Fig 2: Massive ex vivo expansion after seeding LSK SLAM cells for 14 days in S3F11+APU medium. (A) Murine whole bone marrow (WBM) was depleted for cells expressing hematological lin markers (CD5, CD45R, CD11b, Anti-Gr-1, 7-4, and Ter-119) and enriched for LSK (lin-Sca+cKit+) and SLAM (CD48- CD150+) markers. LSK SLAM cells were cultured in a low cell density setting (50 cells/96 well). (B) Expansion factor of HSPCs after cultivating 50 LSK SLAM or 50 lin- cells in different cytokine conditions. Cell number was estimated based on metabolic activity measured with MTT dye. A standard curve with a set number of cells was performed for extrapolation. After 2 weeks of expansion in STIF, SFT, S3F11, or S3F11T medium, only medium containing S3F11(T) cytokines led to significantly higher expansion (over 20,000-fold, n = 3) if HSPCs were purified for an LSK SLAM marker. Statistical analysis performed with two-way ANOVA and Bonferroni multiple comparisons comparing media within sorted population. STIF: SCF, TPO, IGF2, FGF2 in StemSpan SFEMI; SFT: SCF, FLT3-L, TPO in StemSpan SFEMI; S3F11: SCF, IL-3, FLT3-L, IL-11 in IMDM+10% FCS; S3F11T: SCF, IL-3, FLT3-L, IL-11, TPO in IMDM+10% FCS. (C) Mean (n = 3) of surface marker contribution after 7 and 14 days in the respective medium. Adding APU (A83-01, pomalidomide, UM171) to the S3F11 medium maintained the presence of lin- cells, restrained myeloid (CD11b/GR1+) cells on day 7, and attenuated FcεR1α expression until day 14. Statistical analysis performed with Mann–Whitney test. (D) Graphical illustration of the dynamic marker expression during mast cell development in S3F11 medium and the inhibitory influence of A83-01. (E) May-Grünwald Giemsa staining of expanded cells on day 14. The arrows indicate representative cells with dark cytosolic granules typical for mast cells. 20× magnification, 100 μm scale bar. ((F) Expansion factor (number of expanded cells divided by seeded number of 50 LSK SLAM cells) of total, lin-, lin-cKit+, and lin-cKit+Sca1+ cells after 2 weeks in S3F11 or S3F11+APU medium (n = 3). Cell numbers were assessed by Casy. Significantly more total, lin-, lin-cKit+, and LSK+ cells were expanded in S3F11+APU. Two-way ANOVA with Tukey’s correction for multiple testing. “n” is the number of biological replicates; error bars correspond to SD; p < 0.05 = *, p < 0.01 = **, p < 0.01 = ***, p < 0.0001 = ****, and without label if p > 0.05 = not significant.

Fig 3: Immune phenotypic characterization reveals differentiation arrest of early myeloid progenitors after gammaretroviral transduction. Samples from all three sources were analyzed at different time points by flow cytometry for their surface marker expression. (A) Immune phenotype on day 4 revealed a strong similarity of Fresh and S3F11+APU-Exp cells regarding their composition of lin-, myeloid (CD11b/GR1+), CD3/B220/Ter119+, and mast cell (FcεR1α+) progenitors. S3F11-Exp cells harbored an increased amount of lin-FcεR1α+ cells (n = 3 IVIMs). (B) On day 15, in all three cell sources (Fresh, S3F11-Exp, S2F11+APU-Exp), the bulk culture is mostly lin-FcεR1α+ (n = 3 IVIMs). (C) Single sample analyses from IVIM 20220311 showing the development of individual RSF91-transduced samples in comparison with mock and EFS after 21 days in culture. RSF91-transduced Fresh lin- and S3F11+APU-Exp cells differ from mock and EFS. Sample ID is mentioned below the bars. (D) From positively scored plates, individual wells (labeled below) were picked for the expansion of potential clones in culture. Most clones remained lin-FcεR1α- or CD11b/GR1+.