Lineage tracking discloses a contribution of endothelial cells to hematopoiesis in adult BM

(A) Experimental design: tamoxifen was administered to 8- to12-week-old Cdh5-Cre mice to induce fluorescent labeling of VE-Cadherin⁺ cells and their cell progeny. Four weeks later, BM and blood were analyzed. (B) CD31⁺EGFP+ BM ECs in Cre⁻ mice (n=10) and Cre⁺ mice treated with oil (n=13) or tamoxifen (n=10); flow cytometry results. (C and D) CD45+EGFP+ cells in BM and blood from Cre⁻ mice (n=8) and Cre⁺ mice treated with oil (n=6-10) or tamoxifen (n=15-18). Representative flow cytometry gating in Figure S1G. (E) Representative blood smear from a tamoxifen-treated Cdh5-CreERT2(PAC)/ZsGreen mouse showing ZsGreen⁺CD45⁺DAPI⁺ cells (arrows). (F) Kinetics of ZsGreen⁺ cell detection in BM ECs (CD45⁻VE-Cadherin⁺) and blood white blood cells (WBC) post-tamoxifen; mouse n=8-10/group). (G) EGFP+ B and T-lymphocytes, granulocytes, and monocytes in BM of tamoxifen-treated mice (n=14) as percent of total EGFP+ cells; 3 experiments. (H) EGFP⁺ BM LSK, lymphocytes, granulocytes, and monocytes as percent of total EGFP⁺/- cell type; Cdh5-CreERT2(PAC)/mTmG mice (oil n=10; tamoxifen n=15), 3 experiments. (I) UMAP plots of Lin⁻ BM HSPC from tamoxifen-treated Cdh5-CreERT2(PAC)/ZsGreen mice (n=26; 1 femur/mouse) showing FlowSOM clustering of all (ZsGreen⁺/-) and ZsGreen⁺ populations. (J) Violin plots showing ZsGreen⁺ cell distribution across HSPC subsets from (I). Dots represent individual mice; data shown as mean±SD except shown as median in (G). *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test.

BM ECs generate engraftable hematopoietic cells ex vivo

(A) BM cells from tamoxifen-treated mice were cultured on high-attachment Primaria flasks or OP9 cell monolayers. Representative images show ZsGreen⁺ cells at weeks 1, 3, and 8. (B) Workflow for culturing unsorted and sorted BM cell populations. All cells were cultured (8 weeks) on OP9 cell monolayers supplemented with WT BM cells. Culture medium and floating cells were removed twice/week for 7 weeks. At the start of week 8, one final WT BM and medium supplementation was implemented prior to harvest at the end of week 8. Representative image (bottom left) shows sorted ZsGreen⁺ ECs on OP9 monolayer after 4 weeks of culture. (C and D) Representative flow cytometry plots (C) and quantification (D) of CD45⁺ZsGreen⁺ cells from each of the 8-week cultures (n=5). (E) Floating/loosely adherent ZsGreen⁺ cells from unsorted BM 8-week cell cultures were sorted and transplanted (5×104, 2.5×104,1.25×104 or 6.25×103 cells) into lethally irradiated (11 Gy) WT mice (n=2/group). (F - G) Representative image (F) and quantification (G) of low-adherent cells harvested after 8 weeks of culture, showing that >95% of ZsGreen⁺ low-adherent cells are CD45⁺. (H and I) WBC counts (F) and percent ZsGreen⁺ and ZsGreen⁻ cells (G) in blood of transplant recipients 10 weeks post-transplant (n=6); WT controls (no irradiation or transplant; n=5). Dots represent individual mice. Data are shown as mean ± SD. ns, not significant by Student’s t test.

Adult BM endothelial cells give rise to hematopoietic cells following transfer into conditioned recipients

(A) Transplant experiment: donor ECs from BM of tamoxifen-treated mice were FACS-sorted and transplanted into WT C57Bl/6 recipients conditioned with 5-FU or PBS. (B) ZsGreen⁺ ECs detected in BM of 5-FU-conditioned (n=5) or PBS-conditioned (n=15) recipients of ECs 4 weeks post-transplant.(C and D) ZsGreen+CD45+ cells (C) and cell type distribution (D) in the BM and blood of 5-FU-conditioned transplant recipients of BM ECs or no cell controls (n=5/group). (E and F) Age-dependent decline of ZsGreen⁺CD45⁺ cells (E) but not ZsGreen+VE-Cadhenin+ cells (F) in the BM of Cdh5-CreERT2(BAC)/ZsGreen mice (n=35) treated with tamoxifen 4-weeks prior to harvest. (G and H) Cell number (G; mouse n=8-12) and cell type distribution (H; mouse n=6) in the peritoneal cavity (PerC) of PBS- or thioglycolate (TGL)-pretreated (4 hours) mice. (I) ZsGreen⁺ and ZsGreen⁻ PerC cell types in TGL-pretreated mice (n=12). (J) Representative histograms depicting pHrodo Red fluorescence detection of E-Coli phagocytosis. (K and L) E. coli⁺ phagocytosis by ZsGreen⁺ and ZsGreen⁻ PerC neutrophils (K) and macrophages (L) in TGL-pretreated mice (n=4). (M) Representative histograms depicting CellRox Orange fluorescence for cell-associated ROS detection. (N and O) CellRox mean fluorescence intensity (MFI) in ZsGreen⁺ and ZsGreen⁻ PerC neutrophils (N) and macrophages (O) in TGL-pretreated mice (n=4). Dots represent individual mice. Data are shown as mean ± SD. *p < 0.05, ***p < 0.001, ns, not significant by Student’s t test.

Independence of adult EHT from preexisting HSPC

(A) Transplantation experiment: donor LSK sorting, recipient irradiation, transplantation, tamoxifen treatment, and analysis. (B-D) Blood WBC counts (B), percent ZsGreen⁺ PBMC (C), and time course of ZsGreen⁺ PBMC detection (D) in transplant recipients of ZsGreen⁻ LSK (5×104 or 2.5×104 cells/mouse; n=3/group) and ZsGreen-enriched LSKs (2.8×103 cells/mouse; n=2). Results in B and C are from week 24 post-tamoxifen. (E) Experiment: WT BM transplantation (BMTP) into lethally irradiated Cdh5-Cre/mTmG mice (n=9). Four weeks later, tamoxifen was administered; blood was monitored for 16 weeks. (F and G) EGFP⁺ PBMC detection before and after tamoxifen or peanut oil administration (F) and cell type distribution of EGFP⁺ and EGFP⁻ PBMCs at week 12 post-tamoxifen or peanut oil (G) in Cdh5-Cre/mTmG recipients (n=9) of WT BM (5×106 cells). Dots represent individual mice. Data are shown as mean ± SD.

Polylox sc lineage tracing links adult BM ECs to hematopoietic progenitors and mature blood cell progeny

(A) Schematic of Polylox barcode and transcriptome profiling. FACS-enriched ECs (ZsGreen⁺VE-Cadherin+Endomucin+) and EC-depleted (ZsGreen−VE-Cadherin−Endomucin−) BM cells from tamoxifen-treated Cdh5-CreERT2/ZsGreen/PolyloxExpress mice (n=3, 10 week-old at the time of tamoxifen treatment) were mixed (1:1), and encapsulated (147,446 cells loaded; 93,553 processed). Indexed cDNA was used for scRNA-seq and barcode detection by PacBio sequencing after nested PCR enrichment; barcode-transcriptome integration was accomplished via shared cell indices. (B) UMAP clustering and cell type annotation. Clusters 0, 1, 13, and 22 comprise ECs; cluster 14 comprises Mesenchymal-type cells. (C – E) Heatmaps showing “true” Polylox barcodes (pGen < 1×10⁻⁴) linking HSPCs to hematopoietic cells (C), ECs to hematopoietic and other cells (D), and Mesenchymal-type cells to other cells (E). The numbers within the colored boxes identify cell number; the labels at the bottom of each column denote the barcode shared by all cells in that column; the number on the right side the heatmaps reflects the total number of cells in each row. (F) UpSet plot showing cells (identified by colored dots) sharing the same “true” barcode (identified by lines connecting the colored dots); bar graph at the top of the plot reflects (height and number on each bar) the number of “true” barcodes. Colors of dots: EC (red), Mesenchymal-type (orange), ECs connecting with Mesenchymal-type cells (blue), cells other than ECs and Mesenchymal-type cells (black). (G) Violin plots showing selected gene expression profile in Mesenchymal-type cells (cluster 14) and ECs (clusters 0, 1, 13, 22 combined).

Sc transcriptomic analysis of prospective hemogenic ECs

(A) UMAP clustering of 434,810 cells from eight public scRNA-seq datasets. (B) Dot plot showing relative Cdh5 and Runx1 co-expression across clusters; clusters 8 and 50 co-express both genes. (C) UMAP highlighting clusters 8 and 50; all other clusters shown in grey. (D) Violin plots of doublet scores across Leiden clusters. Clusters 50 and 8 show no evidence of doublet enrichment. (E) Datasets proportional contribution to clusters 50 and 8; each dataset is color-coded. (F) Dot plot showing expression of selected marker genes in clusters 50 and 8 (from the public sc RNA-seq datasets listed in Figure 7D) and from clusters 0, 1, 13, 22 and 14 (from Polylox scRNA-seq; Figure 5B). Results reflect mean expression and fraction of cells in group. (G) Cdh5, Runx1 and Col1a2 co-expression in the indicated clusters as a fraction of cells in the cluster. (H and I) t-SNE plot of ECs from 11 murine tissues (G) and Venn diagram (H) showing rare co-expression of Cdh5, Runx1, and Col1a2 in these tissues.

Contribution of Col1a2 and Runx1 expression to ECs hemogenic activity

(A and B) Percent EGFP+CD45+ cells in BM and blood of tamoxifen-treated (n=6) or oil-treated (n=5) Col1a2-CreERT2/mTmG mice (A) and tamoxifen-treated (n=4) or oil-treated (n=3) Col1a2-CreERT2/ZsGreen mice (B). Cre-control mice (n=5 in A, and n=2 in B). (C) Transplant experiment: sorted VE-Cadherin⁺CD45⁻ZsGreen⁺/Col1a2⁺ cells from tamoxifen-treated Col1a2-CreERT2/ZsGreen mice are transplanted into 5-FU-conditioned WT recipients. (D and E) Detection (D) and characterization (E) of ZsGreen⁺CD45⁺ cells in BM and blood of WT 5-FU-conditioned mice (n=5), 4 weeks post-transplant of VE-Cadherin⁺CD45⁻ZsGreen⁺/Col1a2⁺ cells. Control FU-conditioned WT mice (n=4) received no cell transplant (D). (F) Time course of ZsGreen⁺ PBMC detection in control (Cdh5-Cre⁺/ZsGreen⁺) and Runx1EC-KI (Cdh5-Cre⁺/ZsGreen⁺/Runx1-KI) mice (n=10 per group). (G and H) Representative images (G) and quantification (H) of ZsGreen⁺ cells from OP9 cell-supported cultures of BM cells from tamoxifen-treated Cdh5-Cre⁺/ZsGreen⁺ (n=11) and Runx1EC-KI mice (n=5). (I and J) Representative flow cytometry plots (I) and quantification (J) of CD45⁺ZsGreen⁺ cells from OP9 cell-supported BM cell cultures (n=5/group). Dots represent individual mice. Data are shown as mean ± SD. **p<0.01, ***p < 0.001 by Student’s t test.