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1 Supplementary Materials for Thrombopoietin receptor independent stimulation of hematopoietic stem cells by eltrombopag Yun-Ruei Kao, Jiahao Chen, Swathi-Rao Narayanagari, Tihomira I. Todorova, Maria M. Aivalioti, Mariana Ferreira, Pedro M. Ramos, Celine Pallaud, Ioannis Mantzaris, Aditi Shastri, James B. Bussel, Amit Verma, Ulrich Steidl, Britta Will Corresponding author. (B.W.); (U.S.) The PDF file includes: Published September 8, Sci. Transl. Med., eaas956 (8) DOI:.6/scitranslmed.aas956 Fig. S. Effects of on human MPP cells. Fig. S. Effects of on cell cycling of HSCs. Fig. S. Alterations of iron-associated gene expression in human hematopoietic cells after treatment. Fig. S4. -R independent stimulation of HSC self-renewal by. Fig. S5. Reduction of HSCs in c-mpl / mice. Fig. S6. Complete blood counts of c-mpl / mice after in vivo treatment with. Fig. S7. Alterations of glycolysis-associated genes in mouse HSCs after treatment. Fig. S8. FAC dose finding for ex vivo assays. Fig. S9. Intracellular ROS concentrations in HSCs after treatment. Fig. S. Iron-dependent HSC self-renewal upon chelator treatment. Fig. S. Iron-associated gene expression changes in ITP patients treated with. Other Supplementary Material for this manuscript includes the following: (available at Table S (Microsoft Excel format). DEGs in -treated human HSCs. Table S (Microsoft Excel format). DEGs in -treated human HSCs. Table S (Microsoft Excel format). Commonly altered DEGs between - and -treated human HSCs. Table S4 (Microsoft Excel format). IPA analysis with DEGs specifically detected in -treated human HSCs. Table S5 (Microsoft Excel format). Plasma concentrations of in mice. Table S6 (Microsoft Excel format). DEGs in -treated mouse HSCs.
2 Table S7 (Microsoft Excel format). IPA analysis with DEGs in -treated mouse HSCs. Table S8 (Microsoft Excel format). Altered metabolites detected in -treated HPC7 cells. Table S9 (Microsoft Excel format). Pathway analysis with altered metabolites detected in treated HPC7 cells. Table S (Microsoft Excel format). IPA analysis with altered metabolites detected in -treated HPC7 cells. Table S (Microsoft Excel format). Information on primary human-derived specimens. Table S (Microsoft Excel format). List of antibodies used in FACS experiments. Table S (Microsoft Excel format). List of primers for qpcr.
3 Fold change (CFU compared to ) Fold change of CFU Fold change of CFU Fold change of CFU Supplementary Materials A B C Immature Mk Mature Mk BFU-E 5 4 CFU-G D 4 Serial replatings Primary st replate nd replate Fig. S. Effects of on human MPP cells. (A-B) Megakaryocyte (A) and erythroid (B) colony formation in collagen-based culture system (n=4). (C) Granulocytic colony formation in methylcellulose semi-solid medium (n=7). CFU: colony-forming unit; MK: megakaryocyte colonies; BFU-E: erythroid colonies; CFU-G: granulocyte colonies. (D) Colonies from primary methylcellulose cultures were serially replated bi-weekly (n=7). Results are represented as fold change of colonies compared to non-treated () vehicle control (H O). Data are mean ± SEM. p <.5, p <., p <.(Paired Student s t-test).
4 EdU A K K 5K K K K 5K K B 5K K 5K K Propidium iodide 5K K 5K K C Fig. S. Effects of on cell cycling of HSCs. (A) Representative FACS plots of EdU vs. propidium iodide (PI) in human HSCs after 4-hour treatment. (B) Fold change of % human HSCs in different stages of cell cycle (G, S, G /M) after 4-hour treatment compared to the vehicle control. G : EdU PI ; S: EdU + ; G /M: EdU PI +. (C) Fraction of HSCs with at least one completed division at different time points after treatment. Lower panel is showing the statistical significance compared to. Data are
5 mean ± SEM. n.s. non-significant. p <.5 (Paired Student s t-test). (D) Frequency distribution of dividing cells per single HSCs at 7, 96, and hours after treatment. : ng/ml of recombinant human thrombopoietin, : g/ml of eltrombopag, : g/ml of eltrombopag.
6 Fold change (mrna compared to ) Fold change (% GAPDH compared to ) A TFRC SLCA SLC5A8 SLC5A7 FTL HAMP B LDHAL6B PGAM ALDOC PFKL HK PCK Fig. S. Alterations of iron-associated gene expression in human hematopoietic cells after treatment. (A) Quantitative RT-PCR validation of iron-related genes in CD4 cells from healthy donors treated with either vehicle (H O) or ( g/ml) in liquid culture for 6 hours. Results are represented as fold change of % GAPDH compared to non-treated () vehicle control (n=). (B) Expression of glycolysis-related genes in the microarray analysis of human HSCs treated with either vehicle (H O) or ( g/ml) in liquid culture for 6 hours. Glycolysis-related genes that are also HIFA targets are bold; n=4. All data represent mean ± SEM. p <.5, p <., p <. (Paired Student s t-test).
7 Fig. S4. -R independent stimulation of HSC self-renewal by. (A) Serial colony-forming capacity of c-mpl +/+ HSCs after treatment ex vivo. Results are expressed as fold change of colonyforming units compared to non-treated vehicle control (, n=6 independent experiments). (B) HSCs were enumerated by the LTC-IC assay using limiting dilutions of Mpl +/+ LSK cells after ex vivo treatment with either vehicle control (, H O) or ( g/ml). Bar plot (left panel) showing LTC-IC frequency of individual mice expressed as fold change compared to (n= independent experiments). Scatter plot (right panel) showing LTC-IC frequency of ( g/ml) and (H O). The logged fraction of wells that did not form LTC-ICs (nonresponding) is plotted against limiting dilutions of LSK cells (n= independent experiments). LTC-IC frequency was determined using Poisson statistics and p-value was calculated using Chi-square on ELDA online server. All other data are mean ± SEM. p <.5, p <., n.s. (not significant) (Paired Student s t-test).
8 ckit CD48 c-mpl -/- Wild type (WT) Number of phenotypic HSCs per X 6 BMMNCs A Gated on Lin- cells B LSK MPP. HSC.5 5 WT c-mpl-/- LSK.75 MPP.4 HSC.66 Sca- CD5 Fig. S5. Reduction of HSCs in c-mpl / mice. (A) Cell surface expression of phenotypic HSCs (Lin Sca- + c-kit + CD5 + CD48 ) in wildtype (WT) and c-mpl -/- mice. (B) Quantification of the number of phenotypic HSCs within x 6 bone marrow mononuclear cells (BMMNCs) in WT and c-mpl -/- mice. n = 4 or 5. Data are mean ± SEM. p <. (Unpaired Student s t-test).
9 RBC HB MCV WBC NEU LY PLT PCT MPV Fig. S6. Complete blood counts of c-mpl / mice after in vivo treatment with. c-mpl -/- mice were treated with (5 mg/kg) or vehicle control (H O) daily for 4 weeks by oral gavage. Complete blood count was performed using peripheral blood of the mice after 4-week treatment. RBC: red blood cells; HB: hemoglobin; MCV: mean corpuscular volume; WBC: white blood cells; NEU: neutrophils; LY: lymphocytes; PLT: platelets; PCT: plateletcrit; MPV: mean platelet volume. Data are mean ± SEM. p <. (Unpaired Student s t-test).
10 Fold change (mrna compared to ) p=.5 Galm Pfkl Mxi Pgm Ldhb Ldhal6b Igfbp Gmppb Fig. S7. Alterations of glycolysis-associated genes in mouse HSCs after treatment. Expression of glycolysis-related genes in mouse HSCs treated with either vehicle (H O) or ( g/ml) in liquid culture for 6 hours. Glycolytic genes that are also HIFα targets are bold. Results are represented as fold change of % Gapdh compared to non-treated () vehicle control. If not specified otherwise, data are mean ± SEM. p <.5, p <., p <. (Paired Student s t-test).
11 A Primary Serial replatings st replate nd replate B C Fig. S8. FAC dose finding for ex vivo assays. (A) FACS-sorted mouse HSCs were preloaded with,, 5,,, or 5 g/ml of ferric ammonium citrate (FAC) in liquid culture for hour before being subjected to methylcellulose colony-forming assay. Results are represented as fold change of colonies compared to vehicle control (, g/ml of FAC). Colonies from the primary plating were harvested and preloaded with increasing concentrations of FAC for an hour before being subjected to subsequent serial replating in the colony-forming assay (n = 4). (B, C) Calcein staining to measure the concentration of intracellular iron in HPC7 cell line (B) or HSCs (C). Results are presented as mean fluorescence intensity of calcein in HSCs in different treatment conditions relative to the vehicle control (H O, ). If not specified otherwise, data are mean ± SEM. p <.5, p <., p <. (Paired Student s t-test).
12 Fig. S9. Intracellular ROS concentrations in HSCs after treatment. (A) Representative histograms showing mean fluorescence intensity (MFI) of DCF, which directly measures intracellular ROS in mouse BMMNCs treated with vehicle control (), positive control (H O ), negative control (NAC, N-acetyl cysteine), or with (+FAC) or without rescue. Cells were subsequently stained with stem and progenitor cell markers (Lin-Sca+cKit+CD5+CD48-CD4-) to quantify ROS specifically in the HSC compartment. (B) Quantification of MFI of DCF in the HSC compartment after treatment. Mouse BMMNCs were incubated with or g/ml of FAC for hour at 7ºC before being subjected to treatment in liquid culture for hour. H O : µm, NAC: 5 mm, : g/ml of eltrombopag, FAC: g/ml of ferric ammonium citrate. n = -5. Data are mean ± SEM. p <.5, p <., p <. (Paired Student s t-test).
13 log fraction nonresponding.... log fraction nonresponding.... A B C D E F LTC-IC (LSK) G LTC-IC (LSK) p p (/6.6) DFO (/5.) (/6.6) DFX (/.9) dose (number of cells) dose (number of cells) Fig. S. Iron-dependent HSC self-renewal upon chelator treatment. (A) Scheme showing HSCs isolated from wildtype mice preloaded with g/ml of FAC for hour before iron chelator treatment, to examine the effects of iron chelation on HSCs. (B-D) Serial colony-forming capacity of HSCs upon treatment with DFO (B, n=6), DFX (C, n=), or (D, n=5), rescued with FAC. (E) Scheme showing ex
14 vivo treatment with DFO or DFX and functional assessment of HSC frequency in wildtype mice. LSK cells were sorted directly into 96-well plates and treated with DFO ( M), DFX (5 M), or vehicle control (.% DMSO) in LTC-IC assay. (F, G) HSC frequency in LSK cells enumerated by the LTC-IC assay. Left panel: LTC-IC frequency in each individual donor (n=4). Right panel: scatter plot showing LTC-IC frequency of LSK cells in, DFO-treated (F, n=5), or DFX-treated groups (G, n=). HSC frequencies were determined using Poisson statistics and p-value was calculated using Chisquare on ELDA online server. If not specified otherwise, data are mean ± SEM. p <.5, p <., p <. (Paired Student s t-test).
15 Normalized expression Normalized expression % GAPDH % GAPDH % GAPDH % GAPDH % GAPDH % GAPDH A B TFRC Healthy -ITP Normal Acute ITP SLCA Healthy -ITP GSE58 SLC5A8 Healthy -ITP SLC5A7 Healthy -ITP C 4 FTL Healthy -ITP HAMP Healthy -ITP GSE58 AND GSE754 Normal Chronic ITP TFRC FTH FTL SLCA SLC5A7 SLC4A HAMP TFRC FTH FTL SLCA SLC5A7 SLC4A HAMP Fig. S. Iron-associated gene expression changes in ITP patients treated with. (A) Quantitative RT-PCR validation of iron-related genes in CD4 - cells from ITP patients treated in vivo with compared to healthy donors. (B, C) Expression of iron-related genes in primary patients with acute ITP (B) or chronic ITP (C) compared to healthy individuals. We reanalyzed gene expression datasets from GEO database with the accession numbers GSE58 and GSE754. The normalized expression values of iron-related genes are shown. If not specified otherwise, data are mean ± SEM. p <.5, p <., p <. (Unpaired Student s t-test).
(A-B) P2ry14 expression was assessed by (A) genotyping (upper arrow: WT; lower
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Supplemental Information Inventory
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Supplementary Figures Supplementary Figure 1: Phenotypically defined hematopoietic stem and progenitor cell populations show distinct mitochondrial activity and mass. (A) Isolation by FACS of commonly
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Flow cytometry Stained cells were analyzed and sorted by SORP FACS Aria (BD Biosciences).
Mice C57BL/6-Ly5.1 or -Ly5.2 congenic mice were used for LSK transduction and competitive repopulation assays. Animal care was in accordance with the guidelines of Keio University for animal and recombinant
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Supplemental methods Supplemental figure and legend...7. Supplemental table.. 8
Supplemental Digital Content (SDC) Contents Supplemental methods..2-6 Supplemental figure and legend...7 Supplemental table.. 8 1 SDC, Supplemental Methods Flow cytometric analysis of intracellular phosphorylated
Supplemental Table S1. RT-PCR primers used in this study
Supplemental Table S1. RT-PCR primers used in this study -----------------------------------------------------------------------------------------------------------------------------------------------
Supplementary Fig. 1: Characterization of Asxl2 -/- mouse model. (a) HSCs and their
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SUPPLEMENTARY INFORMATION
DOI: 1.138/ncb3342 EV O/E 13 4 B Relative expression 1..8.6.4.2 shctl Pcdh2_1 C Pcdh2_2 Number of shrns 12 8 4 293T mterc +/+ G3 mterc -/- D % of GFP + cells 1 8 6 4 2 Per2 p=.2 p>
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Supplementary Figure 1 Validation of the monoclonal antibody to mouse ACKR1 and expression of ACKR1 by BM hematopoietic cells. (a to d) Comparison of immunostaining of BM cells by anti-mouse ACKR1 antibodies:
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Supporting Information
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