Supplementary Materials for

Transcription

1 Supplementary Materials for Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation Yuki Taya, Yasunori Ota, Adam C. Wilkinson, Ayano Kanazawa, Hiroshi Watarai, Masataka Kasai, Hiromitsu Nakauchi,* Satoshi Yamazaki* *Corresponding author. (S.Y.); (H.N.) This PDF file includes: Materials and Methods Figs. S1 to S13 Published 20 October 2016 on Science First Release DOI: /science.aag3145

2 Supplementary Materials Materials and Methods: Mice C57BL/6 (B6-Ly5.2) mice were purchased from Japan SLC (Shizuoka, Japan). C57BL/6 (B6-Ly5.1) mice and NOD/scid (Ly5.2) mice were bred and maintained at Sankyo Lab Service (Tsukuba, Japan). C57BL/6-Ly5.1/Ly5.2 F 1 mice were used for competitive repopulation assays. NOD/Shi-scid/IL-2R null (NOG) mice were purchased from the Central Institute for Experimental Animals (Kanagawa, Japan). Mice were bred and maintained at the Animal Research Facility of the Institute of Medical Science, University of Tokyo. Animal care was carried out in accordance with the guidance of the University of Tokyo for animal and recombinant DNA experiments. Animal experiments were approved by the Animal Care and Use Committee, Institute of Medical Science, University of Tokyo. Original media and synthetic diets RPMI 1640 R8758 medium (RPMI; Sigma-Aldrich, St. Louis, MO) and DME/F12 D6421 medium (DME/F12; Sigma-Aldrich) without amino acids were specially ordered. Medium consisting of equal volumes of RPMI without amino acids and DME/F12 without amino acids was prepared. Aliquots of 24 amino acids (alanine, arginine HCl, arginine, asparagine, asparagine H 2 O, aspartic acid, cystine 2HCl, cysteine HCl H 2 O, glutamic acid, glutamine, glycine, histidine HCl H 2 O, histidine, trans-4-hydroxyproline, isoleucine, leucine, lysine HCl, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine 2Na 2H 2 O, and valine, except glycine all L-isomers; Sigma-Aldrich) were added individually to yield a variety of individual media deficient in single amino acids (Fig. S1C). Each of these media was identical in composition to S-clone SF-O3 medium (Sanko Junyaku, Tokyo, Japan) with the exception of deficiency in a single amino acid. L-amino acid rodent diets were purchased from Research Diet (New Brunswick, NJ). Measurement of amino acids by high performance liquid chromatography (HPLC) Blood collected by orbital vein sampling was centrifuged to remove cellular components and deproteinized using Amicon ultra-0.5 ml Ultracel-10K centrifugal filters (Merck Millipore, Darmstadt, Germany). BM cells were eluted using water and the eluate was deproteinized using Amicon ultra-0.5 ml Ultracel-10K centrifugal filters (Merck Millipore). Concentrations of amino acids were measured using the Prominence Amino Acid Analysis System (Shimadzu, Kyoto, Japan). Complete blood counts Orbital blood was collected into heparin-containing tubes. Complete blood counts were determined using an automated MEK-6258 hematology analyzer (Nihonkoden, Tokyo, Japan). Analysis and purification of mouse HSCs and hematopoietic progenitor cells CD34 - Kit + Sca1 + Lin - (CD34 - KSL) HSCs and CD34 + KSL cells were purified from BM of 8- to 12-week-old mice. BM cells were stained with allophycocyanin (APC)-conjugated anti-c-kit antibodies (ebioscience, San Diego, CA) and c-kit + cells were enriched using anti-apc magnetic beads and LS columns (both Miltenyi Biotec, Auburn, CA). These cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-cd34 (ebioscience), phycoerythrin

3 (PE)-conjugated anti-sca-1 (ebioscience), and an antibody cocktail consisting of biotinylated anti-gr-1, -Mac-1, -B220/CD45R, -CD4, -CD8, -CD127/IL7Rα, and -Ter-119 monoclonal antibodies (lineage-marker cocktail A; all from ebioscience), followed by staining with streptavidin-apc-efluor 780 (ebioscience) to detect biotinylated antibodies. For isolation of cells using other HSC surface markers (CD150 + CD41 - CD48 - KSL), BM cells isolated from C57BL/6 mice fed synthetic diets were stained with lineage-marker cocktail A, and with APC-conjugated anti-c-kit, FITC-conjugated anti-cd41 (ebioscience), Alexa Fluor 488-conjugated anti-cd48 (BioLegend, San Diego, CA), PE-conjugated anti-cd150 (BioLegend), and Pacific Blue-conjugated anti-sca-1 (BioLegend) antibodies, and with streptavidin-apc-efluor 780. For progenitor cells (CMP, GMP, and MEP) fraction analysis, BM cells isolated from B6 mice fed synthetic diets were stained with lineage-marker cocktail A, and with APC-conjugated anti-c-kit, FITC-conjugated anti-cd34, PE-conjugated anti-cd16/32 (ebioscience), and Pacific Blue-conjugated anti-sca-1 antibodies, and with streptavidin-apc-efluor 780. For CLP fraction analysis, BM cells isolated from B6 mice fed synthetic diets were stained with lineage-marker cocktail B (anti-gr-1, -Mac-1, -B220/CD45R, -CD4, -CD8, -Ter-119, and -CD3e (all ebioscience) and -CD5 (BioLegend), and with APC-conjugated anti-c-kit, FITC-conjugated anti-cd127/il7rα (ebioscience), PE-conjugated anti-cd135/flt3 (ebioscience), and Pacific Blue-conjugated anti-sca-1 antibodies, and with streptavidin-apc-efluor 780. CD34 - KSL and CD34 + KSL analysis and cell sorting were performed on a MoFlo flow cytometer using Summit software (Beckman Coulter, Fullerton, CA). CD150 + CD41 - CD48 - KSL and progenitor fraction cells analysis were performed by FACS Aria-2 flow cytometry (BD Biosciences, San Jose, CA). Results were analyzed with FlowJo software (Tree Star, Ashland, OR). Analysis of mouse PB and BM lineage cells Heparinized peripheral blood samples were stained with FITC-conjugated anti-cd45 (BD Biosciences), APC-conjugated anti-cd3e (Tonbo Science, San Diego, CA), APC-eFluor 780-conjugated anti-b220/cd45r (ebioscience), PE-conjugated anti-gr-1 (ebioscience), and PE-conjugated anti-mac-1 (BD Biosciences) antibodies. BM cells were stained with FITC-conjugated CD45, PE-conjugated anti-cd3e (BD Biosciences), APC-eFluor 780-conjugated anti-b220/cd45r, APC-conjugated anti-gr-1 (BioLegend), and PE/Cy7-conjugated anti-mac-1 (ebioscience) antibodies. Analysis of mouse B cells in BM and T cells in thymus For BM - B cell fraction analysis, BM cells isolated from B6 mice fed synthetic diets were stained with PE/Cy7-conjugated anti-b220/cd45r (BioLegend), APC/Cy7-conjugated anti-immunoglobulin M (IgM) (BioLegend), PE-conjugated anti-cd24 (ebioscience), and PE-conjugated anti-cd25 (BD Biosciences) antibodies. For thymus - T cell fraction analysis, thymocytes isolated from B6 mice fed synthetic diets were stained with lineage-marker cocktail C (anti-gr-1, -Mac-1, -B220/CD45R, -Ter-119, and -CD19 (ebioscience) and -CD11c (BD Biosciences) antibodies), with PB-conjugated anti-cd4 (BioLegend) and Violet500-conjugated anti-cd8a (BD Biosciences) antibodies, and with streptavidin-pe/cy7 (ebioscience). Culture of mouse CD34 - KSL cells and CD34 + KSL cells Purified mouse CD34 - KSL cells were cultured in serum-free S-clone SF-O3 medium or original medium, types listed above (Fig. S1A), supplemented with 1% bovine serum albumin (BSA),

4 mouse SCF (50 ng/ml; PeproTech, Rocky Hill, NJ) and TPO (50 ng/ml; PeproTech). CD34 + KSL cells (40 cells/well) were cultured in serum-free S-clone SF-O3 medium or original medium, types listed above (Fig. S1A), supplemented with 1% BSA, mouse SCF (50 ng/ml), TPO (50 ng/ml), and IL-3 (50 ng/ml; PeproTech). A 10% BSA stock was used for all in vitro culture experiments, which was made up using S-clone SF-03 medium (so final -AA media compositions include AA at ~10%). The cells were incubated at 37 C with 5% CO 2. Cell numbers were counted using a CYTORECON cytometer (GE Healthcare, Amersham, UK) Reactive oxygen species assay 3 x 10 3 KSL cells isolated from B6 mice were cultured with SCF (50 ng/ml), TPO (50 ng/ml), and IL-3 (20 ng/ml) in medium, -Cys medium, and -Val medium in the absence and presence of 2mM NAC (Sigma-Aldrich) for 24 hours. Cells were cultured with 10µM 2,7 -dichlorofluorescein diacetate (Sigma-Aldrich) for 30 min. Oxidation levels were detected by FACS. N-acetyl cysteine (NAC) rescue assay Forty CD34 - KSL cells were cultured for 1 week in original medium (, -Cys, or -Val; listed above, Fig. S1A) with SCF (50 ng/ml) and TPO (50 ng/ml) in the absence or presence of NAC (10µM, 100µM, and 2mM). Cells in each well were counted using a CYTORECON cytometer or under light microscopy. Competitive repopulation assays Competitive repopulation assays were performed using the Ly5 congenic mouse system. 1 x 10 6 BM cells (1-week-cultured CD34 - KSL cells) from B6-Ly5.1 mice and 1 x 10 6 of BM competitor cells from B6-F1 mice of Ly5.1 and 5.2 mice were transplanted into B6-Ly5.2 mice irradiated with a dose of 9.5 Gy. After transplantation, PB cells of the recipients were stained with PE/Cy7-conjugated anti-ly5.1 (Tonbo Science) and FITC-conjugated anti-ly5.2 (BioLegend). The cells were further stained with PE-conjugated anti-mac-1 and -Gr-1 antibodies, APC-conjugated anti-cd4 and -CD8a antibodies (both from ebioscience), and APC-eFluor 780-conjugated anti-b220/cd45r antibodies. The cells were analyzed by FACS Canto II flow cytometry (Becton Dickinson, Franklin Lakes, NJ). The % of chimerism was calculated as (percent Ly5.1 cells) x 100 / (percent Ly5.1 cells + percent F1 cells). Cell cycle analysis BM cells were stained with lineage-marker cocktail A, with APC-conjugated anti-c-kit, FITC-conjugated anti-cd34, and PB-conjugated anti-sca-1antibodies, and with streptavidin-apc-efluor 780. Cells were then were incubated with 0.5 μg/ml Pyronin Y (Sigma-Aldrich) at 37 C for 20 min and analyzed by FACS Aria-2 flow cytometry (Becton Dickinson). Histologic analysis Tissues from mice fed different diets were fixed in 10% neutral buffered formalin and embedded in paraffin. Four-micrometer sections were obtained from each sample. Sections mounted on glass slides were stained with hematoxylin and eosin. BM niche component cells amino acids release assay

5 Primary sciatic nerve cells were prepared from the sciatic nerve of B6 mice using forceps under the stereoscopic microscopy (Leica, Microsystems, Tokyo, Japan). Bones (femur, tibia, and pelvis) were isolated from 12-week-old B6 mice. BM non-hematopoietic cells were isolated from crushed bones by enzymatic digestion with 0.1% trypsin (Sigma-Aldrich). Cells were stained with FITC- or PE-conjugated-anti-CD45 and -Ter-119 (ebioscience) antibodies, Alexa674-labeled anti-alcam (R&D System, Minneapolis, MN) antibody, and PE-conjugated-anti-CD31 (ebioscience) antibody. As defined by phenotype, endothelial (CD31 + CD45 - Ter ) cells, osteoblasts (ALCAM + Ter119 - CD45 - CD31 - ), and PDGFRa + cells (CD140a + Ter119 - CD45 - ) were purified by flow cytometry using a FACS Aria 2. Sciatic nerve cells or isolated osteoblasts, endothelial cells, and PDGFRa + cells (10,000 each) were cultured for 3 days in medium containing no amino acids. Supernatant from each culture was deproteinized using Amicon ultra-0.5 ml Ultracel-10K centrifugal filters and amino acid concentrations were measured by HPLC. Non-irradiation transplantation protocol Autotransplantation: After being fasted for 2 days, recipient mice (B6-Ly5.2) were fed a diet or a -Val diet for 3 weeks. The mice then, without irradiation, received 1x10 7 whole BM cells isolated from donor mice (B6-Ly 5.1) and their diet was simultaneously switched to a diet. PB cells from recipient mice were analyzed 4, 8, and 12 weeks after transplantation. Allotransplantation: After being fasted for 2 days, recipient mice (NOD/scid-Ly5.1) were fed a diet or a -Val diet for 2 weeks. The mice then, without irradiation, received 5x10 3 KSL cells isolated from donor mice (B6-Ly 5.2) and their diet gradually was switched to a diet through addition of valine to the drinking water. Mice were fed 0.12g/L valine for one week and then 1.2g/L valine for one week before being resuming a diet. PB cells from recipient mice were analyzed 4, 8, and 12 weeks after transplantation. Purification of human CD34 + cells and xenogeneic transplantation Human CD34 + cells were purchased from Lonza Japan (Tokyo, Japan). For transplantation, 6-8 week old female NOG mice were irradiated (2-2.5 Gy) and then received CD34 + cells via the tail vein. Three months later, after engraftment of human hematopoietic cells was confirmed, these mice were fed either a diet or a -Val diet (n=4, each group) for 2 weeks.peripheral blood was then evaluated for expression of mouse and human CD45. Culture of human CD34 + CD38 - Lin - cells Human CD34 + cells were stained with APC-conjugated anti-human-cd34 antibodies (BioLegend), PE/Cy7-conjugated anti-human-cd38 antibodies (ebioscience), PB-conjugated anti-human-cd45 antibodies (Biolegend), and an FITC-conjugated anti-human lineage cocktail (anti-cd3, -14, -16, -19, -20, and -56; BioLegend). CD34 + CD38 - Lin - cell analysis and cell sorting were performed by MoFlo flow cytometry using Summit software (Beckman Coulter, Fullerton, CA). Purified human CD34 + CD38 - Lin - cells (300 cells/well) were cultured for 7 days at 37 C with 5% CO2, in serum-free S-clone SF-O3 medium or DME/F12 medium, as listed above (Fig. S1D), supplemented with 1% BSA, human SCF (50 ng/ml), human TPO (50 ng/ml), human Flt3L (50 ng/ml), human IL-3 (20ng/mL), and human IL-6 (20ng/mL; all PeproTech). Cells per well were then counted visually.

6 Autophagy assays For in vitro assays, 4 x 10 3 KSL cells were cultured with SCF (50 ng/ml), TPO (50 ng/ml), and IL-3 (20 ng/ml) in medium, -Val medium, and medium supplemented with rapamycin (500nM) and chloroquinein (10µM; both Enzo Life Sciences, Farmingdale, NY) for 24 hours. Cells were stained with Cyto-ID Green (Enzo Life Sciences) using the manufacturer s protocol. Autophagy levels were detected by FACS. 1 x 10 3 CD34 - KSL cells were cultured with SCF (50 ng/ml) and TPO (50 ng/ml) in medium, -Val medium, and medium supplemented with rapamycin (500nM) and chloroquinein (10µM) for 24 hours. After supernatants were removed, cell suspension droplets were placed onto poly-l-lysine-coated 24-well glass slides (Matsunami Glass, Osaka, Japan). Cells were stained with Cyto-ID Green and Hoechst nuclear stain using manufacturers protocols. Autophagy levels were determined using a Cellomics ArrayScan VTI HCS Reader (ThermoScientific, Pittsburgh, PA), a modular high-content screening instrument designed for high-capacity automated fluorescence cell imaging with quantitative analysis. For in vivo assays, BM cells isolated from mice fed synthetic diets for 3 weeks were stained with lineage-marker cocktail A, with APC-conjugated anti-c-kit, Alexa Fluor 700-conjugated anti-cd34 (ebioscience), and PE-conjugated anti-sca-1antibodies, and with streptavidin-apc-efluor 780. Cells were stained with Cyto-ID Green using the manufacturer s protocol. Autophagy levels were detected by FACS. Statistical analysis Statistical analysis was performed using a two-tailed t-test for two groups and a one-way ANOVA followed by Dunnett s test for multiple groups on Prism 6 software (GraphPad, San Diego, CA). Statistical significance is indicated by *P<0.05, **P<0.01, ***P< All error bars indicate ±SD.

7 Supplementary Figures and Figure Legends: Fig. S1: Physiological amino acid concentrations and media deficient in individual amino acids. (A,B) Amino acid concentrations in PB serum (A) and BM fluid (B) isolated from B6 mice (n=3) and determined by HPLC. Error bars indicate ±SD. (C) Mouse HSPC culture media with composition of S-clone medium but lacking single amino acids. (D) Human HSPC culture media with composition of DME/F12 but lacking single amino acids.

8 Fig.S2. A -Ala -Cys -Asp -Glu -Phe -Gly -His -Ile -Lys -Leu -Met -Asn -Pro -Gln -Arg -Ser -Thr -Val -Trp -Tyr None s-clone SCF 50 ng/ml TPO 50 ng/ml CD34 - KSL (x40) BSA 1 % 40 cells / well culture for 7 days B -Ala -Cys -Asp -Glu -Phe -Gly -His -Ile -Lys -Leu -Met -Asn -Pro -Gln -Arg -Ser -Thr -Val -Trp -Tyr None s-clone SCF 50 ng/ml TPO 50 ng/ml IL-3 20 ng/ml BSA 1% CD34 + KSL (x40) 40 cells / well C Fig. S2: Proliferation and colony-forming ability of CD34 - KSL and CD34 + KSL cells cultured under different amino acid conditions. (A) Photographs of 40 CD34 - KSL cells after incubation with SCF and TPO for 1 week. (B) Photographs of 40 CD34 + KSL cells after incubation with SCF, TPO, and IL-3 for 1 week.

9 (C) Cell counts following 1 week culture of CD34 + KSL cells in various AA medias (as described in Fig. 1A). Experiments were performed in triplicate; error bars indicate ±SD.

10 Fig. S3: The effect of N-acetyl cysteine (NAC) on HSPC cultured in medium, -Cys medium, and -Val medium. (A) Forty CD34 - KSL cells isolated from B6 mice were cultured with SCF and TPO in medium, -Cys medium, and -Val medium in the absence and presence of NAC (10µM, 100µM, and 2mM) for 1 week. Cells in each well were then counted. Experiments were performed in triplicate; error bars indicate ±SD. (B,C) 3 x 10 3 KSL cells isolated from B6 mice were cultured with SCF, TPO, and IL-3 in medium, -Cys medium, and -Val medium for 24 hours. ROS levels in cultured cells were measured using FACS after labeling with 2,7 -dichlorofluorescein diacetate (DCF-DA).

11 Data are presented as means ±SD from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001 (D,E) 3 x 10 3 KSL cells isolated from B6 mice were cultured with SCF, TPO, and IL-3 in -Cys medium in the absence and presence of NAC (2mM) for 24 hours. ROS levels in cultured cells were measured using FACS after labeling with DCF-DA. Data are presented as means ±SD. *P<0.05, **P<0.01, ***P<0.001.

12 Fig. S4: Alterations in autophagy following Val culture (A,B) Autophagy level in cultured KSL cells. 4x10 3 KSL cells isolated from B6 mice were cultured for 24 hours with SCF, TPO, and IL-3 in medium, -Val medium, and medium supplemented with rapamycin (RAPA), 500nM, and chloroquine (CQ), 10µM (RAPA + CQ medium). Autophagy levels in cultured cells were measured using FACS after labeling with Cyto-ID Green. Negative control shows the mean intensity of unstained cells. Data are presented as means ±SD from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001 (C) Autophagy level in cultured HSCs. CD34 - KSL cells isolated from B6 mice were cultured for 24 hours with SCF and TPO in medium, -Val medium, and RAPA+CQ medium,

13 stained with Cyto-ID Green, and automatically assayed by ArrayScan (n=50). Experiments were performed in triplicate; error bars indicate ±SD. (D,E) Autophagy levels, HSCs, after feeding a -Val diet. Freshly isolated BM cells of B6 mice fed a diet or a -Val diet for 3 weeks were stained with Cyto-ID Green. Autophagy levels were measured in HSC fractions (CD34 - KSL). Complete group, n=3; -Val group. n=6. Data are presented as means ±SD from two independent experiments.

14 Fig. S5: Amino acid concentrations in PB and BM were affected in vivo by synthetic diet. (A) Composition table of 4 synthetic diets ( diet, -Val diet, -Cys diet, and -Leu diet). (B) Amino acid levels in PB serum and BM fluid after feeding a -Val diet. B6 mice were fed a diet or a -Val diet. Amino acid concentrations in PB serum and BM fluid were analyzed by HPLC (n=3). Error bars indicate ±SD from two independent measurements.

15 Lineage c-kit CD150 Frequency (%) Lineage c-kit c-kit Pyronin Y Pyronin Y Frequnecy (%) Fig. S6. A B * cystine 0 -cystine D KSL gate c-kit CD34 Sca-1 C CD CD E HSC(CD34 - KSL) in G0 phase c-kit Sca-1 CD41+CD48 0 Fig. S6: Changes in HSC profiles induced by feeding Val and Cys diet. (A) Representative flow cytometry patterns of HSC fraction (CD34 - KSL) cells in mice fed a diet, a -Cys diet, and a -Val diet. (B) Frequency of CD34 - KSL cells in mice fed a diet, a -Cys diet, and a -Val diet (n=3). Data are presented as means ±SD from two independent experiments. *P<0.05 (C) Representative flow cytometry patterns of HSC fraction cells (CD150 + CD41 - CD48 - KSL) from mice fed a diet or a -Val diet for 4 weeks. (D) Representative flow cytometry patterns of CD34 expression and pyronin Y staining intensity in KSL cells from mice fed a diet or a -Val diet for 4 weeks. (E) Frequency of G 0 CD34 - KSL HSCs following dietary modulation for 4 weeks, calculated from (C). Error bars indicate ±SD.

16

17 Fig. S7: Hematopoiesis was affected by feeding a -Val diet. (A) Time-frequency analysis of peripheral blood counts after feeding a -Val diet. B6 mice were fed a diet or a -Val diet and peripheral blood counts (WBC, RBC, PLT) were determined ( group, n=6; -Val group, n=7). (B,C) Time-frequency analysis of lymphoid (B220 + or CD3 + ) and myeloid (Gr1 + Mac1 + ) cells in PB (B) and BM (C) after feeding a -Val diet. B6 mice were fed a diet or a -Val diet for 4 weeks. Lymphoid and myeloid counts were determined ( group, n=8; -Val group. n=9). All data are presented as means ±SD from two independent experiments. *P<0.05, **P<0.01, ***P< (D) Representative flow cytometry patterns of progenitor cells (common myeloid progenitor, CMP; granulocyte-monocyte progenitor, GMP; megakaryocyte-erythrocyte progenitor, MEP) from mice fed a diet or a -Val diet for 4 weeks. (E) Time-frequency analysis of progenitor cell populations after feeding a -Val diet ( group n=3, -Val group n=4). B6 mice were fed a diet or a -Val diet and progenitor cell counts (CMP, GMP, MEP) were determined.

18 B220 CD25 B220 CD25 CD8 % Lineage Flt-3 c-kit Lineage Flt-3 c-kit Fig. S8. A diet 2 weeks diet 2 weeks recovery 1 week diet 3 weeks recovery 2 weeks diet 4 weeks c-kit IL-7Ra Sca-1 c-kit IL-7Ra Sca-1 B CLP ** week C D diet diet 2 weeks weeks recovery 1 week recovery 1 week recovery recovery 2 weeks weeks IgM CD24 IgM CD24 CD4 Fig. S8: Depletion and recovery of lymphoid cells from -Val status (A) Representative flow cytometry patterns of CLP fraction from mice fed a diet or a -Val diet for 2 weeks. Sampling undertaken 1 week and 2 weeks after shift to a diet. (B) Time-frequency analysis of CLP populations after feeding a -Val diet and shifting to a diet ( group n=6, -Val group n=6). (C) Representative flow cytometry patterns of BM-B cells from mice fed a diet or a -Val diet for 2 weeks. Sampling undertaken 1 week and 2 weeks after shift to a diet.

19 (D) Representative flow cytometry patterns of thymus T cells from mice fed a diet or a -Val diet for 2 weeks. Sampling undertaken 1 week and 2 weeks after shift to a diet.

20 mg Fig. S9. x10 7 / femur + tibia A C ** B *** *** week ** Wp *** Spleen Rp Wp Wp Rp Wp Bone marrow 200µm 200µm Wp Wp 400µm 400µm Thymus C M 50µm 50µm 400µm 400µm D Brain Liver 400µm 400µm 50µm 50µm Heart Striated muscle 100µm 100µm 200µm 200µm Lung Adipose tissue 400µm 400µm 100µm 100µm Kidney Testis 200µm 200µm 200µm 200µm Stomach Skin 400µm 400µm 1000µm 1000µm Small intestine Hair follicle 200µm 200µm 100µm 100µm Pancreas 100µm 100µm

21 Fig. S9: Influence of a -Val diet on various organs. (A) Frequency of BM cells in femurs and tibias of mice fed a diet or a -Val diet for 4 weeks (n=3). (B) Time-dependent changes in spleen weight of mice fed a diet or a -Val diet and representative photographs of spleen after feeding for 4 weeks ( group, n=3; -Val group, n=4). (C) BM, spleen, and thymus in mice fed a diet or a -Val diet for 4 weeks. Hematoxylin and eosin stain. Wp, white pulp; Rp, red pulp; C, cortex; M, medulla. (D) Photomicrographs of sections of various organs from mice fed a diet or a -Val diet for 4 weeks. Magnifications differ from tissue to tissue but within a pair of images are the same for each member. Hematoxylin and eosin staining. Arrowheads indicate hair follicles. All data are presented as means ±SD from two independent experiments. *P<0.05, **P<0.01, ***P<0.001.

22 chimerism (%) Body weight (g) Fig. S10. A B Change to diet valine 1wk valine 2wk valine 3wk valine 4wk days Recovery 4 weeks Recovery 4 weeks C D E Bone marrow Lung F G 100 Myeloid cell T cell B cell *** *** *** *** *** *** weeks after transplant Fig. S10: Recovery from -Val status (A) Body weight was measured while feeding a -Val diet for 1-4 weeks and after shifting to a diet for 4 weeks. (B) Representative photographs of spleen and thymus (yellow broken line) 4 weeks after shift to a diet. (C-E) Frequency of HSCs (CD150 + CD41 - CD48 - KSL), HSPCs and Lineage + cells in mice fed a -Val diet for 4 weeks followed by a diet for 4 weeks.

23 (F) Photomicrographs, BM and lung from B6 mice fed a -Val diet for 3 weeks, sampled 3 days after shift to a diet. Hematoxylin and eosin staining. Autopsy finding shown only pulmonary edema, with thickened alveolar walls and alveolar lumina filled with fluid. At this point, hypocellularity in BM was almost recovered. (G) Related to Figure 2C and D. Chimerism of donor PB myeloid cells, T cells, and B cells over 6 months following transplantation (n=6). All error bars are presented as means ±SD.

24

25 Fig. S11: Additional evidence of valine starvation as a successful conditioning protocol for HSC transplantation (A) Above, PB chimerism 16 weeks after non-irradiation transplantation, outlined in Fig. 3A. Below, PB percentages for NOD/scid or B6 mice reared under normal conditions. (B) Representative photographs of hematoxylin and eosin stained spleen in recipient mice fed a -Val diet at 12 weeks after non-irradiation transplantation. Wp, white pulp; Rp, red pulp.(c,d) Chimerism of donor-derived cells 4, 8, 12 (and 56, in D) weeks after non-irradiation transplantation in B6 mice fed a diet or a -Val diet, as outlined in Fig.3D. Results from two independent experiments. Mean values are indicated by bars ( group, n=5; -Val group, n=5). Below, tabular PB chimerism data at 12 months after non-irradiation transplantation for these experiments. (E) Chimerism of donor-derived cells 12 months after secondary transplantation in B6 mice, from Exp. 3 displayed in (D). Complete each group, n=5; -Val each group, n=5. (F) Related to Fig.4. Analysis of PB counts in humanized mice fed a (n=5), -Val (n=4) and -Leu (n=4) diet for 2 weeks. Data are presented as means ±SD. *P<0.05, ***P< (G) Related to Fig.4. BM cell number of humanized mice fed a (n=4), -Leu (n=4), -Val (n=3) diet for 2 weeks.

26 Fig. S12: Successful engraftment of BM transplanted into valine-starved mice but higher re-feeding syndrome-associated mortality when immediately moved to diets (A) Schematic of non-irradiative BM transplantation in mice fed a -Val diet. B6 mice (Ly5.2) fed a diet or a -Val diet for three weeks were transplanted, without irradiation, with 10 7 whole BM cells from donor B6 mice (Ly5.1). The recipient mouse diet was changed at transplantation to a diet. Repopulation activity of donor cells was monitored over the three months after transplantation. 26 mice were used for each cohort, but only 10 mice survived the Val diet followed by the immediate return to the diet following transplantation. (B) PB chimerism of donor-derived cells 12 weeks after non-irradiation transplantation in B6 mice fed a diet or a -Val diet. Mean values are indicated by bars ( group, n=26; -Val group, n=10). (C) PB chimerism 12 weeks after non-irradiation transplantation in recipient mice fed a -Val diet (only data for successfully engrafted mice presented). Engraftment defined as >1% donor chimerism.

27 mg/l mg/l mg/l mg/l Fig. S13. A 20 Osteoblast B 25 Nerve cell C Asp Thr Ser Glu Pro Gly Val Cys N.D. Met Ile CD31 + cell Leu Tyr Phe His Ala Lys Arg D Asp Thr Ser Glu Pro N.D. Gly Val Cys Met Ile PDGFR + cell Leu Tyr Phe His Ala Lys Arg Asp Thr Ser Glu Pro Gly Val N.D. Ile Cys Met Leu Tyr Phe His Ala Lys Arg Fig. S13: Amino acid secretion assay Identification of amino acids secreted by osteoblasts, neuronal cells, vascular endothelial cells, and PDGFRa + cells. By phenotype, osteoblasts (ALCAM + Ter119 - CD45 - CD31 - ), neural cells (sciatic nerve), vascular endothelial cells (CD45 - Ter119 - CD31 + ), and PDGFRa + cells (CD140a + Ter119 - CD45 - ) from B6 mice were incubated in amino acid free medium for 3 days. Concentrations of amino acids in culture supernatants were measured by HPLC (n=3). 0 Asp Thr Ser Glu Pro Gly N.D. Ile Val Cys Met Leu Tyr Phe His Ala Lys Arg