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Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli Are Altered with Age

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Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli Are Altered with Age Cell Reports, Volume 25 Supplemental Information Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli Are Altered with Age Mati Mann, Arnav Mehta, Carl G. de Boer, Monika S. Kowalczyk, Kevin Lee, Pearce Haldeman, Noga Rogel, Abigail R. Knecht, Daneyal Farouq, Aviv Regev, and David Baltimore A B 80 Young 60 Young Aged Aged *** *** 60 40 * * 40 *** *** * *** *** * 20 20 0 0 Myeloid cells (% of CD45+ cells) Granulocytes (% of CD45+ cells) C Young D Young 40 Aged 80 Aged *** 30 *** 60 *** *** *** *** *** 20 40 10 20 T cells (% of CD45+ cells) B cells (% of CD45+ cells) 0 0 0 hr 72 hr 3 wk 6 wk 9 wk 0 hr 72 hr 3 wk 6 wk 9 wk E Spleen F Bone marrow 30 ** 1.5 * 0.3 * ) ) CD45 f ) 1.0 0.2 20 Lin- o f Lin- o (% f s (% o ell (% Cs c 0.5 S 0.1 10 -H SKs eloid L LT My 0 0.0 0.0 35 *** 0.20 * 0.8 NS ) ) 0.15 ) 0.6 Lin- 30 Lin- f f CD45 o o f o (% 0.10 (% 0.4 s (% s SC PPs ell M 25 -H T c ST 0.05 0.2 20 0 0 Young Aged Young Aged Young Aged Figure S1. Aged mice challenged with LPS demonstrate increased myeloid output and an increased frequency of bone marrow HSPCs. Related to Figure 1. (A)-(C) Young (8-12 weeks) and aged (20-24 months) mice were exposed to single sub-lethal dose of LPS and peripheral blood (A) myeloid cell and (B) granulocyte (C) T cell, (D) B cell, frequencies were measured by flow cytometry at the indicated time points after LPS exposure (n = 4-14 per group). (E)-(F) Mice were harvested 2-3 weeks after the second LPS injection. (E) Splenic CD11b+ cell and CD3e+ cell frequencies are shown. (n=4 per group) (F) The frequencies of different bone marrow progenitor cells are shown. LT-HSCs were gated using the markers Lineage-cKit+Sca1+CD150+CD48-, ST-HSCs using the markers Lineage-cKit+Sca1+CD150-CD48-, MPPs using the markers Lineage-cKit+Sca1+CD150-CD48+ and LSKs using the markers Lineage-cKit+Sca1+ (n=5-7 per group). Data represent at least two independent experiments and are presented as mean ± SEM. * denotes p < 0.05, ** denotes p < 0.01 and *** denotes p < 0.001 using 2way ANOVA (A-D), or T test (E,F). A B Young mice primary transplant Aged mice primary transplant 80 80 LT-HSC, no stim 60 60 LT-HSC, stim ST-HSC, no stim merism 40 merism 40 ST-HSC, stim hi hi MPP, no stim % c % 20 c % 20 MPP, stim 0 0 4wk 8wk 12wk 4wk 8wk 12wk Time post-reconstitution Time post-reconstitution C D Young mice secondary transplant Aged mice secondary transplant 50 60 LT-HSC, no stim LT-HSC, stim 40 ST-HSC, no stim 40 30 ST-HSC, stim merism merism MPP, no stim hi hi 20 MPP, stim 20 % c % % c % 10 0 0 4wk 8wk 4wk 8wk Time post-reconstitution Time post-reconstitution E F ns ns 0.6 100 80 0.4 60 40 0.2 LT-HSCs (% of Lin-) 20 CD41+ LT-HSCs (% of Lin-) 0.0 0 ns ns G 3000 H 2500 2000 2000 1500 + LT-HSCs + 41 1000 CD Total LT-HSCs Total 1000 500 Total Total 0 0 No stim Stim No stim Stim No stim Stim No stim Stim Young mice Aged mice Young mice Aged mice Figure S2. Transplantation of LT-HSCs, ST-HSCs and MPPs from young and aged mice, both with and without stimulation, demonstrates only LT-HSCs have long term reconstitution potential. Related to Figure 1. CD45.2 HSPCs were sorted as described in Figure 2 and treated with or without LPS/Pam3csk4 in vitro prior to transplantation into lethally irradiated CD45.1 recipient mice along with CD45.1 bone marrow helper cells. (A)-(D) These panels demonstrate that LT-HSCs from both (A) young and (B) aged mice, had the ability to maintain long term reconstitution even when stimulated in vitro prior to transplantation. All MPPs failed to maintain long term hematopoietic output. (C)-(D) Secondary transplantation was performed using bone marrow cells obtained from mice in parts (A) and (B). Only LT-HSCs were able to maintain long term reconstitution during secondary transplantation. This was the case for both (D) young and (E) aged LT-HSCs, and both initially unstimulated and stimulated LT-HSCs. (E)-(H) The bone marrow compartment for all mice was analyzed 3 months post-reconstitution for the (E) frequency and (G) total number of LT-HSCs, as well as the (F) frequency and (H) total number of CD41+ LT-HSC subsets. Data represent at least two independent experiments (n=7-12 per group) and are presented as mean ± SEM and analyzed using 1way ANOVA (E-H). Young LT-HSCs A Young LT-HSCs Aged LT-HSCs C Young LT-HSCs 15 15B 15 Aged LT-HSCs BMDCs Aged LT-HSCs BMDCs BMDCs 10 10 10 5 5 5 log2(TPM) IL6 for log2(TPM) for IL1b log2(TPM) for TNFa 0 0 0 0 5 10 15 0 5 10 15 0 5 10 15 Time (hours) Time (hours) Time (hours) 40 D E 40 H 20 Tm4sf1 ***** * 20 Myo1e * nostim nostim Gpr183 Zfp773 *** * Wwtr1 Rnd3 0 Mid1 0 Jun *** Plscr2 ** Ptprk ** Matn4 ** −20 Dnm3 **** −20 Ptafr **** Bmp4 * *** Ltbp4 **** Gpr64 −40 log(FC) aged ST-HSCs Gstm2 * aged MPPs Plcl1 * ** 40 40 Gprc5c 5 tSNE2 Thsd4 tSNE2 *** young MPPs young ST-HSCs Cadm1 0 Bmpr1a **** Epas1 * −5 20 Csrnp2 20 Rorb **** Gm9199 stim * stim Ramp2 ***** Gpx3 *** 0 Sbspon ***** * 0 Osmr ***** **** Muc1 Selp ***** ** Dhrs3 −20 Clca1 * −20 Clec1a * ** Vwf ***** Jam2 ** Ehd3 ***** −40 Vmp1 ** Itgb3 Nupr1 *** ** −25 0 25 −40 −20 0 20 Gda Wdfy1 ** tSNE1 tSNE1 Dynlt1b * Serpina3g ** Serinc3 * F Regulation of NF-kB import into nucleus G Plac8 ***** *** Platelet-derived growth factor receptor signaling pathway cluster 2 aged Negative regulation of lymphocyte differentiation Positive regulation of acute inflammatory response vs vs Sterol biosynthetic process Negative regulation of lipid catabolic process cluster 3 young Keratinocyte differentiation G-protein coupled receptor signaling pathway stim LT no-stim LT Negative regulation of T-helper type 1 immune response Positive regulation of B cell differentiation Oncostatin-M-mediated signaling pathway Regulation of lymphocyte activation Regulation of signaling Positive regulation of hematopoiesis Extracellular matrix Regulation of endothelial cell development Negative regulation of cell adhesion Cell proliferation Macrophage proliferation Cell-cell adhesion Regulation of migration Positive regulation of cytokine biosynthtic pathway Regulation of localization Immune response 02461 3 5 02468 -log(p-value) -log(p-value) J I Slamf1 Vwf K Itga2b (CD150) 0.2 (CD41) 25 0.2 0.4 0.2 0.4 0.6 0.4 0.6 0.8 0.6 0.8 1.0 0.8 0 1.0 1.2 1.0 1.2 1.4 1.2 tSNE−2 1.4 1.6 1.4 1.6 1.6 −25 1.8 1.8 2.0 −25 0 25 −25 0 25 −25 0 25 tSNE−1 tSNE−1 tSNE−1 L M Z score 4 2 0 mLT-HSC gene signature 400 200 0 Score Aged Young Individual cells ranked Individual cells ranked Figure S3. Inflammatory challenge HSPCs in vitro leads to induction of inflammatory cytokines and reveals a myeloid biased gene signature in LT-HSCs. Related to Figures 2, 3. (A)-(E) LT-HSCs from young and aged mice were exposed to LPS and Pam3CSK4 in vitro for the indicated time and subjected to bulk RNA-sequencing. Shown are the log2(TPM) values for (A) IL6, (B) IL1β and (C) TNFα over time along with values for BMDCs obtained from (Jovanovic et al., 2015). (D)-(L) LT-HSCs, ST-HSCs and MPPs from young and aged mice were stimulated with LPS and Pam3CSK4 and sorted for single-cell RNA-sequencing. (D)-(E) Single- cell t-SNE plots (as in Figure 3) showing (D) MPPs and (E) ST-HSCs among unstimulated and stimulated cells, and color coded by mouse age. Gene Ontology analysis for all genes differentially upregulated (F) or downregulated (G) in LT-HSCs in cluster 3 vs cluster 2. (H) A gene signature defined in unstimulated LT-HSCs based on differentially expressed genes in stimulated LT-HSCs. Comparably changed genes between unstimulated aged vs young were isolated and used to define this preliminary signature, which was later used to initialize a k-means clustering approach. (I)-(K) t-SNE plots of all LT-HSCs analyzed by single-cell RNA-seq showing the mRNA expression levels, color coded for log2(TPM), of (I) CD150, (J) Vwf and (K) CD41 in each individual cell. (L) A gene signature learned from stimulated LT-HSCs shows the underlying heterogeneity among stimulated LT-HSCs. Heat-map demonstrates the expression of all genes in the inferred gene signature for each single stimulated LT-HSC. The panels below shows a score for each cell based on the expression of genes in this gene signature. The color-coded bar at the bottom shows the age of the mouse of origin. (M) The same signature in Figure 3G was applied to an independent data set of unstimulated young and aged LT-HSCs (Kowalczyk et al., 2015). Data represent at least two independent experiments (n=9) and are presented as mean ± SEM. ** ** ** ** ** B 80 A ** ns 100 60 80 n 40 dow 60 ck no 20 A k A 40 B cells (% of GFP+) RN % m % 0 4 5 4 5 3 3 4 4 20 1 1 MG Klf Klf MG Klf Klf Ikzf Ikzf Stat Stat Zbtb Zbtb HoxA9 HoxA9 0 Young mice Aged mice 1 4 5 4 3 MG Klf Klf Ikzf Stat Zbtb HoxA9 C 40 D 25 ** E 20 ** ** ** * ** ** ** * 20 30 15 15 20 10 10 10 5 T cells (% of GFP+) 5 Myeloid cells (% of GFP+) Granulocytes (% of GFP+) 0 0 0 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 5 5 5 5 5 5 MG MG MG MG MG MG Klf Klf Klf Klf Klf Klf Klf Klf Klf Klf Klf Klf Ikzf Ikzf Ikzf Ikzf Ikzf Ikzf Stat Stat Stat Stat Stat Stat Zbtb Zbtb Zbtb Zbtb Zbtb Zbtb HoxA9 HoxA9 HoxA9 HoxA9 HoxA9 HoxA9 Young mice Aged mice Young mice Aged mice Young mice Aged mice Young, MG Young, MG Young, MG 80 80 80 F Aged, MG Aged, MG Aged, MG Young, shKLF5 Young, shIKZF1 Young, shSTAT3 70 Aged, shKLF5 70 ^ Aged, shIKZF1 70 ^ ^ Aged, shSTAT3 ^ ^ ^ 60 60 60 50 50 50 B cells (% of GFP+) 40 40 40 0 hr 72 hr 1 wk 3 wk 0 hr 72 hr 1 wk 3 wk 0 hr 72 hr 1 wk 3 wk G Young, MG Young, MG Young, MG 30 30 Aged, MG 30 Aged, MG Aged, MG ) Young, shKLF5 Young, shIKZF1 Young, shSTAT3 Aged, shKLF5 Aged,
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