Defined Human Leukemic CD34+ Liquid Cultures to Study HDAC/Transcriptional Repressor Complexes

Windisch, Roland; Kreissig, Sophie; Wichmann, Christian

doi:10.1007/978-1-0716-2788-4_3

Cited by 2 publications

(2 citation statements)

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“…The final construct was verified by sequencing. For viral particle production, HEK293T cells were transfected using a standardized polyethylenimine transfection protocol [11]. The resulting lentiviral supernatant was collected 48 h post-transfection, filtered through a 0.22 μm filter unit and concentrated through centrifugation at 20,000 rpm at 10°C for 2 h. Resuspended lentiviral particles were directly applied to the isolated activated T cells to induce transduction.…”

Section: Methodsmentioning

confidence: 99%

Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets

Xhaxho,

Chen-Wichmann,

Kreissig

et al. 2023

Transfus Med Hemother

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Introduction: Primary human blood cells represent an essential model system to study physiology and disease. However, human blood is a limited resource. During healthy donor plateletpheresis, the leukoreduction system chamber (LRSC) reduces the leukocyte amount within the subsequent platelet concentrate through saturated, fluidized, particle bed filtration technology. Normally, the LRSC is discarded after apheresis is completed. Compared to peripheral blood, LRSC yields 10-fold mononuclear cell concentration. Methods: To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the usually discarded LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells. Results: Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T-cell populations within LRSC, with low CD19+ B cell counts. Magnetic-activated cell sorting (MACS) purified CD3+ T cells were transduced with CD19 CAR-encoding lentiviral self-inactivating vectors using concentrated viral supernatants. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. CD19 CAR T cells were further enriched through anti-CAR MACS, yielding 80% CAR+ T-cell populations. In vitro CAR T cell expansion to clinically relevant numbers was achieved. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell line Nalm6. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 cells. Conclusion: Taken together, we can show that lymphocytes isolated from LRSCs of plateletpheresis sets can be efficiently used for the generation of functional CAR T cells for experimental purposes.

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Section: Methodsmentioning

confidence: 99%

Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets

Xhaxho,

Chen-Wichmann,

Kreissig

et al. 2023

Transfus Med Hemother

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“…Recently, we provided a detailed protocol for KMT2A::ENL-driven immortalization of human HSPCs in vitro. Using a retroviral approach to express the KMT2A::ENL fusion in human HSPCs, long-term ex vivo cultures of KMT2A::ENL+ monocytic progenitor cells could be established this way [ 52 ]. Further, several groups used TALEN and CRISPR/Cas9 genome-editing tools to induce the KMT2A::ENL translocation in primary human HSPCs.…”

Section: Defined Chimeric Transcription Factors Expand Human Cd34+ Pr...mentioning

confidence: 99%

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells

Kreissig,

Windisch,

Wichmann

2023

Cells

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Hemato-oncological diseases account for nearly 10% of all malignancies and can be classified into leukemia, lymphoma, myeloproliferative diseases, and myelodysplastic syndromes. The causes and prognosis of these disease entities are highly variable. Most entities are not permanently controllable and ultimately lead to the patient’s death. At the molecular level, recurrent mutations including chromosomal translocations initiate the transformation from normal stem-/progenitor cells into malignant blasts finally floating the patient’s bone marrow and blood system. In acute myeloid leukemia (AML), the so-called master transcription factors such as RUNX1, KMT2A, and HOX are frequently disrupted by chromosomal translocations, resulting in neomorphic oncogenic fusion genes. Triggering ex vivo expansion of primary human CD34+ stem/progenitor cells represents a distinct characteristic of such chimeric AML transcription factors. Regarding oncogenic mechanisms of AML, most studies focus on murine models. However, due to biological differences between mice and humans, findings are only partly transferable. This review focuses on the genetic manipulation of human CD34+ primary hematopoietic stem/progenitor cells derived from healthy donors to model acute myeloid leukemia cell growth. Analysis of defined single- or multi-hit human cellular AML models will elucidate molecular mechanisms of the development, maintenance, and potential molecular intervention strategies to counteract malignant human AML blast cell growth.

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Defined Human Leukemic CD34+ Liquid Cultures to Study HDAC/Transcriptional Repressor Complexes

Cited by 2 publications

References 61 publications

Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets

Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells

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