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Supplemental Figures 032819.Pptx Summary of Supplemental Figures and Tables 1) Supplemental Figure 1. Frequency of phenotypically defined endothelial cells is unchanged in aged mice. 2) Supplemental Figure 2. Aged LSKs have increased myeloid/megakaryocytic bias 3) Supplemental Figure 3. RBCs are decreased and Platelets are increased in peripheral blood of aged mice 4) Supplemental Figure 4. Aged BMME cultures have increased MSC populations. 5) Supplemental Figure 5. Aged BMME cells increase young LSK cell engraftment following competitive transplantation 6) Supplemental Figure 6. Flow cytometry gating strategy for sorting of young and aged murine Mφs 7) Supplemental Figure 7. Flow cytometry gating strategy for sorting of human Mφs 8) Supplemental Figure 8. Axl-/- LT-HSCs have increased cell engraftment following competitive transplantation 9) Supplemental Figure 9. Schematic representation of mechanisms by which age-dependent defects in marrow Mφs induce megakaryocytic bias in HSC. 10) Table S1. List of antibodies used in the flow cytometric and cell sorting analyses described 11) Table S2. List of cell populations analyzed and their respective immunophenotypes 12) Table S3. List of top GO-BP categories enriched for significantly upregulated genes from murine aged vs young marrow macrophages 13) Table S4. List of significant KEGG Pathways enriched for significantly upregulated genes from murine aged vs young marrow macrophages 1 A Sinusoidal Arteriolar BV786 – CD31 Sca1 – PE-Cy7 B C Arteriolar EC Sinusoidal ECs (Lin-, CD45-, CD31+ Sca1+) (CD45-Lin-CD31+Sca1-) 0.08 0.15 0.06 0.10 0.04 0.05 0.02 PercentLiveof Cells PercentLiveof Cells 0.00 0.00 Young Aged Young Aged Supplemental Figure 1. Frequency of phenotypically defined endothelial cells is unchanged in aged mice. (A) Representative flow cytometry gating for Arteriolar (Lin-, CD45-, CD31+, Sca1+) and Sinusoidal (Lin-, CD45-, CD31+, Sca1-) Endothelial cells from the bone marrow of young and aged mice. (B, C) Quantification of Arteriolar (B) and Sinusoidal (C) endothelial cell populations. 2 Youngà à à à A à à c-Kit-PE-Cy7 CD48-FITC CD48-FITC Flt3-PE Lineage FSC-Aà Sca-1-PerCP- CD150-APCà CD150-APCà CD150-APCà Cy5.5à Agedà à à à à à Lineage c-Kit Flt3 CD48 CD48 FSC-Aà Sca-1à CD150à CD150à CD150à Proportion of LSK B 100 E D LSK Subsets Young 0.5 LSK Subsets 2.0×10 5 80 * ** Aged LT-HSC 0.4 Young 1.5×10 5 60 ST-HSC Aged MPP2 0.3 40 MPP3 *** 1.0×10 5 *** % of% LSK MPP4 0.2 *** *** 20 Cells Live % *** 5.0×10 4 *** 0.1 Number Total 0 Young Aged 0.0 0 LSK LT-HSC ST-HSC MPP2 MPP3 MPP4 LSK LT-HSC ST-HSC MPP2 MPP3 MPP4 C MP and CLP Subsets Marrow Cellularity F 8 MP and CLP Subsets G 4.0×10 6 2.0×10 8 ** Young *** Young × 8 6 ** 3.0×10 6 1.5 10 Aged Aged 1.0×10 8 × 6 4 2.0 10 ** × 7 5.0 10 6 % Live Cells Live % Total Number Total 2 1.0×10 Total Number Total *** 0 Young Aged 0 0 MPs CMP GMP MPs CMP GMP MP and CLP Subsets 0.8 MP and CLP Subsets × 5 H I 5.0 10 *** Young × 5 Young 0.6 4.0 10 Aged *** Aged 3.0×10 5 0.4 *** *** 2.0×10 5 *** % Live Cells Live % 0.2 Total Number Total 1.0×10 5 0.0 0 MEP MkP CLP MEP MkP CLP Supplemental Figure 2. Aged LSKs have increased myeloid/megakaryocytic bias. (a,b) Flow cytometry strategy (a-representative plots from young and aged mouse) and percent contribution (b) of lineage-Sca1+ c-Kit+ (LSK) subsets to HSPC pool of young and aged mice (N=10-15). (c) Marrow cellularity of young and aged mice (N=15). (d-i) Flow cytometry of percent live (d,f,h) and total number (e,g,i) of LSKs and subsets (d,e) and of myeloid (MP, CMP, GMP, MEP), lymphoid (CLP) and megakaryocytic (MkP) progenitors (f-i) in young and aged mice (N=10-15). Data represent mean±s.e.m. (p-values: two-tailed Student’s t-test; *p<0.05; **p<0.01; ***p<0.001). WBC Lymphocytes Monocytes Granulocytes 20 15 1.5 5 4 15 10 1.0 3 /ul /ul /ul /ul 3 3 3 3 10 10 10 10 10 2 5 0.5 5 1 0 0 0.0 0 Young Aged Young Aged Young Aged Young Aged RBC HGB Platelets 14 *** 20 *** 2000 *** 18 12 1500 16 /ul /ul 6/ 10 3 1000 g/dL 10 14 10 8 500 12 6 10 0 Young Aged Young Aged Young Aged Supplemental Figure 3. RBCs are decreased and Platelets are increased in peripheral blood of aged mice. CBC analysis of peripheral blood demonstrates no change in the numbers of (A) WBC, (B) Lymphocytes, (C) Monocytes, (D) Granulocytes, a decrease in the number of (E) RBCs, and (F) HGB, and an increase in the number of (G) Platelets. N=25-30 mice/group. ***P<0.001. 4 Mesenchymal cells ex vivo Endothelial Cells ex vivo Young 60 1.5 * Young Aged *** *** Aged 40 1.0 20 0.5 % of Live Cells % of Live Cells 0 0.0 OBCs MSCs Pα/αV PαS+ LepR+ Arteriolar Sinusoidal Supplemental Figure 4. Increased MSC populations, and decreased osteoblastic cells in aged BMME tissue cultures. Aged BMME cultures are enriched for MSC populations recapitulating what is seen in vivo in aged mice. Arteriolar and Sinusoidal endothelial cells make up less than 1% of total cells in BMME cultures and are not changed in aging. Data represent mean±s.e.m. (p-values: two- tailed Student’s t-test; *p<0.05; ***p<0.001). 5 A B220+ Cell Engraftment 15 *** 10 5 % donor% cells 0 0 5 10 15 20 25 Weeks post transplant B CD3e+ Cell Engraftment 5 4 *** 3 2 % donor% cells 1 0 0 5 10 15 20 25 Weeks post transplant C CD11b+ Cell Engraftment 15 Young MA * Aged MA 10 5 % donor% cells 0 0 5 10 15 20 25 Weeks post transplant Supplemental Figure 5. Aged BMME cells increase cell engraftment following competitive transplantation. (a-c) Aged BMME cells demonstrate increase engraftment of B220+ (a), CD3e+ (b), and CD11b+ (c) cells compared to young BMME cells. (N= 5 Recipients/group). Data represent mean±s.e.m. (p-values: two-tailed Student’s t-test; *p<0.05; **p<0.01; ***p<0.001). A Young murine bone marrow B Aged murine bone marrow Supplemental Figure 6. Flow cytometry gating strategy for young and aged murine Mφs. Representative gating strategy used to analyze murine Mφs. F4/80+, Ly6C-, CD11b+ Mϕs were selected for analysis. A SSC DAPI SSC-H FSC-H FSC FSC-W SSC-W FSC-A 250K 250K 250K 105 200K 200K 200K 104 150K 150K 150K 103 100K 100K 100K 102 50K 50K 50K 0 0 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 250K 105 105 200K 4 10 104 150K 3 CD11b+ 10 103 100K 2 50K 10 0 0 0 0 102 103 104 105 0 103 104 105 0 103 104 105 SSC-A CD11b-FITC CD45-Alexa Fluor 700 CD45-Alexa Fluor 700 CD163-PerCP-Cy5.5 CD235a-PE-Cy7 Supplemental Figure 7. Flow cytometry gating strategy for human Mφs. Representative gating strategy used to analyze Mφs in samples from human volunteers. CD45+, CD163+ and CD45+, CD11b+ Mϕs were selected for analysis. Total Donor Engraftment B Cell Donor Engraftment 90 90 80 *** ** 80 70 70 60 60 50 50 WT 45 WT 50 -/- Donor Cells % of Live of % Cells Donor Axl-/- Axl 0 0 3 5 8 12 16 18 Cells B220+ of % Cells Donor 3 5 8 12 18 Week Week Myeloid Cell Donor Engraftment T Cell Donor Engraftment 80 80 * 70 60 60 50 40 40 WT 20 WT 35 Axl-/- Axl-/- 0 0 Donor Cells % of CD3e+ Cells CD3e+ of % Cells Donor Donor Cells % of CD11b+ Cells CD11b+ of % Cells Donor 3 5 8 12 18 3 5 8 12 16 18 Week Week Supplemental Figure 8. Axl-/- LT-HSCs have increased cell engraftment following competitive transplantation. (a-c) Axl-/- LT-HSCs demonstrate increase engraftment of B220+ (a), CD3e+ (b), and CD11b+ (c) cells compared to wildtype littermates. (N= 5 Recipients/group). Data represent mean±s.e.m. (p-values 2-way ANOVA; *p<0.05; **p<0.01; ***p<0.001). 9 Aging ↓Axl ↓ Efferocytosis ↓PHagocytosis ↑ activated bone marrow Mϕs ↑IL1β ↑HSCs with Megakaryocytic Skewing Supplemental Figure 9. Schematic representation of mechanisms by which age- dependent defects in marrow Mφs induce megakaryocytic bias in HSCs 10 Supplemental Table 1. Frisch, Hoffman et al. Murine Hematopoietic Stem and Progenitor Cell Panel Antibody Clone Fluorophore Supplier and Cat # Lineage α-Ter119 TER-119 BD Biosciences; 560512 α-Gr1 RB6-8C5 Biotin (primary) BD Biosciences; 561103 PE-CF594-Streptavidin (secondary) α-B220 RA3-6B2 BD Biosciences; 561101 α-CD3e 145-2C11 BD Biosciences; 551163 α-cKit 2B8 PE-Cy5 eBioscience; 15-1171 α-Sca1 D7 PerCP-Cy5.5 BD Biosciences; 558162 α-Flt3 A2F10 PE eBioscience; 12-1351 α-CD48 HM48-1 FITC eBioscience; 11-0481 α-CD150 9D1 APC eBioscience; 17-1501 α-CD34 RAM34 Alexa Fluor 700 BD Biosciences; 560518 α- CD41 MWReg30 BUV-395 BD Biosciences; 565980 α-FcγR 2.4G2 APC-Cy7 BD Biosciences; 560541 α-CD127 A7R34 PE-Cy7 Biolegend; 135013 Murine Stromal Cell Panel Antibody Clone Fluorophore Supplier and Cat # Lineage α-Ter119 TER-119 BD Biosciences; 560512 α-Gr1 RB6-8C5 PerCP-Cy5.5 BD Biosciences; 561103 α-B220 RA3-6B2 BD Biosciences; 561101 α-CD3e 145-2C11 BD Biosciences; 551163 α-Sca1 D7 PE-Cy7 BD Biosciences; 558162 α-CD105 MJ7/18 PE-CF594 BD Biosciences; 562762 RMV-7 α-CD51 390 PE BD Biosciences; 551187 α-CD31 FITC BD Biosciences; 558738 α-CD45 30-F11 APC-Cy7 BD Biosciences; 557659 α-CD11b M1/70 Alexa Fluor 700 BD Biosciences; 557960 α-PDGFRα APC BD Biosciences; 562777 α- LepR APA5 Biotin (primary) R&D Systems; BAF497/ BUV395-Streptavidin (secondary) BD Biosciences; 564176 Murine Macrophage Cell Panel (in vitro) Antibody Clone Fluorophore Supplier and Cat # α-CD86 GL-1 PE-Cy7 BD Biosciences; 560582 α-MHCII M5/114.15.2 PerCP-Cy5.5 BD Biosciences; 562363 α-F4/80 BM8 FITC eBioscience; 11-481 α-CD45 30-F11 APC-Cy7 BD Biosciences; 557659 α-CD11b M1/70 Alexa Fluor 700 BD Biosciences; 557960 Supplemental Table 1 (continued).
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