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| Status |
Public on May 22, 2024 |
| Title |
Engineering an inducible leukemia-associated transcription factor enables large-scale ex vivo production of functional human phagocytes |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible transcription factor to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) transcription factor to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 years of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated towards CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established a novel transcription factor that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable transcription factors have the potential to be applied as molecular tools to produce functional immune cells for cell therapy.
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| Overall design |
RNA expression analysis via NanoString nCounter Analysis System with four different cell types (three biological replicates each) using the myeloid innate immunity panel. Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen). The four different cell types include:
- Untransduced hematopoietic stem and progenitor cells (HSPCs) from healthy donors (cultivated for 3 days in media with IL-3, IL-6, SCF, TPO, GM-CSF, FLT3-L)
- HSPCs transduced with a retroviral construct DD-MLL-ENL-ires-GFP. DD is a destabilization domain leading to rapid degradation of MLL-ENL. The small molecule Shield-1 prevents degradation. The used cells were cultivated in the presence of Shield-1 for over 50 days to obtain an MLL-ENL expressing, GFP+ progenitor cell population. Cultivation in media with IL-3, IL-6, SCF, TPO, GM-CSF, FLT3-L.
- Macrophages differentiated from the MLL-ENL expressing progenitor cells after withdrawal of Shield-1 and cultivation in media with GM-CSF, LPS and IFN? for 14 days.
- CD14+ monocytes derived from peripheral blood from healthy donors. PBMCs were isolated via density gradient centrifugation. CD14+ monocytes were isolated using the classical monocyte isolation kit from Miltenyi.
****Submitter states "the raw fastq files are not uploaded because they are patient derived cells and under German law we cannot publicly share the raw sequencing file on GEO"***
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| Contributor(s) |
Windisch R, Danese A, Fischer A, Wichmann C |
| Citation(s) |
38857395 |
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| Submission date |
Jul 20, 2023 |
| Last update date |
Aug 27, 2024 |
| Contact name |
Anna Danese |
| Organization name |
Ludwig Maximilians University
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| Street address |
Grosshaderner strasse 9
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| City |
Planegg |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| Platforms (1) |
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| Samples (14)
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| Relations |
| BioProject |
PRJNA996892 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE237826_RAW.tar |
420.0 Mb |
(http)(custom) |
TAR (of H5) |
| GSE237826_RNAseq_MLL_ENL.raw.counts.txt.gz |
977.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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