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. 2021 Mar 5;11(1):5294.
doi: 10.1038/s41598-021-84468-3.

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory

Maike Stahlhut  1   2 , Teng Cheong Ha  1   2 , Ekaterina Takmakova  1   2 , Michael A Morgan  1   2 , Adrian Schwarzer  1   2   3 , Dirk Schaudien  4 , Matthias Eder  2   3 , Axel Schambach  5   6   7 , Olga S Kustikova  8   9
Affiliations

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory

Maike Stahlhut et al. Sci Rep. .

Abstract

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Generation of conditionally immortalised myeloid progenitor cell lines co-expressing Hoxa9 and Meis1 in the presence of doxycycline. (a) Schematic representation of multimodal retroviral vector and integrated provirus to enable constitutive Hoxa9.EGFP and inducible Meis1.dTomato gene co-expression. RSV, Rous sarcoma virus promoter; Δ, SIN configuration with partially deleted U3 of the 3′ long terminal repeat (LTR); ψ, packaging signal; PRE, post-transcriptional regulatory element; EGFP, dTomato, fluorescent proteins. (b) Experimental design. Murine Rosa26rtTA lineage negative cells were transduced on day 1 under myelomonocytic conditions (36S) without doxycycline (DOX). On the next day, cells were split into DOX untreated (DOX-, OFF) and DOX treated cultures, which were supplemented with 1 µg/mL DOX (DOX+ , ON). HOXA9.EGFP+/MEIS1.dTomato+ double-positive cells (DOX+) and HOXA9.EGFP+ (DOX-) were sorted on days 13–23 after transduction followed by a limiting dilution assay to select stable H9M-ciMPs. (c) The number of potential cell lines for DOX+ and DOX- conditions (~ day 45 after transduction). The experiment was performed independently for multimodal retroviral vectors with (MRV) or without (MRV*) LM-PCR linker in three biological replicates for DOX+ and two biological replicates for DOX- conditions (one biological replicate / one 96-well plate). (d-e) The number of generated, pre-selected for LM-PCR analysis and selected cell lines for MRV and MRV* vectors under DOX supplementation. (f) Average vector copy number (VCN) determined for pre-selected (n = 10 for MRV, n = 12 for MRV*) H9M-ciMPs. (g) Gene expression levels of EGFP+dTomato+ double-positive cells in selected (n = 8) H9M-ciMPs after cryopreservation (pre-cultured ~ 3 months in 36S conditions before cryopreservation). Horizontal lines represent the mean values. Each data point represents the result of an individual cell line. LM-PCR, ligation-mediated PCR. H9M-ciMPs, conditionally immortalised myeloid progenitor cell lines engineered to co-express Hoxa9 and Meis1. (d–g) generated in Prism 5 (GraphPad software, San Diego, CA).
Figure 2
Figure 2
Characterisation of H9M-ciMPs under myelomonocytic conditions (36S) and different doxycycline application. (a) Schematic presentation of 1 µg/mL doxycycline application (DOX+ , ON), removal (DOX-, OFF) and reapplication (DOX+ , re-ON). 5 × 105 DOX+ cells were taken for parallel ON and OFF experiments (initial day (D) 0); 1/4 DOX- cells were taken for parallel re-ON experiment (initial D7). Cell numbers for growth curves were calculated based on initial days. Indicated days correspond to time points taken for analysis. H9M-ciMPs #1,5,40 are marked in red, light blue and blue, respectively; #11,12,24,25,29 are marked in black. The H9M-ciMP colour coding applies to all parts of the figure. (b) Exponential growth equation under ON conditions. R2, R-squared value. (c) Percentages of viable cell numbers under different DOX conditions, n = 8. (d) Dot plot overlays to present expression of EGFP+dTomato+ and CD11b+Gr-1+ (frequency percentages are given for H9M-ciMPs #1). Arrows indicate percentages of CD11bhighGr-1high cells. (e) Percentages of EGFP+dTomato+ and CD11b+Gr-1+ cells for D0/ON and D21/ON, n = 8. (f) Relative transcriptional expression of Meis1 and Hoxa9-total in H9M-ciMPs upon DOX ON/OFF/re-ON conditions. Expression levels in non-transduced non-cultured lineage negative cells (Mock) were set to 1. The mean values are represented by horizontal lines, n = 8. (g) Cytospin analysis (May-Grünwald/Giemsa staining, magnification × 80) of H9M-ciMP #1 under different DOX supplementation. Dashed black arrows indicate immature myeloid cells; solid black arrows indicate neutrophils or macrophages. (h) Histogram presentation of differential cell counts for H9M-ciMPs under different DOX conditions. #1,5,11,12,40, n = 5; #24,25,29, n = 3. IMM/MO, immature cells: myeloblast-, promyelocyte-, monoblast-, promonocyte- and monocyte-like cells; MY/MM, myelocyte-, metamyelocyte-like; Nph, band neutrophil, segmented neutrophil; Mph, macrophage. Data are represented as mean ± SD. (i) Exponential growth equations and linear regression after DOX removal. (j) Exponential growth equation after DOX reapplication. The values R2 and r2 are indicated as a measure of goodness-of-fit of the exponential and linear growth models. Each data point represents the result of an individual cell line. ns, not significant, P>0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (non-parametric two-tailed Mann–Whitney test). H9M-ciMPs, conditionally immortalised myeloid progenitors co-expressing Hoxa9/Meis1. Figure 2c,e,f, generated in Matplotlib 3.1.1 https://matplotlib.org; (d) generated in PowerPoint using plots from Flowjo 10 (Tree Star, Ashland, OR); (g) generated in PowerPoint using pictures from NDP.view 2.8.24 viewing software U12388-01, https://www.hamamatsu.com/eu/en/product/type/U12388-01/index.html.
Figure 3
Figure 3
Selection and characterisation of H9M-ciMPs under stem cell maintaining conditions and doxycycline application. (a) Schematic presentation of H9M-ciMP selection under stem cell maintaining conditions (STIF) and 1 µg/mL doxycycline (ON) supplementation. H9M-ciMPs resulting from MRV or MRV* experiments were cultured under STIF conditions. MRV, MRV*, multimodal retroviral vectors with or without LM-PCR linker, respectively. Each data point represents the result of an individual cell line. Horizontal lines represent the mean values. (b) Exponential growth equation for H9M-ciMPs cultured under STIF/ON conditions. R2, the R-squared value. (c) Dot plot overlays to present expression of CD11b+Gr-1+ in H9M-ciMPs at day 10 of culture under STIF/ON conditions (frequency percentages are given for H9M-ciMP cell line #1). (d) Percentages of EGFP+dTomato+ and CD11b+Gr-1+ cells for D10/ON and D17/ON STIF conditions, n = 3. Each data point represents the result of an individual cell line. Horizontal lines represent the mean values. ns, not significant, p > 0.05 (non-parametric two-tailed Mann–Whitney test). (e) Histogram presentation of relative transcriptional expression of Meis1 and Hoxa9 (corresponds to Hoxa9-total) in H9M-ciMPs. Expression levels in non-transduced non-cultured lineage negative cells (Mock) were set to 1. Data are represented as mean ± SD, n = 3. (f) Histogram presentation of differential cell count for H9M-ciMPs under STIF/ON conditions. IMM/MO, immature cells: myeloblast-, promyelocyte-, monoblast-, promonocyte- and monocyte-like cells; MY/MM, myelocyte-, metamyelocyte-like; Nph, band neutrophil, segmented neutrophil; Mph, macrophage. Data are represented as mean ± SD, n = 3. H9M-ciMPs, conditionally immortalised myeloid progenitor cell lines engineered to co-express Hoxa9 and Meis1. D, day. For (b) and (c) H9M-ciMPs #1, 5 and 40 are marked in red, light blue and blue, respectively. (a, d) generated in Prism 5 (GraphPad software, San Diego, CA); (c) generated in PowerPoint using plots from Flowjo 10 (Tree Star, Ashland, OR).
Figure 4
Figure 4
Drug-controlled murine serial transplantation of H9M-ciMPs. (a) Experimental design: H9M-ciMPs cultured in myelomonocytic (36S) or stem cell maintaining (STIF) cytokine conditions were transplanted into primary (1°) recipient mice and symptomatic mice were analysed 8 weeks after transplantation. Bone marrow (BM) cells from selected 1° 36S and STIF recipients were transplanted into secondary (2°) recipients and analysed after 2 weeks. 1° and 2° recipients received doxycycline (DOX). (b) Spleen weight of 1° and 2° 36S and STIF recipients. Dashed lines indicate the normal spleen weight range. (c) Analysis of white blood cell (WBC) counts in 1° and 2° recipients (WBC data for 1° STIF recipient #227 are not presented due to a technical problem). (d) Histogram presentation of Meis1 and Hoxa9 transcriptional expression in BM cells of 1° and 2° 36S and STIF recipients. The expression level of the control non-transplanted DOX-treated murine cells (Mock) was set to 1. Data are represented as mean ± SD for the animals in each group, n = 4. *, P < 0.05 (non-parametric two-tailed Mann–Whitney test). (e) Average vector copy number (VCN) in total BM cells of individual 1° and 2° 36S and STIF recipients. (f) Histogram presentation of percentages of EGFP and/or dTomato expression in BM cells of 1° and 2° 36S and STIF recipients. Data are represented as mean ± SD for the animals in each group, n = 4. Percentages of EGFP+dTomato+ cells were compared (1° 36S versus 1° STIF, 2° 36S versus 2° STIF). The differences are not statistically significant, P > 0.05. For (b) and (c), horizontal lines indicate median values; for (e), horizontal lines indicate mean values. Each data point represents the result of an individual animal, n = 4, *, P < 0.05. Comparisons were made using the non-parametric two-tailed Mann–Whitney test. H9M-ciMPs, conditionally immortalised myeloid progenitor cell lines engineered to co-express Hoxa9 and Meis1; W, week. (b, c, e) generated in Prism 5 (GraphPad software, San Diego, CA).
Figure 5
Figure 5
Immunophenotype, morphology and insertional analysis of doxycycline-dependent H9M-ciMP induced AMLs. (a) Strategy of bone marrow (BM) immunophenotype analysis of doxycycline-treated primary (1°) recipients transplanted with H9M-ciMPs cultured under myelomonocytic (36S) or stem cell maintaining (STIF) conditions. Flow cytometry plot overlays to present expression of CD11b/Gr-1 and CD44/c-KIT in the EGFP+dTomato+ and EGFP−dTomato+ cells. Frequency percentages are given for recipients #224 (36S) and #228 (STIF). (b–c) Histogram presentation of percentages of CD11b+ and/or Gr-1+ (b) and CD44+ and/or c-KIT+ (c) positive cells in gated EGFP+dTomato+ and EGFP−dTomato+ cells from BM of 1° and secondary (2°) 36S and STIF recipients. Data are represented as mean ± SD for animals in each group, n = 4. (d) Cytospin analysis (May-Grünwald/Giemsa staining, magnification × 60) of BM cells from selected 1° and 2° 36S and STIF recipients. (e) Ligation-mediated PCR analysis demonstrating insertion sites in BM cells of 1° 36S and STIF recipients. W, water; M, 100 bp marker; bp, base pairs; ic, internal control; mock, BM cells from non-transplanted doxycycline-treated mice. (f) Histogram presentation of transcriptional expression for genes located in the Ninj2 locus in BM cells of 1° 36S and STIF recipients. The expression level of the control non-transplanted DOX-treated murine cells (Mock) was set to 1. Data are represented as mean ± SD, n = 4 (for Ninj2), n = 3 (for other genes). (g) Genes located within ± 250 kb from retroviral insertion in the Ninj2 locus. Scale based on the Ensembl database, release 98—August 2020. H9M-ciMPs, conditionally immortalised myeloid progenitor cell lines engineered to co-express Hoxa9 and Meis1; AML, acute myeloid leukaemia.
Figure 6
Figure 6
Characterisation of H9M-ciMPs cultured under T-lymphoid conditions and doxycycline application. (a) Experimental design describing the co-culture of H9M-ciMPs with OP9-DL1 stromal cells under T-lymphoid cytokine conditions following 36S (36S.T-Ly) and STIF (STIF.T-Ly) conditions, respectively, in the presence of 1 µg/mL doxycycline (DOX+ , ON). (b) Exponential growth equation for H9M-ciMPs under 36S.T-Ly and STIF.T-Ly conditions. R2, R-squared value. (c) Percentages of viable cell number under 36S.T-Ly (n = 8) and STIF.T-Ly (n = 3) conditions. (d) Dot plot overlays to present expression of EGFP+dTomato+, CD11b+Gr-1+ and CD44+CD25+ under 36S.T-Ly (n = 8) and STIF.T-Ly (n = 3) conditions (frequency percentages are given for H9M-ciMP #1). (e) Percentages of EGFP+dTomato+ under initial 36S and STIF conditions and under 36S.T-Ly (n = 8) and STIF.T-Ly (n = 3) conditions at D0/ON and D17/ON. D, day. (f) Histogram presentation of relative transcriptional expression of Meis1 and Hoxa9 (corresponds to Hoxa9-total) in H9M-ciMPs under 36S.T-Ly (n = 8) and STIF.T-Ly (n = 3) conditions. Expression levels in non-transduced non-cultured lineage negative cells (Mock) were set to 1. Data are represented as mean ± SD. (g) Histogram presentation of differential cell count for H9M-ciMPs under 36S.T-Ly (n = 8) and STIF.T-Ly (n = 3) conditions at D0/ON and D17/ON. IMM/MO, immature cells: myeloblast-, promyelocyte-, monoblast-, promonocyte- and monocyte-like cells; MY/MM, myelocyte-, metamyelocyte-like; Nph, band neutrophil, segmented neutrophil; Mph, macrophage. Data are represented as mean ± SD. ***, P < 0.001, statistically significant differences revealed for IMM/MO and MY/MM. (h) Cytospin analysis (May-Grünwald/Giemsa staining, magnification × 80) of selected cell line #1 for D0/ON and D17/ON. Dashed black arrows indicate immature myeloid cells; solid black arrows indicate more differentiated cells. (i) Percentages of CD11b+Gr-1+ cells in H9M-ciMPs cultured under 36S.T-Ly (n = 8) and STIF.T-Ly (n = 3) conditions at D0/ON and D17/ON. H9M-ciMPs, conditionally immortalised myeloid progenitor cell lines co-expressing Hoxa9/Meis1. H9M-ciMPs #1,5,40 are marked in red, light blue and blue, respectively, and #11,12,24,25,29 are marked in black as “others”. Each data point represents the result of an individual cell line. For (c, e, i), horizontal lines indicate mean values. ns, not significant, p > 0.05; *, P < 0.05; **, P < 0.01. Comparisons were made using non-parametric two-tailed Mann–Whitney test; (c) generated in Matplotlib 3.1.1 https://matplotlib.org; (d) generated in PowerPoint using plots from Flowjo 10 (Tree Star, Ashland, OR); (e, i) generated in Prism 5 (GraphPad Software, San Diego, CA); (h) generated in PowerPoint using pictures from NDP.view2.8.24 viewing software U12388-01, https://www.hamamatsu.com/eu/en/product/type/U12388-01/index.html.
Figure 7
Figure 7
ciLICs co-expressing Hoxa9/Meis1 under different microenvironmental conditions ex vivo upon switching doxycycline ON/OFF/re-ON. Doxycycline (DOX) application (DOX+ , ON), removal (DOX-, OFF) reapplication (DOX+ , re-ON); ciLIC, conditionally immortalised leukaemia initiating cells; STIF, stem cell maintaining conditions; T-lymphoid, co-culture with OP9-DL1 stromal cells under T-lymphoid conditions; neg, negative. Red arrows indicate ciLICs; blue arrows indicate committed cells, e.g. neutrophils.
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References

    1. Stahlhut M, et al. Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression. Biomaterials. 2015;63:189–201. doi: 10.1016/j.biomaterials.2015.06.022. - DOI - PubMed
    1. Stahlhut M, Schambach A, Kustikova OS. Multimodal lentiviral vectors for pharmacologically controlled switching between constitutive single gene expression and tetracycline-regulated multiple gene collaboration. Hum. Gene Ther. Methods. 2017;28:191–204. doi: 10.1089/hgtb.2017.073. - DOI - PMC - PubMed
    1. Kustikova OS, et al. Dose response and clonal variability of lentiviral tetracycline-regulated vectors in murine hematopoietic cells. Exp. Hematol. 2014;42:505–515.e7. doi: 10.1016/j.exphem.2014.03.004. - DOI - PubMed
    1. Horton SJ, Huntly BJP. Recent advances in acute myeloid leukemia stem cell biology. Haematologica. 2012;97:966–974. doi: 10.3324/haematol.2011.054734. - DOI - PMC - PubMed
    1. Konopleva MY, Jordan CT. Leukemia stem cells and microenvironment: biology and therapeutic targeting. J. Clin. Oncol. 2011;29:591–599. doi: 10.1200/JCO.2010.31.0904. - DOI - PMC - PubMed

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