) and moderately correlated (p = 0.004, r = 0.571) with PA and MT, respectively, while MMGRMS was moderately correlated with PA (p = 0.018, r = 0.500) and MT (p = 0.014, r = 0.515). The greater mechanical behavior of individuals possessing a larger PA and MT of the VL may reflect increased cross-bridge activity within the muscle fibers. Additionally, PA may help explain sex-related differences in MMGRMS between sexes.
- Published
- 2023
- Full Text
- View/download PDF
3. CRISPR/Cas9 mediated CXCL4 knockout in human iPS cells of polycythemia vera patient with JAK2 V617F mutation
- Author
-
Janik Boehnke, Salim Atakhanov, Marcelo A.S. Toledo, Herdit M. Schüler, Stephanie Sontag, Nicolas Chatain, Steffen Koschmieder, Tim H. Brümmendorf, Rafael Kramann, and Martin Zenke
- Subjects
Induced pluripotent stem cell ,iPS cells ,Hematopoiesis ,CXCL4 ,PF4 ,JAK2 V617F ,Biology (General) ,QH301-705.5 - Abstract
The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases.
- Published
- 2021
- Full Text
- View/download PDF
4. Human DC3 Antigen Presenting Dendritic Cells From Induced Pluripotent Stem Cells
- Author
-
Taiki Satoh, Marcelo A. S. Toledo, Janik Boehnke, Kathrin Olschok, Niclas Flosdorf, Katrin Götz, Caroline Küstermann, Stephanie Sontag, Kristin Seré, Steffen Koschmieder, Tim H. Brümmendorf, Nicolas Chatain, Yoh-ichi Tagawa, and Martin Zenke
- Subjects
induced pluripotent stem cell ,iPS cells ,hematopoiesis ,dendritic cells ,DC3 ,JAK2 ,Biology (General) ,QH301-705.5 - Abstract
Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8+ T cells into CD8+ CD103+ tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8+ T cells into CD8+ CD103+ tissue-resident memory T cells in vitro. To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617Fhet and JAK2 V617Fhom mutations into JAK2 V617Fhet and JAK2 V617Fhom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias toward erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics.
- Published
- 2021
- Full Text
- View/download PDF
5. Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells
- Author
-
Olivia Cypris, Joana Frobel, Shivam Rai, Julia Franzen, Stephanie Sontag, Roman Goetzke, Marcelo A. Szymanski de Toledo, Martin Zenke, and Wolfgang Wagner
- Subjects
DNA methylation ,Epigenetic ,Hematopoiesis ,Induced pluripotent stem cells ,Mesenchymal stromal cells ,Stromal support ,Medicine ,Genetics ,QH426-470 - Abstract
Abstract Background Differentiation 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. Results In 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. Conclusion Differentiation 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.
- Published
- 2019
- Full Text
- View/download PDF
6. Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets
- Author
-
Stephanie Sontag, Malrun Förster, Kristin Seré, and Martin Zenke
- Subjects
Biology (General) ,QH301-705.5 - Abstract
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+ CD304+ (Jongbloed et al., 2010; Joffre et al., 2012; Swiecki and Colonna, 2015). To investigate subset specification and function of human DC, we established a protocol to generate cDC1, cDC2 and pDC in vitro from human iPS cells (or ES cells) (Sontag et al., 2017). Therefore, we differentiated iPS cells (or ES cells), via mesoderm commitment and hemato-endothelial specification, into CD43+ CD31+ hematopoietic progenitors. Subsequently, those were seeded onto inactivated OP9 stromal cells with FLT3L, SCF, GM-CSF and IL-4 or FLT3L, SCF and GM-CSF to specify cDC1 and cDC2, or cDC1 and pDC, respectively.
- Published
- 2017
- Full Text
- View/download PDF
7. Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing.
- Author
-
Martin M J Kirschner, Mirle Schemionek, Claudia Schubert, Nicolas Chatain, Stephanie Sontag, Susanne Isfort, Nadina Ortiz-Brüchle, Karla Schmitt, Luisa Krüger, Klaus Zerres, Martin Zenke, Tim H Brümmendorf, and Steffen Koschmieder
- Subjects
Medicine ,Science - Abstract
In order to assess the feasibility of amplicon-based parallel next generation sequencing (NGS) for the diagnosis of myeloproliferative neoplasms (MPN), we investigated multiplex-PCR of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes. Samples from 64 patients with MPN and five controls as well as seven (myeloid) cell lines were analyzed. Healthy donor and reactive erythrocytosis samples showed several frequent single-nucleotide polymorphisms (SNPs) but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of the known mutations. In the patient samples, JAK2 V617F was present in all PV, 4 of 10 ET, and 16 of 19 MF patients. The JAK2 V617F allele burden was different in the three groups (ET, 33+/-22%; PV 48+/-28% and MF 68+/- 29%). Further analysis detected both previously described and undescribed mutations (i.e., G12V NRAS, IDH1 R132H, E255G ABL, and V125G IDH1 mutations). One patient with lymphoid BC/Ph+ ALL who harbored a T315I ABL mutation and was treated with ponatinib was found to have developed a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation might have caused ponatinib resistance in this patient. In conclusion, amplicon-sequencing-based NGS allows simultaneous analysis of multiple MPN associated genes for diagnosis and during treatment and measurement of the mutant allele burden.
- Published
- 2015
- Full Text
- View/download PDF
8. To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture.
- Author
-
Charlotte A Willmann, Hatim Hemeda, Lisa A Pieper, Michael Lenz, Jie Qin, Sylvia Joussen, Stephanie Sontag, Paul Wanek, Bernd Denecke, Herdit M Schüler, Martin Zenke, and Wolfgang Wagner
- Subjects
Medicine ,Science - Abstract
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.
- Published
- 2013
- Full Text
- View/download PDF
9. Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF
- Author
-
Kai Ling Liang, Juliette Roels, Marieke Lavaert, Tom Putteman, Lena Boehme, Laurentijn Tilleman, Imke Velghe, Valentina Pegoretti, Inge Van de Walle, Stephanie Sontag, Jolien Vandewalle, Bart Vandekerckhove, Georges Leclercq, Pieter Van Vlierberghe, Claude Libert, Filip Van Nieuwerburgh, Roman Fischer, Roland E. Kontermann, Klaus Pfizenmaier, Gina Doody, Martin Zenke, and Tom Taghon
- Subjects
Immunology ,Immunology and Allergy - Published
- 2023
10. Abstract 3203: EVOcells Oncology: Tailored genetic engineering of iPSC-derived immune effector cells and combination with the right biologic therapeutics result in optimal killing of primary tumor cells from patients
- Author
-
Michael Esquerré, Audrey Holtzinger, Nadja Wagner, Monika Braun, Mélanie Pichery, Stefanie Pfaender, Stephanie Sontag, Kathrin Haake, Michela Mirenda, Michael Paillasse, Davide Grandolfo, Chloé Beuraud, Mandy Richter, Philip Hublitz, Julien Bousquet, Marion Fabre, Mylène Gador, Daniel Sommermeyer, Tanja Schneider, Oriane Bombarde, Camille Esquerré, Loic Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, and Markus Dangl
- Subjects
Cancer Research ,Oncology - Abstract
Current autologous cell therapies, with blockbuster products on the market, have been leading for a decade to unprecedented clinical successes in patients with hematological malignancies. However, these patient-derived T-cell therapies are facing many challenges. The use of GMP iPSC lines to produce immune effector cells will reduce the complexity of the manufacturing process and will provide an unlimited source of starting material. The goal of the EVOcells Oncology platform is to offer a truly allogeneic cell therapy platform to treat a broad number of cancer patients with consistent quality and scalability of the final product. Besides, the versatility of our platform to produce different immune cell types combined to customized genetic engineering strategies will bring cell therapy to the level of personalized medicine. Our “off-the-shelf” cell therapy platform has already validated two pillars: iPSC-derived NK cells (iNK) and iPSC-derived Macrophages (iMACs). Through multiple genetic engineering strategies specific to each immune cell type, we are developing a comprehensive portfolio of cell therapy products to address specific tumor escape mechanism in liquid and solid tumors. Our initial effort aimed to develop these two innate immune cell types to propose efficacious cell therapies with an increased safety profile as they have low risk of graft-versus-host disease (GvHD) or CRS (Cytokine Release Syndrome). Thanks to the expression of a broad pattern of activatory receptors, iNK cells form Immunological Synapses with tumor cells leading in turn to efficient killing with and without addition of a CAR construct. Besides, we demonstrated the possibility to combine “naked” iNK cells with marketed therapeutic monoclonal antibodies (mAb) to further improve their efficacy. At the end of the differentiation process, iMACs are showing a M0 like phenotype with high plasticity allowing the in vitro differentiation of the cells towards either a M1 or a M2 polarization in response to the appropriate stimulations. iMACs produce key macrophages cytokines and are able to kill tumor cells via ADCP (Antibody-Dependent-Cell-Phagocytosis) mechanism when combined to a therapeutic mAb. Thanks to our collaboration with clinicians at the IUCT-Oncopole (Toulouse Cancer Hospital), we were able to identify appropriate cancer indications and further demonstrate in a translational fashion that both iNK and iMACs are able to kill primary resistant tumor cells which were isolated from patient’ samples. Taken together, these results are showing the versatility and the breadth of our EVOcells Oncology platform to produce a true arsenal of cell therapies and its potential for future clinical development. Citation Format: Michael Esquerré, Audrey Holtzinger, Nadja Wagner, Monika Braun, Mélanie Pichery, Stefanie Pfaender, Stephanie Sontag, Kathrin Haake, Michela Mirenda, Michael Paillasse, Davide Grandolfo, Chloé Beuraud, Mandy Richter, Philip Hublitz, Julien Bousquet, Marion Fabre, Mylène Gador, Daniel Sommermeyer, Tanja Schneider, Oriane Bombarde, Camille Esquerré, Loic Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. EVOcells Oncology: Tailored genetic engineering of iPSC-derived immune effector cells and combination with the right biologic therapeutics result in optimal killing of primary tumor cells from patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3203.
- Published
- 2023
11. Nintedanib targets KIT D816V neoplastic cells derived from induced pluripotent stem cells of systemic mastocytosis
- Author
-
Ahmed Emad Ibrahim Hamouda, Peter Ettmayer, Gregor Eisenwort, Satu Mustjoki, Giulia Rossetti, Kristina Feldberg, Jens Panse, Frank Hilberg, Anne Kaiser, Marcelo A. S. Toledo, Karoline V. Gleixner, Malrun Gatz, Peter Valent, Angela Maurer, Andreas Reiter, Nicolas Chatain, Herdit M. Schüler, Wolfgang Wagner, Olli Dufva, Riccardo Guareschi, Tim H. Brümmendorf, Roman Goetzke, Martin Zenke, Steffen Koschmieder, Mohamad Jawhar, Frederick Kluge, Till Braunschweig, Antonio Sechi, Stephanie Sontag, TRIMM - Translational Immunology Research Program, Doctoral Programme in Clinical Research, Department of Clinical Chemistry and Hematology, Digital Precision Cancer Medicine (iCAN), HUS Comprehensive Cancer Center, Doctoral Programme in Drug Research, Doctoral Programme in Biomedicine, and Research Programs Unit
- Subjects
MIDOSTAURIN ,Indoles ,3122 Cancers ,Induced Pluripotent Stem Cells ,Immunology ,Antineoplastic Agents ,IMATINIB ,Biochemistry ,CLASSIFICATION ,Receptor tyrosine kinase ,MASITINIB ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Mastocytosis, Systemic ,Growth factor receptor ,Tumor Cells, Cultured ,medicine ,Humans ,Point Mutation ,ddc:610 ,WILD-TYPE ,Systemic mastocytosis ,Induced pluripotent stem cell ,030304 developmental biology ,0303 health sciences ,biology ,MUTATIONS ,Chemistry ,INHIBITOR ,Cell Biology ,Hematology ,Mast cell leukemia ,medicine.disease ,Embryonic stem cell ,3. Good health ,C-KIT ,Proto-Oncogene Proteins c-kit ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,MAST-CELLS ,GROWTH ,Nintedanib ,Tyrosine kinase - Abstract
The KIT D816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KIT D816V 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 and mast cell leukemia 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, a US Food and Drug Administration–approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, 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 adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V–targeted therapy of advanced SM.
- Published
- 2021
12. Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy
- Author
-
Alexandra Brahmer, Markus P. Radsak, Keito F.A. Philippi, Elmo W. I. Neuberger, Stephanie Sontag, Perikles Simon, and Wolfgang Wagner
- Subjects
Cell type ,Myeloid ,Lymphocyte ,Bisulfite sequencing ,610 Medizin ,796 Athletic and outdoor sports and games ,570 Life sciences ,610 Medical sciences ,medicine ,Genetics ,Humans ,Liquid biopsy ,Exercise ,Molecular Biology ,Genetics (clinical) ,Acute leukemia ,796 Sport ,business.industry ,Liquid Biopsy ,Methylation ,DNA Methylation ,medicine.anatomical_structure ,Cell-free fetal DNA ,Immunology ,business ,Cell-Free Nucleic Acids ,Granulocytes ,570 Biowissenschaften ,Developmental Biology - Abstract
Clinical epigenetics 14, 29 (2022). doi:10.1186/s13148-022-01245-3, Published by BioMed Central, [Erscheinungsort nicht ermittelbar]
- Published
- 2022
13. Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms
- Author
-
Tim H. Brümmendorf, Nicolas Chatain, Bernd Denecke, Caroline Küstermann, Klaus Strathmann, Tiago Maié, Stephanie Sontag, Martin Zenke, Annika Hubrich, Ivan G. Costa, Julian Baumeister, Andreas Schuppert, Steffen Koschmieder, Deniz Gezer, Kamil R. Kranc, Lijuan Han, and Kristin Seré
- Subjects
Adult ,Male ,0301 basic medicine ,Cancer Research ,Induced Pluripotent Stem Cells ,Apoptosis ,Echinomycin ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Polycythemia vera ,Biomarkers, Tumor ,Tumor Cells, Cultured ,medicine ,Humans ,Progenitor cell ,Myelofibrosis ,Aged ,Cell Proliferation ,Aged, 80 and over ,Antibiotics, Antineoplastic ,Myeloproliferative Disorders ,Essential thrombocythemia ,Cell Cycle ,Hematology ,Janus Kinase 2 ,Middle Aged ,Hypoxia-Inducible Factor 1, alpha Subunit ,Prognosis ,medicine.disease ,Warburg effect ,Gene Expression Regulation, Neoplastic ,Haematopoiesis ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Mutation ,Cancer research ,Female ,Bone marrow ,Follow-Up Studies - Abstract
Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.
- Published
- 2019
14. Toward Clinical Application of Leukocyte Counts Based on Targeted DNA Methylation Analysis
- Author
-
Stephanie Sontag, Ledio Bocova, Wouter H G Hubens, Selina Nüchtern, Matthis Schnitker, Thomas Look, Kema M Schröder, Birgit Plümäkers, Vithurithra Tharmapalan, Martina Wessiepe, Thomas Kraus, Jan Kramer, Lothar Rink, Steffen Koschmieder, and Wolfgang Wagner
- Subjects
Epigenomics ,Leukocyte Count ,Biochemistry (medical) ,Clinical Biochemistry ,Leukocytes ,Humans ,DNA Methylation ,Granulocytes - Abstract
Clinical chemistry 68(5), 646-656 (2022). doi:10.1093/clinchem/hvac006, Published by Oxford University Press, Oxford
- Published
- 2021
15. The Effects Of 10-weeks Of Continuous Cycling On Pennation Angle Of The Vastus Lateralis
- Author
-
Alex Olmos, Stephanie Sontag, Sergio Perez, Sunggun Jeon, Shawn Allen, Jeremy Lippman, and Michael Trevino
- Subjects
Physical Therapy, Sports Therapy and Rehabilitation ,Orthopedics and Sports Medicine - Published
- 2022
16. Nintedanib Targets KIT D816V Neoplastic Cells Derived from Induced Pluripotent Stem cells of Systemic Mastocytosis
- Author
-
Marcelo A. S. Toledo, Malrun Gatz, Stephanie Sontag, Karoline V. Gleixner, Gregor Eisenwort, Kristina Feldberg, Frederick Kluge, Riccardo Guareschi, Giulia Rossetti, Antonio S. Sechi, Olli M. J. Dufva, Satu M. Mustjoki, Angela Maurer, Herdit M. Schüler, Roman Goetzke, Till Braunschweig, Anne Simonowski, Jens Panse, Mohamad Jawhar, Andreas Reiter, Frank Hilberg, Peter Ettmayer, Wolfgang Wagner, Steffen Koschmieder, Tim H. Brümmendorf, Peter Valent, Nicolas Chatain, and Martin Zenke
- Subjects
0303 health sciences ,Biology ,Mast cell leukemia ,medicine.disease ,Embryonic stem cell ,Receptor tyrosine kinase ,3. Good health ,03 medical and health sciences ,Haematopoiesis ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Cancer research ,medicine ,biology.protein ,Nintedanib ,Systemic mastocytosis ,Induced pluripotent stem cell ,Tyrosine kinase ,030304 developmental biology - Abstract
TheKITD816V 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.KITD816V therefore represents a prime therapeutic target for SM. Here we generated a panel of patient-specificKITD816V 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.KITD816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineeredKITD816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed forKITD816V iPSC hematopoiesis.KITD816V 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 as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derivedKITD816V 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.
- Published
- 2020
- Full Text
- View/download PDF
17. CRISPR/Cas9 mediated CXCL4 knockout in human iPS cells of polycythemia vera patient with JAK2 V617F mutation
- Author
-
Marcelo A. S. Toledo, Herdit M. Schüler, Janik Boehnke, Nicolas Chatain, Steffen Koschmieder, Tim H. Brümmendorf, Martin Zenke, Stephanie Sontag, Rafael Kramann, and Salim Atakhanov
- Subjects
Chemokine ,QH301-705.5 ,Induced Pluripotent Stem Cells ,medicine.disease_cause ,JAK2 V617F ,Exon ,Polycythemia vera ,hemic and lymphatic diseases ,medicine ,Humans ,CRISPR ,Biology (General) ,Induced pluripotent stem cell ,Myelofibrosis ,Polycythemia Vera ,Mutation ,biology ,CXCL4 ,Cell Biology ,General Medicine ,Janus Kinase 2 ,medicine.disease ,Hematopoiesis ,iPS cells ,PF4 ,Haematopoiesis ,biology.protein ,Cancer research ,CRISPR-Cas Systems ,Developmental Biology - Abstract
Stem cell research 55, 102490 (2021). doi:10.1016/j.scr.2021.102490, Published by Elsevier, Amsterdam [u.a.]
- Published
- 2021
18. Current developments in human stem cell research and clinical translation
- Author
-
Martin Zenke and Stephanie Sontag
- Subjects
Computer science ,Translation (biology) ,Current (fluid) ,Stem cell ,Neuroscience - Published
- 2019
19. The role of Nav1.7 in human nociceptors: insights from human induced pluripotent stem cell-derived sensory neurons of erythromelalgia patients
- Author
-
Roman Goetzke, Herdit M. Schüler, Marc Rogers, Ellen Jørum, Angelika Lampert, Martin Hampl, Elisangela Bressan, Anthony M. Rush, Martin Zenke, Zacharias Kohl, Clara M. Kerth, Thi Kim Chi Le, Barbara Namer, Alec Foerster, Petra Hautvast, Martin Schmelz, Corinna Rösseler, Wolfgang Wagner, Kim Le Cann, Inge Petter Kleggetveit, Beate Winner, Jannis E. Meents, and Stephanie Sontag
- Subjects
Patch-Clamp Techniques ,Action potential ,Action Potentials ,Membrane Potentials ,0302 clinical medicine ,030202 anesthesiology ,Ganglia, Spinal ,pharmacology [Tetrodotoxin] ,pain ,Induced pluripotent stem cell ,Prepulse inhibition ,genetics [NAV1.7 Voltage-Gated Sodium Channel] ,NAV1.7 Voltage-Gated Sodium Channel ,Nociceptors ,Afterhyperpolarization ,cytology [Induced Pluripotent Stem Cells] ,Erythromelalgia ,metabolism [NAV1.7 Voltage-Gated Sodium Channel] ,iPS cells ,Neurology ,genetics [Pain] ,genetics [Erythromelalgia] ,Nociceptor ,sodium channel ,Research Paper ,physiology [Nociceptors] ,Sensory Receptor Cells ,Induced Pluripotent Stem Cells ,Pain ,Sensory system ,Tetrodotoxin ,physiopathology [Erythromelalgia] ,patch clamp ,03 medical and health sciences ,methods [Patch-Clamp Techniques] ,stem cells ,medicine ,Voltage-gated sodium channel ,metabolism [Sensory Receptor Cells] ,Humans ,ddc:610 ,business.industry ,drug effects [Action Potentials] ,cytology [Ganglia, Spinal] ,Sodium channel ,drug effects [Membrane Potentials] ,medicine.disease ,diagnosis [Pain] ,Electric Stimulation ,methods [Electric Stimulation] ,Anesthesiology and Pain Medicine ,nervous system ,Neurology (clinical) ,Inherited pain syndrome ,business ,Action potential firing ,Neuroscience ,Patch-clamp ,030217 neurology & neurosurgery - Abstract
Supplemental Digital Content is Available in the Text. Human sodium channel NaV1.7 in induced pluripotent stem cell–derived sensory neurons sets the action potential threshold but does not support subthreshold depolarizations., The chronic pain syndrome inherited erythromelalgia (IEM) is attributed to mutations in the voltage-gated sodium channel (NaV) 1.7. Still, recent studies targeting NaV1.7 in clinical trials have provided conflicting results. Here, we differentiated induced pluripotent stem cells from IEM patients with the NaV1.7/I848T mutation into sensory nociceptors. Action potentials in these IEM nociceptors displayed a decreased firing threshold, an enhanced upstroke, and afterhyperpolarization, all of which may explain the increased pain experienced by patients. Subsequently, we investigated the voltage dependence of the tetrodotoxin-sensitive NaV activation in these human sensory neurons using a specific prepulse voltage protocol. The IEM mutation induced a hyperpolarizing shift of NaV activation, which leads to activation of NaV1.7 at more negative potentials. Our results indicate that NaV1.7 is not active during subthreshold depolarizations, but that its activity defines the action potential threshold and contributes significantly to the action potential upstroke. Thus, our model system with induced pluripotent stem cell–derived sensory neurons provides a new rationale for NaV1.7 function and promises to be valuable as a translational tool to profile and develop more efficacious clinical analgesics.
- Published
- 2019
20. Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells
- Author
-
Shivam Rai, Roman Goetzke, Marcelo A. S. Toledo, Julia Franzen, Martin Zenke, Joana Frobel, Wolfgang Wagner, Olivia Cypris, and Stephanie Sontag
- Subjects
0301 basic medicine ,Stromal cell ,lcsh:QH426-470 ,Induced Pluripotent Stem Cells ,Mesenchymal stromal cells ,lcsh:Medicine ,Biology ,Epigenesis, Genetic ,Cell therapy ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,Humans ,Epigenetics ,Induced pluripotent stem cell ,Molecular Biology ,Genetics (clinical) ,Cells, Cultured ,Cell Proliferation ,Hematopoietic differentiation ,DNA methylation ,Research ,lcsh:R ,Mesenchymal stem cell ,dNaM ,Epigenetic ,Cell Differentiation ,Mesenchymal Stem Cells ,Hematopoietic Stem Cells ,Coculture Techniques ,Cell biology ,Hematopoiesis ,lcsh:Genetics ,Haematopoiesis ,030104 developmental biology ,030220 oncology & carcinogenesis ,Leukocyte Common Antigens ,Stromal support ,Developmental Biology - Abstract
Background Differentiation 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. Results In 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. Conclusion Differentiation 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 material The online version of this article (10.1186/s13148-019-0617-1) contains supplementary material, which is available to authorized users.
- Published
- 2019
21. Women who take n-3 long-chain polyunsaturated fatty acid supplements during pregnancy and lactation meet the recommended intake
- Author
-
Xiaoming Jia, Linda J. McCargar, Fatheema B. Subhan, Murphy Andrews, Jamie Wildgrube, Mohammadreza Pakseresht, Stephanie Sontag, Nour Wattar, and Catherine J. Field
- Subjects
Adult ,Physiology ,Endocrinology, Diabetes and Metabolism ,Recommended Dietary Allowances ,Alberta ,Cohort Studies ,Pregnancy ,Physiology (medical) ,Lactation ,Fatty Acids, Omega-3 ,medicine ,Humans ,Prospective Studies ,Food science ,chemistry.chemical_classification ,Analysis of Variance ,Nutrition and Dietetics ,business.industry ,General Medicine ,medicine.disease ,Eicosapentaenoic acid ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Docosahexaenoic acid ,Dietary Supplements ,Female ,business ,Long chain ,Polyunsaturated fatty acid ,Recommended Intake - 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.
- Published
- 2015
22. Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets
- Author
-
Martin Zenke, Kristin Seré, Malrun Förster, and Stephanie Sontag
- Subjects
0301 basic medicine ,Induced stem cells ,Strategy and Management ,Mechanical Engineering ,Cellular differentiation ,Metals and Alloys ,Dendritic cell ,Biology ,Embryonic stem cell ,Industrial and Manufacturing Engineering ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Methods Article ,Stem cell ,Induced pluripotent stem cell ,Cell potency ,Adult stem cell - Abstract
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(+) CD304(+) ( Jongbloed et al., 2010 ; Joffre et al., 2012 ; Swiecki and Colonna, 2015). To investigate subset specification and function of human DC, we established a protocol to generate cDC1, cDC2 and pDC in vitro from human iPS cells (or ES cells) ( Sontag et al., 2017 ). Therefore, we differentiated iPS cells (or ES cells), via mesoderm commitment and hemato-endothelial specification, into CD43(+) CD31(+) hematopoietic progenitors. Subsequently, those were seeded onto inactivated OP9 stromal cells with FLT3L, SCF, GM-CSF and IL-4 or FLT3L, SCF and GM-CSF to specify cDC1 and cDC2, or cDC1 and pDC, respectively.
- Published
- 2017
23. Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells
- Author
-
Paul Wanek, Herdit M. Schüler, Saskia Mitzka, Stefan Rose-John, Jie Qin, Kristin Seré, Martin Zenke, Steffen Koschmieder, Malrun Förster, and Stephanie Sontag
- Subjects
0301 basic medicine ,Cellular differentiation ,Antigen presentation ,Induced Pluripotent Stem Cells ,Stem cell factor ,Biology ,Models, Biological ,03 medical and health sciences ,Humans ,Progenitor cell ,Induced pluripotent stem cell ,Cell Engineering ,Cell Biology ,Dendritic cell ,Dendritic Cells ,Embryonic stem cell ,Cell biology ,Hematopoiesis ,030104 developmental biology ,Immunology ,Interferon Regulatory Factors ,Molecular Medicine ,Stem cell ,CRISPR-Cas Systems ,Gene Deletion ,Developmental Biology ,Granulocytes - Abstract
Stem cells 35(4), 898-908 (2017). doi:10.1002/stem.2565, Published by Wiley-Blackwell, Hoboken, NJ
- Published
- 2017
24. Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)
- Author
-
Caroline Küstermann, Steffen Koschmieder, Tim H. Brümmendorf, Julian Baumeister, Annika Hubrich, Kristin Seré, Martin Zenke, Deniz Gezer, Nicolas Chatain, Kamil Kranc, and Stephanie Sontag
- Subjects
Ruxolitinib ,Chemistry ,Immunology ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Echinomycin ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Haematopoiesis ,0302 clinical medicine ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,medicine ,Cancer research ,Stem cell ,Progenitor cell ,Protein kinase B ,PI3K/AKT/mTOR pathway ,030215 immunology ,medicine.drug - Abstract
Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.
- Published
- 2018
25. Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing
- Author
-
Nadina Ortiz-Brüchle, Claudia Schubert, Nicolas Chatain, Susanne Isfort, Steffen Koschmieder, Martin Kirschner, Karla Schmitt, Luisa Krüger, Mirle Schemionek, Klaus Zerres, Tim H. Brümmendorf, Martin Zenke, and Stephanie Sontag
- Subjects
Neuroblastoma RAS viral oncogene homolog ,Quality Control ,Science ,Single-nucleotide polymorphism ,Biology ,medicine.disease_cause ,Polymorphism, Single Nucleotide ,Frameshift mutation ,chemistry.chemical_compound ,hemic and lymphatic diseases ,Cell Line, Tumor ,medicine ,Humans ,Alleles ,Mutation ,Multidisciplinary ,ABL ,Myeloproliferative Disorders ,Genome, Human ,Ponatinib ,High-Throughput Nucleotide Sequencing ,Amplicon ,Janus Kinase 2 ,Molecular biology ,chemistry ,Bone marrow neoplasm ,Medicine ,ddc:500 ,Bone Marrow Neoplasms ,Multiplex Polymerase Chain Reaction ,Research Article ,Follow-Up Studies - Abstract
PLoS one 10(4), e0123476 (2015). doi:10.1371/journal.pone.0123476, Published by PLoS, Lawrence, Kan.
- Published
- 2015
- Full Text
- View/download PDF
26. Cell fusion enhances mesendodermal differentiation of human induced pluripotent stem cells
- Author
-
Qiong Lin, Michael Peitz, Isabelle Leisten, Robert Chunhua Zhao, Saskia Mitzka, Stephanie Sontag, Rebekka K. Schneider, Jie Qin, Anna Jauch, Martin Zenke, Oliver Brüstle, Xiaoying Wang, and Wolfgang Wagner
- Subjects
Cell fusion ,animal structures ,Cellular differentiation ,Induced Pluripotent Stem Cells ,Nodal signaling ,Cell Differentiation ,Cell Biology ,Hematology ,Anatomy ,Biology ,Hematopoietic Stem Cells ,Embryonic stem cell ,Antigens, Differentiation ,Cell biology ,Cell Fusion ,Mesoderm ,Haematopoiesis ,Original Research Reports ,embryonic structures ,Humans ,Stem cell ,NODAL ,Induced pluripotent stem cell ,Developmental Biology - Abstract
Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSCs) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias toward hematopoietic lineages but also toward other mesendodermal lineages. Additionally, during differentiation of iPS hybrids, expression of early mesendodermal markers-Brachyury (T), MIX1 Homeobox-Like Protein 1 (MIXL1), and Goosecoid (GSC)-appeared with faster kinetics than in parental iPS cells. Following iPS hybrid differentiation there was a prominent induction of NODAL and inhibition of NODAL signaling blunted mesendodermal differentiation. This indicates that NODAL signaling is critically involved in mesendodermal bias of iPS hybrid differentiation. In summary, we demonstrate that iPS cell fusion with HSCs prominently enhances iPS cell differentiation.
- Published
- 2014
27. Dissecting Genomic Aberrations In CML and Bcr-Abl Negative Myeloproliferative Neoplasms By The Use Of Multiplex-PCR and Parallel Resequencing
- Author
-
Susanne Isfort, Martin Zenke, Claudia Schubert, Nadina Ortiz-Brüchle, Karla Bennemann, Mirle Schemionek, Nicolas Chatain, Stephanie Sontag, Martin Kirschner, Klaus Zerres, Tim H. Bruemmendorf, and Steffen Koschmieder
- Subjects
Genetics ,Neuroblastoma RAS viral oncogene homolog ,Oncology ,medicine.medical_specialty ,Mutation ,business.industry ,Immunology ,Ponatinib ,Cell Biology ,Hematology ,medicine.disease_cause ,Biochemistry ,Frameshift mutation ,Dasatinib ,chemistry.chemical_compound ,Imatinib mesylate ,chemistry ,hemic and lymphatic diseases ,Internal medicine ,medicine ,KRAS ,business ,Bosutinib ,medicine.drug - Abstract
Introduction Next-Generation Sequencing holds the promise of comprehensive analysis of molecular aberrations in human malignancies and therapeutic approaches individually tailored to each patient. Methods We investigated the use of a multiplex-PCR (TruseqAmplicon Cancer Panel, Illumina) of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes to identify mutations in a cohort of patients with myeloproliferativeneoplasms (MPN). After signed informed consent, samples from 59 patients with MPN (19 MF [8 PMF, 11 post-PV/ET-MF], 14 PV, 10 ET, 10 CML, 4 HES, and 2 SM), two patients with reactive erythrocytosis, and two anonymized healthy controls as well as six myeloid cell lines (K562, HEL, HMC1, SUPB15, HL60, U937) were analyzed on a Miseq sequencer (Illumina), using 250 ng of genomic DNA from peripheral blood -derived cells. Results Altogether, the quality of the sequencing runs was very good, with Q30 values above 90%. 151 bidirectional cycles were performed, yielding between 2 and 6 Gigabases of sequencing data.Healthy donor and reactive erythrocytosis samples showed several SNPs but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of a TP53 frameshift mutation (c.405_406insC; in 98% of transcripts) in K562, JAK2 V617F (100%) and TP53 M133K (99%) mutations in HEL, two heterozygous KIT mutations (V560G in 51% and D816V in 52%) and a TP53 C277F (16%) mutation in HMC1, while SUP-B15, HL60, and U937 showed no abnormality in the tested gene set.JAK2 V617F was present in all PV, 4 of 10 ET, and 14 of 19 MF patients.The JAK2 V617F allele burden was significantly higher in MF than ET (p=0.026) but not PV (71+/-27% vs. 33+/-22% vs. 55+/-29%, respectively). Further analysis detected a previously described G12V NRAS mutation (13% of transcripts) in a patient with JAK2 V617F negative PMF and an additional IDH1 R132H mutation (24%) in a JAK2 V617F positive (46%) MF patient with 20% basophils and hyperhistaminemia. Another JAK2 V617F positive (31%) MF sample showed an E255G ABL mutation (10%). One patient with JAK2 V617F negative ET showed an ERBB2 A847D sequence variant (50%). Moreover, an S935N CSF1R mutation (17%) and a V125G IDH1 mutation (9%) were each detected in one case of PV, but the biological relevance remains unclear so far. Four patients with CML-CP (n=3) or –AP (n=1) showed subclones with sequence variants in the HNF1A gene, with two S304P changes (9 and 10% of transcripts) and two 872delC mutations (6 and 5%), the latter of which have already been implicated in colon cancer. Two patients with CML-CP showed KIT mutations (a V532I mutation and a known oncogenic mutation V530I). This latter patient also harbored the known E255K ABL mutation – leading to imatinib resistance. Interestingly, this patient showed a good response to dasatinib (which is also active against KIT) but not to bosutinib (which has no activity against KIT). These data suggest that HNF1A and KIT may play a role in CML pathogenesis. One patient with lymphoid BC/Ph+ ALL who had a T315I ABL mutation and was treated with ponatinib, was found to harbor a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib had led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation may have caused ponatinib resistance in this patient. Additionally, other not yet defined aberrations may have been responsible for the observed resistance. Finally, while both SM patients were negative for KIT D816V, one of them harbored a KRAS 436G>A(146A>T) mutation (34%) which is a known oncogene in colorectal cancer and may thus also play a role in SM pathogenesis. We are currently generating induced pluripotent stem cells from patients harboring selected mutations described above in order to better be able to study the functional properties of genetically unstable malignant stem cell populations. Conclusion Amplicon-based next-generation sequencing may uncover additional oncogenic mutations in patients with MPN, potentially explaining therapy resistance and opening new therapeutic options for individual patients. Disclosures: Off Label Use: Two individual patients mentioned that were treated with ponatinib or bosutinib within compassionate use trials before these drugs were approved for the indication. Bruemmendorf:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria. Koschmieder:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.
- Published
- 2013
28. To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture
- Author
-
Michael Lenz, Charlotte A. Willmann, Hatim Hemeda, Lisa A. Pieper, Sylvia Joussen, Bernd Denecke, Paul Wanek, Stephanie Sontag, Wolfgang Wagner, Herdit M. Schüler, Martin Zenke, and Jie Qin
- Subjects
Cellular differentiation ,Clone (cell biology) ,Cell Culture Techniques ,lcsh:Medicine ,Mesodermal Cells ,Mice ,Molecular Cell Biology ,lcsh:Science ,Induced pluripotent stem cell ,Cells, Cultured ,Oligonucleotide Array Sequence Analysis ,Microscopy ,Multidisciplinary ,Cultured ,Mesenchymal Stromal Cells ,Reverse Transcriptase Polymerase Chain Reaction ,Stem Cells ,Cell Differentiation ,Genomics ,Flow Cytometry ,Cellular Types ,Research Article ,Biotechnology ,Pluripotent Stem Cells ,Stromal cell ,Cells ,Induced Pluripotent Stem Cells ,Biomedical Engineering ,Bioengineering ,Germ layer ,Biology ,Fluorescence ,Animals ,Humans ,Embryonic Stem Cells ,Cell Proliferation ,Tissue Engineering ,Gene Expression Profiling ,lcsh:R ,Mesenchymal stem cell ,Mesenchymal Stem Cells ,Fibroblasts ,Molecular biology ,Embryonic stem cell ,Clone Cells ,Microscopy, Fluorescence ,Cell culture ,Karyotyping ,lcsh:Q ,Genome Expression Analysis ,Developmental Biology - Abstract
PLoS one 8(5), e65324 (2013). doi:10.1371/journal.pone.0065324, Published by PLoS [u.a.], Lawrence, Kan.
- Published
- 2013
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.