Unique transcriptional and cell surface protein characteristics of extramedullary HSCs/MPPs. (A-E,G) Analysis of 10X scRNA-seq data from 16 651 transcriptionally defined HSCs/MPPs (sum of clusters 0, 4, 5, 11, and 21 from Figure 1A) combining matched BM and spleen (SPL) from the same individuals (OD1, 3812 cells; OD2, 3460 cells; OD4, 9379 cells). (A) Uniform Manifold Approximation and Projections (UMAPs) of HSCs/MPPs clustered according to SAM (k-means = 2; top panels colored according to SAM cluster, bottom panels according to tissue). (B) Bar graphs of the proportions of BM- and SPL-derived HSCs/MPPs in the SAM0-med and SAM1-extramed clusters. (C) Proportions of SAM0-med and SAM1-extramed HSCs/MPPs in the HSC/MPP space of each tissue. (D) Analysis of genes differentially expressed between the SAM0-med (n = 7068 cells) and SAM1-extramed (n = 9583 cells) clusters. Pre-ranked Gene Set Enrichment Analysis  (GSEA) of population-specific signatures (left; cord blood long-term HSC [CB LT-HSC] and short-term HSC [ST-HSC] from Laurenti et al,⁴⁷ BM LT-HSC (unpublished), other from Laurenti et al³⁹) and lineage-priming modules (right; from Velten et al⁴³) comparing SAM0-med with SAM1-extramed HSCs/MPPs. Selected lineage-priming modules are shown. All gene sets are listed in supplemental Table 4g. (E) Volcano plot of differentially expressed surface proteins (P < .05; log-fold change >0.5) in SAM0-med and SAM1-extramed HSCs/MPPs from CITE-seq data of OD4. (F) Pseudo-time of all transcriptionally defined HSCs/MPPs in each tissue. Kruskal-Wallis test with multiple comparison. (G) GSEA of C2 curated MSigDB pathways (false discovery rate <0.05 by preranked GSEA) on differentially expressed genes between SAM0-med (n = 7068 cells) and SAM1-extramed (n = 9583 cells) HSCs/MPPs. Selected gene sets are shown. All gene sets are listed in supplemental Table 4h. (H) scRNA-seq data from four mPB CD19^–CD34⁺ HSPCs (28 026 cells) were integrated with the same BMs and nonmobilized PBs as in Figure 2. The cell density across the UMAP coordinates of each tissue is displayed as contours filled by a color gradient. Different HSPC groups are indicated by dashed lines. (I-J) Gene signatures of medullary- and extramedullary-type HSCs/MPPs were used to compute a BM- or SPL-type identity score for each HSC/MPP cell of the multi-tissue landscape. Box plots show the ratio between BM- and SPL-type scores for each sample (“identity ratio”). Notches indicate the 95% confidence interval of the median (middle line). (I) The identity ratio was calculated for BM and SPL HSCs/MPPs taken from our 10X multi-tissue landscape and then validated by using transcriptionally defined HSCs/MPPs from the Human Cell Atlas BM data set (HCA BM)⁴⁰ as well as Smart-seq2 (SS2) data from single-cell sorted phenotypic HSCs/MPPs (CD19^–CD34⁺CD38^–CD45RA^–) from BM (SS2 BM) and SPL (SS2 SPL) of OD1 and OD2. (J) Boxplots show the identity ratio for BM (10X BM), nonmobilized PB (10X PB), and mPB (10X mPB) calculated using only the data integration containing these tissues. Mean ± standard deviation is shown in panels B and C. Two-tailed paired t test. *P = .02. LyP, lymphoid progenitor; MDP, monocyte/dendritic cell progenitor; MEMBP, MEMB progenitor; MLP, multilymphoid progenitor; MyP, My progenitor; NES, normalized enrichment score; PID, Pathway Interaction Database; WP, WikiPathways.