Research ArticleBlood Disorders

Thrombopoietin receptor–independent stimulation of hematopoietic stem cells by eltrombopag

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Science Translational Medicine  12 Sep 2018:
Vol. 10, Issue 458, eaas9563
DOI: 10.1126/scitranslmed.aas9563
  • Fig. 1 EP stimulates multilineage differentiation and self-renewal capacity of human HSCs.

    (A) Scheme showing isolation of human HSCs for colony-forming assays. BM, bone marrow; HSPC, hematopoietic stem and progenitor cell. (B and C) MK (B) and erythroid (C) colony formation in collagen-based culture system (n = 4). (D) Granulocytic colony formation in methylcellulose semi-solid medium (n = 7). CFU, colony-forming unit; MK, MK colonies; BFU-E, burst-forming unit erythroid colonies; CFU-G, granulocyte colonies. Representative morphology for each type of colony is also shown. Scale bars, 100 μm. (E) Colonies from primary methylcellulose cultures were serially replated biweekly (n = 7). Results are represented as fold change of colonies compared to nontreated (NT) vehicle control (water). (F) Fold change of percentage of dividing HSCs compared to vehicle-treated control 24 hours after treatment. Percentage of dividing HSCs is expressed as the number of HSCs with at least one completed division out of total HSCs scored. (G) Representative fluorescence-activated cell sorting (FACS) plots of Ki-67 and Hoechst staining of HSCs (LinCD34+CD38CD49f+) exposed to rhTPO and EP at low and high concentrations for 24 hours. (H) Quantification of percentage of cells in G0 versus G1 of cell cycle after stimulation with TPO, EP-lo, and EP-hi for 24 hours in liquid culture compared to vehicle control (NT). rhTPO, 100 ng/ml of rhTPO; EP-lo, EP (3 μg/ml); EP-hi, EP (10 μg/ml). n = 4. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (paired Student’s t test).

  • Fig. 2 EP triggers molecular pathways in human HSCs distinct from TPO signaling.

    (A and B) Volcano plots showing the distribution of gene expression changes in human HSCs upon treatment with TPO (A) and EP (B). Up-regulated genes are indicated in red, whereas down-regulated genes are shown in green. (C) Supervised hierarchical clustering of expression profiles of HSCs treated with TPO, EP, or vehicle control (NT) using the genes differentially expressed among groups. (D) Venn diagram comparing DEGs between TPO- and EP-treated HSCs. (E) Enrichment analysis of DEGs containing iron-responsive elements (IREs) in TPO- or EP-treated HSCs. Hypergeometric test was used to calculate the significance (P value) of IRE enrichment. (F and G) Ingenuity pathway analysis (IPA) of upstream regulators (F) and canonical pathways (G) for DEGs specific to EP-treated HSCs. Top 10 pathways with significance P < 0.05 are shown. The yellow and blue bars represent positive and negative Z scores, respectively. Black bars represent the −log10 of the significance (P value). The red line represents the cutoff of significance (P = 0.05). Iron-related pathways are shown in bold. (H) Quantitative real-time polymerase chain reaction (qRT-PCR) validation of DEGs in EP-treated HSCs. Fold changes of HIF1A, HMOX1, and GADD45B mRNA expression are shown. If not specified otherwise, then data are means ± SEM. *P < 0.05, **P < 0.01 (paired Student’s t test).

  • Fig. 3 EP increases HSC self-renewal independently of the TPO-R.

    (A) Serial colony-forming capacity of Mpl−/− HSCs treated with EP (10 μg/ml) compared to NT (water; n = 4). (B) LTC-IC assay of Mpl−/− LinSca-1+c-Kit+ (LSK) cells after ex vivo treatment with vehicle control (NT) or EP. Left: Bar plot showing the fold change of LTC-IC frequency compared to NT, estimated for each of the individual mice (n = 4, paired t test). Right: Scatter plot showing LTC-IC frequency for EP and NT. (C) Scheme showing in vivo treatment schedule of Mpl−/− mice. Mice were treated with EP daily for 28 days, followed by immunophenotypic and functional assessment of HSC frequency. KO, knockout. (D) Frequency of phenotypic HSCs within the bone marrow after EP (n = 14 mice) or vehicle treatment (NT; n = 12 mice). *P < 0.05 (unpaired t test). (E and F) Representative images (E) and fold change of stem cell frequency (F) assessed by LTC-IC assay for Mpl−/− LSK cells. Scale bars, 1 mm. (G) HSC frequency in EP-treated mice compared to control determined by limiting dilution transplantation. Results are pooled from five independent experiments (n = 74 and 70 recipient mice for NT and EP, respectively). If not specified otherwise, then data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (paired Student’s t test).

  • Fig. 4 EP induces molecular changes consistent with intracellular iron reduction.

    (A) Distribution of expression changes in HSCs upon EP treatment. (B) Enrichment analysis of DEGs containing IREs in EP-treated HSCs. (C) IPA analysis of upstream regulators of DEGs. (D) Fold change of Tfrc, Ftl1, Slc40a1, Hamp, and Hif1a in EP-treated HSCs by qPCR. (E and F) Change of CD71/TFRC mean fluorescence intensity (MFI) in HSCs (E) or HPC7 cell line (F) after EP treatment [n = 6 and 2, respectively, for (E) and (F)]. (G) Calcein staining to measure the concentration of intracellular iron. Results are presented as MFI of calcein in HSCs treated with EP relative to NT. (H) IPA of canonical pathways with DEGs in EP-treated HSCs. Top 10 pathways with significance of P < 0.05 are shown. (I and J) IPA of canonical pathways (I) and upstream regulators (J) with altered metabolites (fold change > 1.5 or <−1.5) in the metabolite profiling of HPC7 cells with EP treatment (n = 4). The yellow and blue bars represent positive and negative Z scores, respectively. Black bars represent the −log10 of the significance (P value). The red line represents the cutoff of significance (P = 0.05). If not specified otherwise, then data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (paired Student’s t test).

  • Fig. 5 EP increases the number of functional long-term HSCs in patients with hematopoietic deficiency.

    (A to C) HSC frequency in healthy donors enumerated by the LTC-IC assay. Left: LTC-IC frequency in each individual donor (n = 4). Right: Scatter plot showing LTC-IC frequency of CD34+ cells in NT versus those treated with iron chelators DFO (10 μM) (A), DFX (5 μM) (B), or EP with or without rescue (C). n.s., not significant. (D) Representative images of LTC-IC assay showing limiting dilutions of CD34+ cells from healthy donors treated ex vivo with either vehicle (NT) or EP with or without rescue (EP + FAC). (E) FACS analysis of HSCs (LinCD34+ CD38CD49f+) in ITP patients treated with either romiplostim (control) or EP. (F and G) Quantification of phenotypic HSCs within BMMNC (F) and CD34+ CD38 (G) compartments. (H) Representative images of LTC-IC assay showing CD34+ cells from ITP patients treated in vivo with romiplostim (control) or EP. (I) HSC frequency in ITP patients treated in vivo with romiplostim (n = 2) or EP (n = 4). Scale bars, 1 mm. LTC-IC assay. HSC frequencies were estimated by Poisson statistics using extreme limiting dilution analysis (ELDA). If not specified otherwise, then data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (paired Student’s t test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/458/eaas9563/DC1

    Fig. S1. Effects of EP on human MPP cells.

    Fig. S2. Effects of EP on cell cycling of HSCs.

    Fig. S3. Alterations of iron-associated gene expression in human hematopoietic cells after EP treatment.

    Fig. S4. TPO-R–independent stimulation of HSC self-renewal by EP.

    Fig. S5. Reduction of HSCs in c-Mpl−/− mice.

    Fig. S6. Complete blood counts of c-Mpl−/− mice after in vivo treatment with EP.

    Fig. S7. Alterations of glycolysis-associated genes in mouse HSCs after EP treatment.

    Fig. S8. FAC dose finding for ex vivo assays.

    Fig. S9. Intracellular ROS concentrations in HSCs after EP treatment.

    Fig. S10. Iron-dependent HSC self-renewal upon chelator treatment.

    Fig. S11. Iron-associated gene expression changes in ITP patients treated with EP.

    Table S1. DEGs in TPO-treated human HSCs.

    Table S2. DEGs in EP-treated human HSCs.

    Table S3. Commonly altered DEGs between EP- and TPO-treated human HSCs.

    Table S4. IPA analysis with DEGs specifically detected in EP-treated human HSCs.

    Table S5. Plasma concentrations of EP in mice.

    Table S6. DEGs in EP-treated mouse HSCs.

    Table S7. IPA analysis with DEGs in EP-treated mouse HSCs.

    Table S8. Altered metabolites detected in EP-treated HPC7 cells.

    Table S9. Pathway analysis with altered metabolites detected in EP-treated HPC7 cells.

    Table S10. IPA analysis with altered metabolites detected in EP-treated HPC7 cells.

    Table S11. Information on primary human-derived specimens.

    Table S12. List of antibodies used in FACS experiments.

    Table S13. List of primers for qPCR.

  • The PDF file includes:

    • Fig. S1. Effects of EP on human MPP cells.
    • Fig. S2. Effects of EP on cell cycling of HSCs.
    • Fig. S3. Alterations of iron-associated gene expression in human hematopoietic cells after EP treatment.
    • Fig. S4. TPO-R–independent stimulation of HSC self-renewal by EP.
    • Fig. S5. Reduction of HSCs in c-Mpl−/− mice.
    • Fig. S6. Complete blood counts of c-Mpl−/− mice after in vivo treatment with EP.
    • Fig. S7. Alterations of glycolysis-associated genes in mouse HSCs after EP treatment.
    • Fig. S8. FAC dose finding for ex vivo assays.
    • Fig. S9. Intracellular ROS concentrations in HSCs after EP treatment.
    • Fig. S10. Iron-dependent HSC self-renewal upon chelator treatment.
    • Fig. S11. Iron-associated gene expression changes in ITP patients treated with EP.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). DEGs in TPO-treated human HSCs.
    • Table S2 (Microsoft Excel format). DEGs in EP-treated human HSCs.
    • Table S3 (Microsoft Excel format). Commonly altered DEGs between EP- and TPO-treated human HSCs.
    • Table S4 (Microsoft Excel format). IPA analysis with DEGs specifically detected in EP-treated human HSCs.
    • Table S5 (Microsoft Excel format). Plasma concentrations of EP in mice.
    • Table S6 (Microsoft Excel format). DEGs in EP-treated mouse HSCs.
    • Table S7 (Microsoft Excel format). IPA analysis with DEGs in EP-treated mouse HSCs.
    • Table S8 (Microsoft Excel format). Altered metabolites detected in EP-treated HPC7 cells.
    • Table S9 (Microsoft Excel format). Pathway analysis with altered metabolites detected in EP-treated HPC7 cells.
    • Table S10 (Microsoft Excel format). IPA analysis with altered metabolites detected in EP-treated HPC7 cells.
    • Table S11 (Microsoft Excel format). Information on primary human-derived specimens.
    • Table S12 (Microsoft Excel format). List of antibodies used in FACS experiments.
    • Table S13 (Microsoft Excel format). List of primers for qPCR.

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