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Article ; Online: An Improved Lentiviral Fluorescent Genetic Barcoding Approach Distinguishes Hematopoietic Stem Cell Properties in Multiplexed

Lieske, Anna / Ha, Teng Cheong / Schambach, Axel / Maetzig, Tobias

2021 Volume 32, Issue 19-20, Page(s) 1280–1294

Abstract: Hematopoietic stem cells (HSCs) represent a rare cell population of particular interest for biomedical research and regenerative medicine. Various marker combinations enable the isolation of HSCs, but fail to reach purity in transplantation assays. To ... ...

Abstract	Hematopoietic stem cells (HSCs) represent a rare cell population of particular interest for biomedical research and regenerative medicine. Various marker combinations enable the isolation of HSCs, but fail to reach purity in transplantation assays. To reduce animal consumption, we developed a multiplexing system based on lentiviral fluorescent genetic barcoding (FGB) to enable the parallel characterization of multiple HSC samples within single animals. While previous FGB-mediated HSC multiplexing experiments achieved high
MeSH term(s)	Animals ; Genetic Vectors/genetics ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Immunophenotyping ; Mice
Language	English
Publishing date	2021-08-26
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1028152-6
ISSN	1557-7422 ; 1043-0342
ISSN (online)	1557-7422
ISSN	1043-0342
DOI	10.1089/hum.2021.042
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Zs.A 2988: Show issues

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Article ; Online: Correction for Gupta et al., "Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration".

Gupta, Saumya Shree / Maetzig, Tobias / Maertens, Goedele N / Sharif, Azar / Rothe, Michael / Weidner-Glunde, Magdalena / Galla, Melanie / Schambach, Axel / Cherepanov, Peter / Schulz, Thomas F

Journal of virology

2024 Volume 98, Issue 4, Page(s) e0013524

Language	English
Publishing date	2024-03-13
Publishing country	United States
Document type	Journal Article ; Published Erratum
ZDB-ID	80174-4
ISSN	1098-5514 ; 0022-538X
ISSN (online)	1098-5514
ISSN	0022-538X
DOI	10.1128/jvi.00135-24
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Zs.A 609: Show issues

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Article ; Online: A 19-color single-tube full spectrum flow cytometry assay for the detection of measurable residual disease in acute myeloid leukemia.

Fokken, Hendrik / Waclawski, Julian / Kattre, Nadine / Kloos, Arnold / Müller, Sebastian / Ettinger, Max / Kacprowski, Tim / Heuser, Michael / Maetzig, Tobias / Schwarzer, Adrian

Cytometry. Part A : the journal of the International Society for Analytical Cytology

2023 Volume 105, Issue 3, Page(s) 181–195

Abstract: Multiparameter flow cytometry (MFC) has emerged as a standard method for quantifying measurable residual disease (MRD) in acute myeloid leukemia. However, the limited number of available channels on conventional flow cytometers requires the division of a ...

Abstract	Multiparameter flow cytometry (MFC) has emerged as a standard method for quantifying measurable residual disease (MRD) in acute myeloid leukemia. However, the limited number of available channels on conventional flow cytometers requires the division of a diagnostic sample into several tubes, restricting the number of cells and the complexity of immunophenotypes that can be analyzed. Full spectrum flow cytometers overcome this limitation by enabling the simultaneous use of up to 40 fluorescent markers. Here, we used this approach to develop a good laboratory practice-conform single-tube 19-color MRD detection assay that complies with recommendations of the European LeukemiaNet Flow-MRD Working Party. We based our assay on clinically-validated antibody clones and evaluated its performance on an IVD-certified full spectrum flow cytometer. We measured MRD and normal bone marrow samples and compared the MRD data to a widely used reference MRD-MFC panel generating highly concordant results. Using our newly developed single-tube panel, we established reference values in healthy bone marrow for 28 consensus leukemia-associated immunophenotypes and introduced a semi-automated dimensionality-reduction, clustering and cell type identification approach that aids the unbiased detection of aberrant cells. In summary, we provide a comprehensive full spectrum MRD-MFC workflow with the potential for rapid implementation for routine diagnostics due to reduced cell requirements and ease of data analysis with increased reproducibility in comparison to conventional FlowMRD routines.
MeSH term(s)	Humans ; Flow Cytometry/methods ; Reproducibility of Results ; Leukemia, Myeloid, Acute/diagnosis ; Bone Marrow/metabolism ; Neoplasm, Residual/diagnosis
Language	English
Publishing date	2023-12-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2099868-5
ISSN	1552-4930 ; 0196-4763 ; 1552-4922
ISSN (online)	1552-4930
ISSN	0196-4763 ; 1552-4922
DOI	10.1002/cyto.a.24811
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Zs.A 1688: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Development of Inducible Molecular Switches Based on All-in-One Lentiviral Vectors Equipped with Drug Controlled FLP Recombinase.

Maetzig, Tobias / Schambach, Axel

Methods in molecular biology (Clifton, N.J.)

2016 Volume 1448, Page(s) 23–39

Abstract: Drug-inducible recombination based on flippase (FLP) is frequently used in animal models and in transgenic cell lines to initiate or to abrogate gene expression. Although the system is highly efficient, functional gene analyses depend on the availability ...

Abstract	Drug-inducible recombination based on flippase (FLP) is frequently used in animal models and in transgenic cell lines to initiate or to abrogate gene expression. Although the system is highly efficient, functional gene analyses depend on the availability of suitable animal models. In contrast, lentiviral vectors are readily available and versatile tools for the transfer of genetic information into a wide variety of target cells, and can be produced at high titer in a timely manner. To combine the advantages of both approaches, we generated a tight, drug-controlled FLP recombinase consisting of a 5' FKBP12 derived conditional destruction domain and a 3' estrogen receptor ligand binding (ERT2) domain. We successfully constructed lentiviral vectors expressing drug-controlled FLP in combination with a fluorescent reporter for recombination of FLP recognition target (FRT) sites located in trans as well as with target alleles located in cis (all-in-one configuration). In this chapter, we describe the design of the drug controlled FLP recombinase, the construction of molecular switches consisting of FLP expressing lentiviral vectors for inducible recombination of target sites located in cis and in trans, as well as the details for the characterization of lentiviral FLP vectors in cell lines.
Language	English
Publishing date	2016
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-3753-0_2
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Article ; Online: A pro B cell population forms the apex of the leukemic hierarchy in Hoxa9/Meis1-dependent AML.

Lieske, Anna / Agyeman-Duah, Eric / Selich, Anton / Dörpmund, Nicole / Talbot, Steven R / Schambach, Axel / Maetzig, Tobias

Leukemia

2022 Volume 37, Issue 1, Page(s) 79–90

Abstract: Relapse is a major challenge to therapeutic success in acute myeloid leukemia (AML) and can be partly associated with heterogeneous leukemic stem cell (LSC) properties. In the murine Hoxa9/Meis1-dependent (H9M) AML model, LSC potential lies in three ... ...

Abstract	Relapse is a major challenge to therapeutic success in acute myeloid leukemia (AML) and can be partly associated with heterogeneous leukemic stem cell (LSC) properties. In the murine Hoxa9/Meis1-dependent (H9M) AML model, LSC potential lies in three defined immunophenotypes, including Lin
MeSH term(s)	Animals ; Mice ; Basic-Leucine Zipper Transcription Factors ; Cell Differentiation ; Leukemia, Myeloid, Acute/genetics ; Leukemia, Myeloid, Acute/drug therapy ; Myeloid Ecotropic Viral Integration Site 1 Protein/genetics ; Neoplastic Stem Cells ; Precursor Cells, B-Lymphoid
Chemical Substances	Basic-Leucine Zipper Transcription Factors ; Myeloid Ecotropic Viral Integration Site 1 Protein ; homeobox protein HOXA9 ; Meis1 protein, mouse
Language	English
Publishing date	2022-12-14
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	807030-1
ISSN	1476-5551 ; 0887-6924
ISSN (online)	1476-5551
ISSN	0887-6924
DOI	10.1038/s41375-022-01775-y
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Zs.A 2295: Show issues

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Article ; Online: Fluorescent genetic barcoding for cellular multiplex analyses.

Maetzig, Tobias / Morgan, Michael / Schambach, Axel

Experimental hematology

2018 Volume 67, Page(s) 10–17

Abstract: Hematopoiesis depends on the controlled differentiation of hematopoietic stem cells to mature cells with defined functions. Although each cell population within the hematopoietic hierarchy can be described by phenotypic markers, isolation of marker pure ... ...

Abstract	Hematopoiesis depends on the controlled differentiation of hematopoietic stem cells to mature cells with defined functions. Although each cell population within the hematopoietic hierarchy can be described by phenotypic markers, isolation of marker pure populations does not necessarily result in cells with homogeneous functionality. However, techniques that enable the efficient characterization of cell behavior with high resolution are limited. Although single-cell transplantation assays demand high mouse numbers and workload, sequencing-based fate tracking techniques require the destruction of the host cell, substantial financial resources, and bioinformatics expertise and suffer from a delay between sample acquisition and data interpretation. To make analyses more efficient, several laboratories recently developed flow cytometry-driven, fluorescence-based multiplexing approaches that enable parallel analysis of longitudinal behavior from multiple clonally derived cells or polyclonal populations. Although these fluorescent genetic barcoding systems are still in their infancy, their power lies in the use of retroviral vectors for gene marking of multiple populations with unique fluorescent color codes. Tracing of color-coded cells by flow cytometry guarantees the accessibility of information on population behavior in real time and at low cost, supports the prospective isolation of cells for downstream analyses, and can be applied to cell line models as well as to human- and animal-derived primary cells. Here, we discuss recent progress in the emerging field of fluorescent genetic barcoding for longitudinal multiplex cell tracking in biomedical research and how this technique will help to uncover mechanisms regulating cell behavior with clonal resolution in a reduced number of experimental samples.
MeSH term(s)	Animals ; Animals, Genetically Modified ; Cell Line ; Cell Lineage ; Cell Separation/methods ; Cell Tracking/methods ; Clone Cells ; Computer Systems ; Flow Cytometry/methods ; Genes, Reporter ; Genetic Vectors ; Glioblastoma/pathology ; Hematopoiesis ; Humans ; Lentivirus/genetics ; Luminescent Proteins/analysis ; Luminescent Proteins/genetics ; Mice ; Microscopy, Fluorescence
Chemical Substances	Luminescent Proteins
Language	English
Publishing date	2018-08-08
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	185107-x
ISSN	1873-2399 ; 0531-5573 ; 0301-472X
ISSN (online)	1873-2399
ISSN	0531-5573 ; 0301-472X
DOI	10.1016/j.exphem.2018.08.001
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Zs.A 922: Show issues

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ab Jg. 2022: Lesesaal (EG)

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Article ; Online: Real-Time Characterization of Clonal Fate Decisions in Complex Leukemia Samples by Fluorescent Genetic Barcoding.

Maetzig, Tobias / Lieske, Anna / Dörpmund, Nicole / Rothe, Michael / Kleppa, Marc-Jens / Dziadek, Violetta / Hassan, Jacob Jalil / Dahlke, Julia / Borchert, Dorit / Schambach, Axel

Cells

2022 Volume 11, Issue 24

Abstract: Clonal heterogeneity in acute myeloid leukemia (AML) forms the basis for treatment failure and relapse. Attempts to decipher clonal evolution and clonal competition primarily depend on deep sequencing approaches. However, this prevents the experimental ... ...

Abstract	Clonal heterogeneity in acute myeloid leukemia (AML) forms the basis for treatment failure and relapse. Attempts to decipher clonal evolution and clonal competition primarily depend on deep sequencing approaches. However, this prevents the experimental confirmation of the identified disease-relevant traits on the same cell material. Here, we describe the development and application of a complex fluorescent genetic barcoding (cFGB) lentiviral vector system for the labeling and subsequent multiplex tracking of up to 48 viable AML clones by flow cytometry. This approach allowed the visualization of longitudinal changes in the in vitro growth behavior of multiplexed color-coded AML clones for up to 137 days. Functional studies of flow cytometry-enriched clones documented their stably inherited increase in competitiveness, despite the absence of growth-promoting mutations in exome sequencing data. Transplantation of aliquots of a color-coded AML cell mix into mice revealed the initial engraftment of similar clones and their subsequent differential distribution in the animals over time. Targeted RNA-sequencing of paired pre-malignant and de novo expanded clones linked gene sets associated with Myc-targets, embryonic stem cells, and RAS signaling to the foundation of clonal expansion. These results demonstrate the potency of cFGB-mediated clonal tracking for the deconvolution of verifiable driver-mechanisms underlying clonal selection in leukemia.
Language	English
Publishing date	2022-12-14
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells11244045
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Article ; Online: Competitive sgRNA Screen Identifies p38 MAPK as a Druggable Target to Improve HSPC Engraftment.

Klatt, Denise / Ha, Teng-Cheong / Schinke, Maximilian / Selich, Anton / Lieske, Anna / Dahlke, Julia / Morgan, Michael / Maetzig, Tobias / Schambach, Axel

Cells

2020 Volume 9, Issue 10

Abstract: Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused ...

Abstract	Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy. The candidates were validated in a competitive transplantation assay and tested in a disease context using IL1B-challenged or X-CGD HSPCs. The sgRNA screen identified
MeSH term(s)	Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Disease Models, Animal ; Genetic Diseases, X-Linked/genetics ; Genetic Diseases, X-Linked/pathology ; Genetic Diseases, X-Linked/therapy ; Genetic Therapy/methods ; Granulomatous Disease, Chronic/genetics ; Granulomatous Disease, Chronic/pathology ; Granulomatous Disease, Chronic/therapy ; Hematopoietic Stem Cell Transplantation/methods ; Hematopoietic Stem Cells/metabolism ; Hematopoietic Stem Cells/pathology ; Humans ; Inflammation/genetics ; Inflammation/pathology ; Inflammation/therapy ; Interleukin-1beta/genetics ; Mice ; RNA/genetics ; RNA/therapeutic use ; Receptors, CXCR4/genetics ; Signal Transduction/genetics ; p38 Mitogen-Activated Protein Kinases/genetics
Chemical Substances	CXCR4 protein, mouse ; IL1B protein, mouse ; Interleukin-1beta ; Receptors, CXCR4 ; RNA (63231-63-0) ; p38 Mitogen-Activated Protein Kinases (EC 2.7.11.24)
Language	English
Publishing date	2020-09-29
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells9102194
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Article ; Online: A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.

Hassan, Jacob Jalil / Lieske, Anna / Dörpmund, Nicole / Klatt, Denise / Hoffmann, Dirk / Kleppa, Marc-Jens / Kustikova, Olga S / Stahlhut, Maike / Schwarzer, Adrian / Schambach, Axel / Maetzig, Tobias

International journal of molecular sciences

2021 Volume 22, Issue 17

Abstract: ... ...

Abstract	HOXA9
MeSH term(s)	Apoptosis ; Biomarkers, Tumor/genetics ; Biomarkers, Tumor/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Group IV Phospholipases A2/antagonists & inhibitors ; Group IV Phospholipases A2/genetics ; High-Throughput Screening Assays ; Homeodomain Proteins/genetics ; Homeodomain Proteins/metabolism ; Humans ; Leukemia, Myeloid, Acute/genetics ; Leukemia, Myeloid, Acute/metabolism ; Leukemia, Myeloid, Acute/pathology ; Myeloid Ecotropic Viral Integration Site 1 Protein/genetics ; Myeloid Ecotropic Viral Integration Site 1 Protein/metabolism ; Tumor Cells, Cultured
Chemical Substances	Biomarkers, Tumor ; Homeodomain Proteins ; MEIS1 protein, human ; Myeloid Ecotropic Viral Integration Site 1 Protein ; homeobox protein HOXA9 ; Group IV Phospholipases A2 (EC 3.1.1.4) ; PLA2G4A protein, human (EC 3.1.1.4)
Language	English
Publishing date	2021-08-30
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms22179411
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Article ; Online: Competitive sgRNA Screen Identifies p38 MAPK as a Druggable Target to Improve HSPC Engraftment

Denise Klatt / Teng-Cheong Ha / Maximilian Schinke / Anton Selich / Anna Lieske / Julia Dahlke / Michael Morgan / Tobias Maetzig / Axel Schambach

Cells, Vol 9, Iss 2194, p

2020 Volume 2194

Abstract	Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy. The candidates were validated in a competitive transplantation assay and tested in a disease context using IL1B-challenged or X-CGD HSPCs. The sgRNA screen identified Mapk14 ( p38 ) as a potential target to increase HSPC engraftment. Knockout of p38 prior to transplantation was sufficient to induce a selective advantage. Inhibition of p38 increased expression of the HSC homing factor CXCR4 and reduced apoptosis and proliferation in HSPCs. For potential clinical translation, treatment of IL1B-challenged or X-CGD HSPCs with a p38 inhibitor led to a 1.5-fold increase of donor cell engraftment. In summary, our findings demonstrate that p38 may serve as a potential druggable target to restore engraftment of HSPCs in the context of X-CGD gene therapy.
Keywords	HSPCs ; X-linked chronic granulomatous disease (X-CGD) ; p38 MAPK ; CRISPR-Cas9 ; bone marrow transplantation ; interleukin-1 beta ; Biology (General) ; QH301-705.5
Subject code	610
Language	English
Publishing date	2020-09-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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