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. 2019 Jan 21;10(1):366.
doi: 10.1038/s41467-018-08166-x.

A highly efficient and faithful MDS patient-derived xenotransplantation model for pre-clinical studies

Yuanbin Song  1 , Anthony Rongvaux  2   3 , Ashley Taylor  1 , Tingting Jiang  4 , Toma Tebaldi  1   5 , Kunthavai Balasubramanian  1 , Arun Bagale  1   6 , Yunus Kasim Terzi  1   7 , Rana Gbyli  1 , Xiaman Wang  1   8 , Xiaoying Fu  1   9 , Yimeng Gao  1 , Jun Zhao  4 , Nikolai Podoltsev  1 , Mina Xu  4 , Natalia Neparidze  1 , Ellice Wong  10 , Richard Torres  11 , Emanuela M Bruscia  12 , Yuval Kluger  4   13   14 , Markus G Manz  15 , Richard A Flavell  16   17 , Stephanie Halene  18
Affiliations

A highly efficient and faithful MDS patient-derived xenotransplantation model for pre-clinical studies

Yuanbin Song et al. Nat Commun. .

Abstract

Comprehensive preclinical studies of Myelodysplastic Syndromes (MDS) have been elusive due to limited ability of MDS stem cells to engraft current immunodeficient murine hosts. Here we report a MDS patient-derived xenotransplantation model in cytokine-humanized immunodeficient "MISTRG" mice that provides efficient and faithful disease representation across all MDS subtypes. MISTRG MDS patient-derived xenografts (PDX) reproduce patients' dysplastic morphology with multi-lineage representation, including erythro- and megakaryopoiesis. MISTRG MDS-PDX replicate the original sample's genetic complexity and can be propagated via serial transplantation. MISTRG MDS-PDX demonstrate the cytotoxic and differentiation potential of targeted therapeutics providing superior readouts of drug mechanism of action and therapeutic efficacy. Physiologic humanization of the hematopoietic stem cell niche proves critical to MDS stem cell propagation and function in vivo. The MISTRG MDS-PDX model opens novel avenues of research and long-awaited opportunities in MDS research.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Enhanced engraftment of adult healthy bone marrow (BM)-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) in human cytokine-knockin MISTRG mice. a Universal experimental setup. Human BM-derived CD34+ HSPCs were pre-incubated with anti-CD3 antibody (OKT3) and injected intrahepatically into newborn (D2–3) NSG or MISTRG mice conditioned with the respective maximum tolerated irradiation doses (NSG 100 cGy, MISTRG 2 × 150 cGy). Mice were analyzed 10–17 (healthy BM), 13–30 (myelodysplastic syndrome (MDS)), and 9−24 (acute myeloid leukemia (AML)) weeks post transplantation. b, c Comparison of overall human CD45+ engraftment in peripheral blood (PB) and BM in NSG versus MISTRG mice. Individual mice are represented by symbols. d Relative distribution of myeloid CD33+ (red), B-lymphoid CD19+ (blue), and T-lymphoid CD3+ (gray) cells as % of human CD45+ cells in NSG vs. MISTRG mice. e BM histology of representative NSG and MISTRG mice from (d). Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) stains for huCD45, huCD15, huCD68 in NSG (top) and MISTRG BM (bottom row) (scale bars 10 µm, original magnification 60×). f, g Comparison of erythroid and megakaryocytic lineage engraftment in BM of NSG and MISTRG mice. h BM histology of representative NSG and MISTRG mice from (d). H&E and IHC stains for huCD235 and huCD61 as in (e). For detailed sample information see Supplementary Table 1. In (c, d, e, f, g) data are represented as means ± S.E.M.; Mann–Whitney test: n.s. not significant, *p < 0.05, **p < 0.01
Fig. 2
Fig. 2
Enhanced engraftment of lower- and higher-risk myelodysplastic syndrome (MDS) in MISTRG mice. a–c Analysis of huCD45 engraftment was performed as detailed in Fig. 1a at >12 weeks post transplantation. a Analysis of MDS-5q-, -SLD-, -MLD-, and -MLD-RS-engrafted NSG and MISTRG mice. b Analysis of MDS/MPN and MDS-EB-1-engrafted NSG and MISTRG mice. c Analysis of MDS-EB-2-engrafted NSG and MISTRG mice. MISTRG afford significantly higher engraftment than NSG in lower- and higher-grade MDS. d–f Relative distribution of myeloid CD33+ (red), B-lymphoid CD19+ (blue), and T-lymphoid CD3+ (gray) cells as % of human CD45+ cells in NSG vs. MISTRG mice. Stacked bar graphs represent means ± S.E.M. Mann–Whitney test; n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. g Split-donor huCD45+ BM engraftment in NSG (black) versus MISTRG (red) mice plotted against CD34+ cell number injected/mouse. Individual mice are represented by symbols. Linear regression, Pearson's correlations and p values of % engraftment to CD34+ cell number in NSG (r = 0.39, p < 0.0001) vs. MISTRG (r = 0.42, p < 0.0001) are displayed. h Percentage of transplanted mice with huCD45+ bonemarrow (BM) engraftment levels >0.01% < 1%, 1–10%, and >10% for split-donor grafts in NSG (59/111, 44/111, and 8/111, respectively) and MISTRG (20/154, 51/154, and 83/154, respectively) mice (Fisher’s exact test, ****p < 0.0001 for NSG vs. MISTRG). For detailed patient sample information see Supplementary Table 1. SLD single lineage dysplasia; MLD multi lineage dysplasia; RS Ringsideroblasts; MPN myeloproliferative neoplasm
Fig. 3
Fig. 3
Erythroid and megakaryocytic lineage representation in myelodysplastic syndrome (MDS) MISTRG xenografts. a Analysis of human erythroid lineage output in NSG versus MISTRG mice engrafted with lower- and higher-risk MDS (as in Fig. 2) via determination of CD71+/− huCD235−/+ expression in hu/muCD45− muTer119− bone marrow (BM). b Analysis of human megakaryocytic lineage output (MK and platelets) in NSG versus MISTRG mice (engrafted as in Fig. 2) via determination of huCD41+ in hu/muCD45− BM. c NSG and MISTRG xenografted with MDS-EB-2 (Y025) BM with inverted myeloid/erythroid ratio. d Patient BM aspirate (top) and sorted human erythroblasts from engrafted NSG and MISTRG BM (bottom) (for overall engraftment see Fig. 2c, Y025). e Representative BM histology from representative NSG and MISTRG recipients engrafted >1% stained with hematoxylin and eosin (H&E), huCD45, huCD235, and huCD61. f Representative fluorescence-activated cell sorting (FACS) plots of erythroid lineage differentiation based on huCD71 and huCD235 expression in huCD45− muCD45− mTer119− cells (huCD71hihuCD235a− (pro-erythroblasts (EB)), huCD71hihuCD235a+ (basophilic EB/normoblasts), huCD71−huCD235a+ (reticulocytes, RBC))
Fig. 4
Fig. 4
MISTRG replicate myelodysplasia and clonal evolution upon disease progression. a NSG and MISTRG were engrafted with low and int-1 risk Sf3B1 mutant myelodysplastic syndrome (MDS) with ring sideroblasts (see Figs. 2, 3) and patient and NSG and MISTRG xenografts were stained with Prussian blue iron stain (scale bars 10 µm, original magnification 60×). b SF3B1 mutation was verified in the patient’s and representative NSG and MISTRG xenografts by Sanger sequencing. c–e MISTRG engrafted with consecutive MDS-EB-1 and secondary acute myeloid leukemia (sAML) samples from the same patient (Y019 and Y028, respectively). c Overall (huCD45+) engraftment in peripheral blood (PB) and bone marrow (BM). Individual mice are represented by symbols, with means ± S.E.M. d Histology from MDS-EB-1 (Y019) diagnostic BM and representative engrafted MISTRG BM. Hematoxylin and eosin (H&E) and huCD61 stains reveal human megakaryocytic dysplasia and reticulin stain reveals bone marrow fibrosis (high-power magnification scale bars 20 µm). e Targeted exome sequencing results from MISTRG xenografted with same patient’s primary MDS-EB-1 diagnosis samples and sAML at the time of disease progression. For each mutation, variant allele frequencies (VAFs) are shown for the patient (black) and representative MISTRG (red) mice with engraftment levels >1%. Mean VAF values between MDS-EB-1 and sAML are connected by lines
Fig. 5
Fig. 5
MISTRG support phenotypic and functional clonal myelodysplastic syndrome (MDS) stem cells with long-term multi-lineage engraftment potential in serial transplantation. a Representative immunohistochemistry (IHC) for huCD45 and huCD34 distribution in NSG (of n = 5) and MISTRG (of n = 12) bone marrow (BM) engrafted with MDS-EB-1 (Y014; scale bars for low-power field: 100 µm, original magnification 10×; high-power field: 10 µm, original magnification 60×). b, c MISTRG engraft phenotypic MDS stem cells. b Representative fluorescence-activated cell sorting (FACS) plots and c quantification of hematopoietic stem cell (HSC) representation (lin−CD34+CD38−CD45RA−CD90+ of huCD45+) of corresponding patient (high-risk MDS-EB2, Y023), and NSG and MISTRG xenografts. d–j MISTRG engraft functional MDS stem cells. d Secondary xenotransplantation experimental setup. e–h Primary and secondary transplantation of MPN/MDS sample with 3% blasts (Y013) comparing e overall huCD45+ engraftment in peripheral blood (PB) and BM, f phenotypic HSC % in BM, and g relative distribution of myeloid CD33+ (red), B-lymphoid CD19+ (blue), and T-lymphoid CD3+ (gray) cells as % of human CD45+ cells in NSG vs. MISTRG mice. In scatter plots individual mice are represented by symbols with means ± S.E.M.; symbols for corresponding 1° and 2° recipient mice are color coded; statistics represent Mann–Whitney test; n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Stacked bar graphs represent means ± S.E.M. Mann–Whitney test; n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. h Clonality was determined in representative primary and secondary MISTRG recipients with engraftment levels >1% via targeted exome sequencing. Variant allele frequencies (VAFs) in primary and secondary recipients were plotted against the corresponding patient’s. Individual mice are represented by symbol shape and mutations are color coded. Linear regression, Pearson's correlations, and p values between patient and xenograft VAF are displayed. i, j Primary and secondary transplantation of a low-risk MDS-RS-SLD sample (Y007) comparing i overall engraftment in PB and BM and j multi-lineage representation in BM of primary and secondary NSG and MISTRG recipients. Individual mice are represented by symbols with means ± S.E.M.; symbols for corresponding 1° and 2° recipient mice are color coded; statistics represent Mann–Whitney test; n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For patient information see Supplementary Table 1
Fig. 6
Fig. 6
MISTRG replicate granulocytic and megakaryocytic differentiation in response to inhibition of mutant isocitrate dehydrogenase 2 (IDH2) in vivo. a In vivo treatment of mutant IDH2 R140Q in MDS-EB-2 (Y021)-engrafted MISTRG mice with the IDH2MUT inhibitor enasidenib. Representative histologic images of vehicle-treated (n = 8, left) and enasidenib-treated (n = 6, right) mice engrafted with MDS-EB-2 (Y021). Immunohistochemistry (IHC) stains for huCD45, huCD68, huCD15, and huCD61 (scale bars 100 µm, original magnification 10×; high-power field 10 µm, original magnification 60×). b Representative fluorescence-activated cell sorting (FACS) plots showing myeloid maturation in response to enasidenib and quantitation of huCD15+ and huCD11b+ expression in vehicle- versus enasidenib-treated MISTRG mice. c Comparison of human engraftment in bone marrow (BM) from vehicle-treated (n = 8) and enasidenib-treated (n = 6) MISTRG mice. d Quantitation of huCD41+ expression in peripheral blood (PB) and BM from vehicle-treated (n = 8) and enasidenib-treated (n = 6) MISTRG mice. e Quantitation of D-2-HG in plasma of pre- and post-administration of vehicle or enasidenib. Individual mice are represented by symbols with mean ± S.E.M.; statistics represent Mann–Whitney test; n.s. not significant, *p < 0.05, **p < 0.01 for aggregate NSG vs. MISTRG. f Representation of variant allele frequencies (VAFs) of driver mutations in vehicle-treated (left) or enasidenib-treated (right) MISTRG (y-axis) plotted against the patient’s VAFs (x-axis). Individual mice represented by symbol shape, mutations color coded. Linear regressors, Pearson's correlations, and p values between patient and xenograft VAFs are displayed
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References

    1. Haase D, et al. New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood. 2007;110:4385–4395. doi: 10.1182/blood-2007-03-082404. - DOI - PubMed
    1. Papaemmanuil E, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013;122:3616–3627. doi: 10.1182/blood-2013-08-518886. - DOI - PMC - PubMed
    1. Cazzola M, Della Porta MG, Malcovati L. The genetic basis of myelodysplasia and its clinical relevance. Blood. 2013;122:4021–4034. doi: 10.1182/blood-2013-09-381665. - DOI - PMC - PubMed
    1. Tohyama K, Tsutani H, Ueda T, Nakamura T, Yoshida Y. Establishment and characterization of a novel myeloid cell line from the bone marrow of a patient with the myelodysplastic syndrome. Br. J. Haematol. 1994;87:235–242. doi: 10.1111/j.1365-2141.1994.tb04904.x. - DOI - PubMed
    1. Drexler HG, Dirks WG, Macleod RA. Many are called MDS cell lines: one is chosen. Leuk. Res. 2009;33:1011–1016. doi: 10.1016/j.leukres.2009.03.005. - DOI - PubMed

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