HSC-independent definitive hematopoiesis persists into adult life

Abstract

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Keywords: AGM region; B-1a cells; CP: Developmental biology; HSC precursors; HSC-independent hematopoiesis; adaptive immune lymphocytes; hemogenic endothelial cells; lineage tracing mouse models; mouse embryo; multi-potent progenitors.

Figure 1.. HSCs start lymphopoiesis in mice after 4 weeks of age and do not completely replace fetus-derived lymphocytes

(A) Experimental design. TAM was injected once on P2 into iFgd5 mice to label HSCs. Tomato⁺ blood cells in the BM, spleen, and peritoneal cavity were examined at different time points. (B) The Tomato ratio of each blood cell subset to HSCs (<4 weeks, n = 3; 8–12 weeks, n = 9; >6 months, n = 5–6). (C–E) “Fate mapping scatterplots” showing the correlation between Tomato% in HSCs (x axis) and Tomato% of a target cell population (y axis). The equation and R² values were calculated using Prism. (C) Scatterplots of MPP (n = 18) and spleen macrophages (n = 15). A strong correlation was seen as early as 8 weeks after birth (late, red dots). Blue dots represent correlation when analyzed before 4 weeks of age. (D) Scatterplots of spleen FO (n = 18), CD4 (n = 18), and CD8 T cells (n = 18). Blue dots, less than 4 months; red dots, more than 6 months. (E) Scatterplots of peritoneal B-1a cells (n = 18) and mast cells (n = 17). Blue dots, less than 4 months; red dots, more than 6 months. (F) The Tomato ratio of T progenitor populations in the thymus at 4 weeks (n = 4), 8–12 weeks (n = 9), and 5–12 months (n = 5) after birth. (G) Actual Tomato% in various hematopoietic populations in the P0 liver, thymus, and spleen when TAM was injected at E14.5 and E15.5 (n = 4). **p < 0.01, *p < 0.05 (unpaired Student’s t test).

Figure 2.. HECs produce MPP and B and T lymphocytes that persist in old mice

(A) Experimental design. A single dose of TAM was injected into iCdh5 pregnant mice at E7.5–E11.5, respectively, and Tomato⁺ HSPCs were examined at different time points after birth. (B) Cdh5Cre marks HSC-independent and HSC-dependent lineages and defined “adjusted EC-derived lymphocytes.” The estimated Tomato% of HSC-derived cells was determined using the equations in each fate mapping plot in Figure 1. (C–I) Adjusted EC-derived spleen FO B cells (C), CD4 (D), CD8 (E), peritoneal B-1a cells (F), mast cells (G), BM MPPs (H), and spleen macrophages (mac) (I) when TAM was injected at each embryonic time point. Shown are (E) early time points (<4 months old) and (L) late time points (>6 months old). The numbers of analyzed mice and actual Tomato% of these cells are shown in Figure S3. ***p < 0.001, **p < 0.01, *p < 0.05, unpaired Student’s t test. The numbers of analyzed mice are listed in Figure S3A.

Figure 3.. HECs produce the majority of HPCs in the FL

(A) Experimental design. TAM was injected at E7.5, E9.5, or E11.5, and FL progenitors were examined at E15.5. (B) Tomato% in CD45⁺c-kit⁺ HPCs and CD45⁻VC⁺ ECs in the YS and AGM region at E10.5 following TAM injection at E7.5 (n = 5). (C) Tomato% in HSCs and HPCs in the E15.5 FL following TAM injection at E7.5 (n = 6). (D) Representative fluorescence-activated cell sorting (FACS) plots of (C). (E) Tomato% in HPCs and ECs in the YS and AGM region at E10.5 following TAM injection at E9.5 (n = 3). (F) Tomato% in HSCs and HPCs in the E15.5 FL following TAM injection at E9.5 (n = 4). (G) Tomato% in HSCs and MPPs in the E15.5 FL following TAM injection at E11.5 (n = 3). (H) B-1 and/or B-2 progenitor colony-forming cell numbers. Tomato⁺ MPPs in E15.5 FL following TAM injection at E7.5 were sorted and used for the B-progenitor colony formation assay. Eight days after plating, all colonies were individually picked up and subjected to FACS analysis for B-1 (CD93⁺CD19⁺B220⁻) and/or B-2 progenitors (CD93⁺CD19⁻B220⁺). **p < 0.01, *p < 0.05, unpaired Student’s t test.

Figure 4.. LT-HSCs, MPPs, and B-1 repopulating cells arise independent from VC⁺c-kit⁺EPCR⁺ cells in the E11.5 AGM region

(A–C) Five to 50 pre-HSCs from the E11.5 AGM region were injected into sublethally irradiated NSG neonates. Shown is the CD45.2⁺ donor cell percentage in the peripheral blood (PB) of the recipient mice 4–6 weeks after transplantation (left panels). Donor cell percentage and their composition within the lymphoid subsets in the peritoneal cells, spleen, and BM are depicted. The engraftment patterns were categorized into three groups: (A) multi-lineage engraftment with BM LSK repopulation (HSC engraftment), (B) multi-lineage engraftment without BM LSK repopulation (MPP engraftment), and (C) only B-1 with minimal B-2 cell engraftment without BM repopulation (B-1 cell engraftment). (D) Donor-derived cell lineage distribution in the PB of the recipient mice in each group. HSC, HSC-engraftment group (A), n = 5; MPP, multi-lineage engraftment group (B), n = 6; B-1 only, only B-1 with minimal B-2 cell engraftment group (C), n = 4. (E) Representative FACS plots of the BM of an HSC-engrafted mouse (mouse 2). (F) Representative FACS plots of the BM of the secondary transplanted mice with BM cells from mouse 2. (G) Representative FACS plots of the BM of an MPP engrafted mouse (mouse 9).

Figure 5.. scRNA-seq analysis showed HSC and B lymphoid signatures in E11.5 pre-HSCs and E12 and E14 FL HSCs

(A) Dimensionality reduction of scRNA-seq data using PCA colored by cell type. E11A, E11.5 AGM pre-HSCs; E11Y, E11.5 YS pre-HSCs; E12F, E12.5 FL HSCs; and E14F, E14.5 FL HSCs. (B) A heatmap depicting the expression of HSC-, B cell-, T cell-, and ILC-related genes in the E11 AGM and YS pre-HSC populations and E12.5 and E14.5 FL HSCs. Red, blue, and yellow intensities indicate high, low, and intermediate expression levels, respectively. (C) Pseudo-time analysis of our scRNA-seq data. (D) Velocity analysis of the 96-cell data, indicating two directions (right arrow and left arrow). (E) Genes that were highly expressed at both ends of the arrows in Figure 1D were applied to the gene expression subsets of HSC and B progenitors in the ImmGen database (https://www.immgen.org/Databrowser19/DatabrowserPage.html). (F and G) Pseudo-time analysis of the public data on E11.5 pre-HSCs (types I and II), intra-aortic clusters (IACs), and E14.5 FL HSCs.^, Each cell type was also mapped in the pseudo-time analysis. (H) Selected HSC- and lymphoid cell-related genes were plotted in the pseudo-time analysis of the public data. Genes expressed and/or essential for HSC/MPP (top panel), HSC/B progenitors (center panel), and B progenitors (bottom panel) are depicted. (I) Velocity analysis of the public datasets.

Figure 6.. Our working model proposing EC-derived multiple waves of hematopoiesis

(A) EC-derived (HSC-independent) and HSC-derived lymphopoiesis show how we can estimate the percentages of each wave. (B) Multiple waves of hematopoiesis produce MPPs, LPs, B-1a cells, and HSCs during E7.5–E11.5. EC-derived HSPCs are the major cell populations in young animals. While postnatal HSC-derived lymphoid cells replace these embryonic EC-derived blood cells with age, a significant percentage of embryonic EC-derived lymphocytes persist in old mice.