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TGFβ Inhibitor A83-01 Enhances Murine HSPC Expansion for Gene Therapy

Authors :
Jenni Fleischauer
Antonella Lucia Bastone
Anton Selich
Philipp John-Neek
Luisa Weisskoeppel
Dirk Schaudien
Axel Schambach
Michael Rothe
Source :
Cells, Vol 12, Iss 15, p 1978 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

Murine hematopoietic stem and progenitor cells (HSPCs) are commonly used as model systems during gene therapeutic retroviral vector development and preclinical biosafety assessment. Here, we developed cell culture conditions to maintain stemness and prevent differentiation during HSPC culture. We used the small compounds A83-01, pomalidomide, and UM171 (APU). Highly purified LSK SLAM cells expanded in medium containing SCF, IL-3, FLT3-L, and IL-11 but rapidly differentiated to myeloid progenitors and mast cells. The supplementation of APU attenuated the differentiation and preserved the stemness of HSPCs. The TGFβ inhibitor A83-01 was identified as the major effector. It significantly inhibited the mast-cell-associated expression of FcεR1α and the transcription of genes regulating the formation of granules and promoted a 3800-fold expansion of LSK cells. As a functional readout, we used expanded HSPCs in state-of-the-art genotoxicity assays. Like fresh cells, APU-expanded HSPCs transduced with a mutagenic retroviral vector developed a myeloid differentiation block with clonal restriction and dysregulated oncogenic transcriptomic signatures due to vector integration near the high-risk locus Mecom. Thus, expanded HSPCs might serve as a novel cell source for retroviral vector testing and genotoxicity studies.

Details

Language :
English
ISSN :
20734409
Volume :
12
Issue :
15
Database :
Directory of Open Access Journals
Journal :
Cells
Publication Type :
Academic Journal
Accession number :
edsdoj.b5b2afbbff114c7087cce1d60b31eee8
Document Type :
article
Full Text :
https://doi.org/10.3390/cells12151978