Ebf3+ niche-derived CXCL12 is required for the localization and maintenance of hematopoietic stem cells – Nature Communications

CXCL12 attracts HSCs to CAR cells within bone marrow

We generated CXCL12^f/- mice (Supplementary Fig. 1) and crossed them with mice expressing the CreERT2 transgene under the control of the Ebf3 gene, in which Cre recombinase can be activated in CAR cells but not in other bone marrow cell populations when injected with tamoxifen (Ebf3-CreERT2 mice)¹⁰. CXCL12 was deleted in a portion of CAR cells when Ebf3-CreERT2;CXCL12^f/- mice were injected with tamoxifen once and analyzed 10–14 weeks after tamoxifen treatment. Quantitative real-time polymerase chain reaction with reverse transcription (qRT-PCR) analysis of sorted PDGFRβ⁺Sca-1^–CD31^–CD45^–Ter119^– CAR cells⁹ revealed that CXCL12 was deleted from about 70% of the CAR cells in the mutant mice (Fig. 1a). Flow cytometric analysis revealed that the bone marrow from the mutant mice contained normal total hematopoietic cell counts, normal numbers of c-kit⁺CD19⁺IgM^– pro-B cells and c-kit^–CD19⁺IgM^– pre-B cells, and only modestly reduced numbers of the CD34^–CD150⁺CD48^– or CD150⁺CD48^– subset of Lin^–Sca-1⁺c-kit⁺ (LSK) cells, which are highly enriched for long-term repopulating HSCs (LT-HSCs) compared with control animals (Fig. 1b–d). These results raise the possibility that CXCL12 production from a subset of CXCL12-intact CAR cells contributes to HSC maintenance in the mutants.

a–d Bone marrow from 21- to 25-week-old Ebf3-CreERT2;CXCL12^+/+ control (n = 8) and Ebf3-CreERT2;CXCL12^f/- conditional knockout mice (n = 6) injected once with tamoxifen was analyzed. CXCL12 mRNA levels in CAR cells (a), the total hematopoietic cell counts (b), and the numbers of CD34^–CD150⁺CD48^–LSK (HSCs), CD150⁺CD48^–LSK (LSK-SLAM) HSCs (c), pro-B cells and pre-B cells (d) in the bone marrow. e Schematic illustrating generation of the CXCL12-tdTomato^f/f mouse line. f–l CXCL12-tdTomato^f/f control and Ebf3-CreERT2;CXCL12-tdTomato^f/f conditional knockout mice transplanted with bone marrow cells from Evi1-GFP mice were injected with tamoxifen three times and analyzed 17 to 23 weeks after treatment. Frequencies of CXCL12-tdTomato⁺ cells in S100⁺ CAR cells (f) and frequencies of random spots within each distance from CXCL12-intact CAR cells (g) in the bone marrow sections (n = 4). The numbers of phenotypic LT-HSCs in the bone marrow (h) (n = 6). Immunohistochemical analysis of Evi1-GFP^hic-kit⁺ HSCs (arrow) in contact with CXCL12-intact CAR cells (arrowhead) (i). The frequencies of Evi1-GFP^hic-kit⁺ HSCs within each distance from CXCL12-intact CAR cells in the bone marrow sections (n = 4) (j). Immunohistochemical analysis of Evi1-GFP^hic-kit⁺ HSCs (white arrows) in contact with the CXCL12-tdTomato⁺PDGFRβ⁺ CXCL12-intact CAR cells (white arrowheads) and distant from the CXCL12-tdTomato^–PDGFRβ⁺ CXCL12-deficient CAR cells (yellow arrowheads) in the mutants (k). The frequencies of Evi1-GFP^hic-kit⁺ HSCs within each distance from CXCL12-tdTomato⁺PDGFRβ⁺ CXCL12-intact CAR cells or CXCL12-tdTomato^–PDGFRβ⁺ CXCL12-deficient CAR cells in the bone marrow sections of the mutants (n = 4) (l). In (j), total of 960 and 1030 Evi1-GFP^hic-kit⁺ HSCs in 50 and 52 bone marrow sections were analyzed in CXCL12-tdTomato^f/f control and Ebf3-CreERT2;CXCL12^f/- conditional knockout mice, respectively, in 4 independent experiments. In (l), total 789 Evi1-GFP^hic-kit⁺ HSCs in 36 bone marrow sections were analyzed in 4 independent experiments. All error bars represent SE of the mean. Statistical significances were calculated using the two-tailed unpaired Student’s t test. Source data are provided as a Source data file.

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To address this possibility, we analyzed the localization of HSCs relative to CXCL12-intact CAR cells in these HSC-intact CXCL12 conditionally deficient mice. To visualize HSCs, we generated HSC-reporter mice, in which three EGFP genes were knocked into the HSC-specific Evi1 gene (Evi1-GFP mice) (Supplementary Fig. 2a). Evi1 is a transcription factor, which is specifically expressed in HSCs in the hematopoietic system and essential for the maintenance of HSCs^25,26. In these mice, the majority of phenotypic and functional HSCs were Evi1-GFP^hic-kit⁺ (Supplementary Fig. 2b, c). Conversely, the majority of Evi1-GFP^hic-kit⁺ cells comprised CD150⁺CD48^– LSK (LSK-SLAM) HSCs (Supplementary Fig. 2d). Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the Evi1-GFP^hic-kit⁺ cells was 1 in 3.8 cells (Supplementary Fig. 2e). Flow cytometric analysis revealed that Lin^–Sca-1⁺c-kit⁺CD150^–CD48^– multipotent progenitors (MPPs) expressed lower levels of Evi1-GFP compared to HSCs and other primitive hematopoietic progenitors, including Lin^–Sca-1^–c-kit⁺CD34⁺FcγRII/III^hi granulocyte/macrophage progenitors (GMPs) and Lin^–Sca-1^–c-kit⁺CD34^–FcγRII/III^lo megakaryocyte/erythrocyte progenitors (MEPs), did not express Evi1-GFP (Supplementary Fig. 2b). We could detect the fluorescence signals in fixed HSCs but not in fixed MPPs isolated from Evi1-GFP mice by histology (Supplementary Fig. 3). Flow cytometric analysis revealed HSC numbers were unaltered in Evi1-GFP mice (Supplementary Fig. 4). In addition, the mRNA levels of Evi1 in HSCs were unaltered in the absence of CXCL12 in CAR cells as described later. Together, Evi1-GFP mice allowed visualization of HSCs in the bone marrow sections of control and CXCL12 conditionally deficient mice. To visualize CXCL12-intact and CXCL12-deficient CAR cells, we generated another conditional CXCL12-targeted mouse line by inserting loxP sites that flank exons 2 and 3 of the Cxcl12 gene and a linked tandem dimer Tomato (tdTomato) gene to act as a CXCL12-specific reporter (CXCL12-tdTomato^f/f mice) (Fig. 1e), and we crossed them with Ebf3-CreERT2 mice. Control CXCL12-tdTomato^f/f and Ebf3-CreERT2;CXCL12-tdTomato^f/f mice were transplanted with bone marrow cells from Evi1-GFP HSC-reporter mice, injected with tamoxifen three times after 16 weeks following transplantation, and analyzed 17 to 23 weeks after tamoxifen treatment. In these chimeric mice, the red fluorescent protein tdTomato was expressed in CXCL12-intact CAR cells but not in CXCL12-deficient CAR cells. Immunohistochemical analysis with antibodies against S100, which is specifically expressed in CAR cells in bone marrow cavities⁹, showed that about 42% of the S100⁺ CAR cells lacked CXCL12-tdTomato expression in mutant chimeric mice, indicating that CXCL12-intact CAR cells were reduced by about 2-fold (Fig. 1f). Consistent with this, frequencies of random spots located within 5 μm of CXCL12-intact CAR cells were reduced compared with control chimeras (Fig. 1g). However, frequencies and numbers of HSCs were unaltered (Fig. 1h and Supplementary Fig. 5) and frequencies of Evi1-GFP^hic-kit⁺ HSCs located within 5 μm distance from CXCL12-intact CAR cells were unaltered in the mutant chimeras (Fig. 1i, j and Supplementary Fig. 6a). We next compared the distribution of HSCs with the distribution of CXCL12-intact and CXCL12-deficient CAR cells using antibodies against PDGFRβ, which is preferentially expressed in CAR cells in the bone marrow. Although the numbers of CXCL12-intact and CXCL12-deficient CAR cells were similar (Fig. 1f), frequencies of HSCs located within 5 μm distance from CXCL12-tdTomato^–PDGFRβ⁺ CXCL12-deficient CAR cells were markedly reduced compared to those from CXCL12-tdTomato⁺PDGFRβ⁺ CXCL12-intact CAR cells in the mutant chimeras (Fig. 1k, l and Supplementary Fig. 6b). These results reveal that HSCs detached from CXCL12-deficient CAR cells and attached to CXCL12-intact CAR cells in the mutants, indicating that CXCL12 attracts HSCs to CAR cells within bone marrow. On the other hand, few HSCs are located within 5 μm distance from the bone surface, suggesting that few HSCs were maintained in the endosteal niches in the mutants.

CAR/LepR⁺ cell-derived CXCL12 is essential for the maintenance of HSCs in aged mice

Because CXCL12 production from a small subset of CAR cells might contribute to HSC maintenance in 16-week-old LepR-Cre;CXCL12^f/- mice, we examined HSCs and their progeny in 90-week-old LepR-Cre;CXCL12^f/- mice, in which LepR was expressed in almost all the CAR cells⁴. qRT-PCR analysis of sorted PDGFRβ⁺Sca-1^–CD31^–CD45^–Ter119^– CAR cells revealed that CXCL12 was deleted in about 99.5% of the CAR cells (Fig. 2a). Flow cytometric analysis revealed that the total hematopoietic cell counts and numbers of LT-HSCs, Lin^–IL-7Rα⁺Flt3⁺ common lymphoid progenitors (CLPs), pro-B cells, pre-B cells, mature B cells, pDCs, NK cells, GMPs, Gr-1^hiCD11b⁺ granulocytes, MEPs, and c-kit⁺CD71⁺Ter119^lo proerythroblasts were severely reduced in the bone marrow of the mutant mice compared with control animals (Fig. 2b–d). The magnitude of the reduction was increased in B cell progenitors compared with LT-HSCs, GMPs, and MEPs in aged LepR-Cre;CXCL12^f/- mice. Consistent with this, frequencies of B cell progenitors were severely reduced in the bone marrow of the mutant mice compared with control animals (Supplementary Fig. 7b). In addition, we estimated the repopulating potential in HSCs using repopulating units (RUs), based on a competitive repopulation assay, where short-lived peripheral blood myeloid cells were analyzed, and found that RUs were markedly reduced in bone marrow of the mutants (Fig. 2e). These results indicate that CAR/LepR⁺ cell-derived CXCL12 is essential for the maintenance of HSCs and hematopoietic progenitors in the bone marrow of aged mice.

a CXCL12 mRNA levels in CAR cells from 90-week-old LepR-Cre;CXCL12^+/+ control (n = 9) and LepR-Cre;CXCL12^f/- conditional knockout (n = 10) mice. b–e The total hematopoietic cell counts (b) and the numbers of CD34^–CD150⁺CD48^–LSK HSCs, LSK-SLAM HSCs (c), CLPs, pro-B cells, pre-B cells, mature B cells, pDCs, NK cells, GMPs, granulocytes, MEPs, proerythroblasts (pro-E) (d), and repopulating units (RUs) (e) in the bone marrow from 90-week-old LepR-Cre;CXCL12^+/+ control (n = 9) and LepR-Cre;CXCL12^f/- conditional knockout (n = 10) mice. All error bars represent SE of the mean. Statistical significances were calculated using the two-tailed unpaired Student’s t test. Source data are provided as a Source data file.

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CAR cell-derived CXCL12 is essential for the maintenance of HSCs and hematopoietic progenitors

To examine the role of CXCL12 produced by CAR cells in younger adults, Ebf3-CreERT2;CXCL12^f/- mice were injected with tamoxifen eight times every other day beginning at 10 weeks of age and analyzed at 10 to 14 weeks after tamoxifen treatment. qRT-PCR analysis of sorted PDGFRβ⁺Sca-1^–CD31^–CD45^–Ter119^– CAR cells revealed that CXCL12 was deleted from more than 99.5% of the CAR cells in the majority of tamoxifen-treated Ebf3-CreERT2;CXCL12^f/- mice (CXCL12^ΔCAR mice) (Fig. 3a). Flow cytometric analysis of CXCL12^ΔCAR mice revealed that the total hematopoietic cell counts and frequencies and numbers of LT-HSCs, EPCR⁺CD150⁺CD48^–LSK (LSK-ESLAM) HSCs, LSK-SLAM HSCs, MPPs, CLPs, pro-B cells, pre-B cells, mature B cells, pDCs, NK cells, GMPs, granulocytes, MEPs, and proerythroblasts were reduced in the bone marrow of the mutants compared with control animals (Fig. 3b–e and Supplementary Fig. 8a–c). Of note, the magnitude of the reduction was increased in B cell progenitors compared with LT-HSCs, GMPs, and MEPs in the bone marrow of CXCL12^ΔCAR mice. In addition, the numbers of Lin^–Sca-1⁺c-kit⁺CD150^–CD48⁺flt3⁺ MPP4s with high lymphoid and low megakaryocyte/erythroid potential were reduced; however, the numbers of Lin^–Sca-1⁺c-kit⁺CD150^–CD48⁺flt3^– MPP3s with high myeloid potential were unaltered and Lin^–Sca-1⁺c-kit⁺CD150⁺CD48⁺flt3^– MPP2s with low lymphoid and high megakaryocyte/erythroid potential²⁷ were modestly increased (Fig. 3d and Supplementary Fig. 8b). We next analyzed RUs and the frequency and number of functional HSCs, which were determined using CRUs based on limiting-dilution of competitive repopulation assays, where short-lived peripheral blood myeloid cells were analyzed. The numbers of CRUs and RUs were markedly reduced in the bone marrow of CXCL12^ΔCAR mice (Fig. 3f). To confirm this, we performed secondary transplantations. Mice competitively reconstituted with 1500 donor-derived Lin^–Sca-1⁺c-kit⁺ primitive hematopoietic stem and progenitor cells from primary recipients transplanted with 200 cells in the HSC population from CXCL12^ΔCAR mice exhibited markedly reduced long-term multilineage reconstitution by donor-derived HSCs in secondary recipients (Supplementary Fig. 9). Cyclin-dependent kinase inhibitor p57 is essential for quiescence and maintenance of HSCs²⁸. The mRNA levels of p57 were reduced but those of the transcription factor Evi1 essential for HSC maintenance were unaltered in LT-HSCs in the marrow of CXCL12^ΔCAR mice (Fig. 3g). These results indicate that CAR cell-derived CXCL12 is essential for the maintenance of HSCs and hematopoietic progenitors within bone marrow.

Bone marrow from 21- to 25-week-old Ebf3-CreERT2;CXCL12^+/+ control and Ebf3-CreERT2;CXCL12^f/- CXCL12^ΔCAR mice injected with tamoxifen eight times was analyzed. a–e CXCL12 mRNA levels in CAR cells (a), the total hematopoietic cell counts (b), numbers of CD34^–CD150⁺CD48^–LSK HSCs, LSK-ESLAM HSCs, LSK-SLAM HSCs (c), MPPs, MPP2s, MPP3s, MPP4s (d), CLPs, pro-B cells, pre-B cells, mature B cells, pDCs, NK cells, GMPs, granulocytes, MEPs and proerythroblasts (pro-E) (e) in the bone marrow of Ebf3-CreERT2;CXCL12^+/+ control (n = 31) and CXCL12^ΔCAR (n = 21) mice. f RUs of Ebf3-CreERT2;CXCL12^+/+ control (n = 7) and CXCL12^ΔCAR (n = 6) mice and the numbers of functional LT-HSCs in the bone marrow, which were determined by measuring the competitive repopulating units (CRUs) (n = 3). g The mRNA levels of p57 and Evi1 in LT-HSCs in the bone marrow of Ebf3-CreERT2;CXCL12^+/+ control (n = 8) and CXCL12^ΔCAR (n = 5) mice. All error bars represent SE of the mean. Statistical significances were calculated using the two-tailed unpaired Student’s t test. Source data are provided as a Source data file.

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The ability of HSCs to generate B cell progenitors was markedly reduced in mice lacking CXCL12 in CAR cells

Since it was previously demonstrated that distinct HSCs exist that are stably biased towards the generation of lymphoid or myeloid cells^{19,20,21,22,23,24}, we examined the ability of HSCs from CXCL12^ΔCAR and control mice to generate B cell or myeloid progenitors within wild-type bone marrow. Sorted 200 cells in the HSC population from CXCL12^ΔCAR or control mice were transplanted with 5×10⁵ competitor bone marrow cells into wild-type mice. The percentages of donor-derived LT-HSCs, pro-B cells, pre-B cells, immature B cells, mature B cells, GMPs, and MEPs in the bone marrow were analyzed at 16 weeks after transplantation (Fig. 4a, c–e and Supplementary Fig. 10a–c) and the percentages of donor-derived B220⁺ B cells and Gr-1^hi granulocytes in the peripheral blood were analyzed at 4, 8, 12, 16 weeks after transplantation (Fig. 4b, f and Supplementary Fig. 9 and 10b, c). In the bone marrow, donor B and myeloid cell chimerism was reduced in recipients of HSCs from CXCL12^ΔCAR mice (Fig. 4a and Supplementary Fig. 10a), but donor contribution into B cell progenitors, i.e. % donor B cell progenitors divided by % donor myeloid progenitors (donor pro-B/GMP and pre-B/GMP reconstitution ratios), in recipients of HSCs from CXCL12^ΔCAR mice was markedly lower than that in recipients of HSCs from control animals (Fig. 4c), indicating a stable myeloid bias of HSCs from CXCL12^ΔCAR mice. In addition, the donor B cell progenitor/erythro-megakaryocytic progenitor (pro-B/MEP and pre-B/MEP) reconstitution ratios (Fig. 4d) and % donor B cell progenitors divided by % donor LSK-SLAM HSCs (pro-B/LSK-SLAM and pre-B/LSK-SLAM) in recipients of HSCs from CXCL12^ΔCAR mice (Fig. 4e) were markedly lower than those in recipients of HSCs from control animals. Consistent with this, the donor B cell/granulocyte reconstitution ratios in the peripheral blood were lower in recipients of HSCs from CXCL12^ΔCAR mice than those in recipients of HSCs from control animals (Fig. 4f). These results confirmed the attenuated ability of HSCs from CXCL12^ΔCAR mice to generate B lineage progeny. Consistent with this, frequencies of the CD150^lo or CD229^hi subset, which gave a higher level of lymphoid reconstitution^23,29, in the LT-HSC population were reduced in CXCL12^ΔCAR mice (Fig. 4g and Supplementary Fig. 11). Interestingly, the magnitudes of the decreases in the donor B cell progenitor/myeloid progenitor and B cell progenitor/erythro-megakaryocytic progenitor reconstitution ratios in mutant HSC chimeric mice were not smaller when compared to ratios of B cell progenitor numbers to myeloid or erythro-megakaryocytic progenitor numbers in CXCL12^ΔCAR mice (Compare Fig. 4c, d with 4h). Similar results were obtained when we performed secondary transplantations. We transplanted 1500 donor-derived Lin^–Sca-1⁺c-kit⁺ primitive hematopoietic stem and progenitor cells from primary recipients transplanted with 200 cells in the HSC population. The ability of HSCs from CXCL12^ΔCAR mice to generate B cell progenitors was markedly reduced after transplantation in secondary recipients (Supplementary Fig. 12).

a–f Sorted 200 cells in the HSC population from 21- to 25-week-old tamoxifen-treated CD45.2⁺ Ebf3-CreERT2;CXCL12^+/+ control or Ebf3-CreERT2;CXCL12^f/- CXCL12^ΔCAR mice, in which CXCL12 was deleted from more than 99.5% of the CAR cells (CXCL12^ΔCAR mice) were transplanted with 5 × 10⁵ CD45.1⁺ competitor bone marrow cells into CD45.1⁺CD45.2⁺ wild-type mice. The percentages of donor-derived cells were analyzed at 16 weeks after transplantation. a, b Donor chimerism of hematopoietic stem and progenitor cells in the bone marrow of recipients transplanted with HSCs from Ebf3-CreERT2;CXCL12^+/+ control (n = 6) or CXCL12^ΔCAR (n = 5) mice (a) and CD45⁺ cells, granulocytes, and B cells in the peripheral blood of recipients transplanted with HSCs from Ebf3-CreERT2;CXCL12^+/+ control (n = 6) or CXCL12^ΔCAR (n = 3) mice (b). c–e % donor B cell progenitors divided by % donor myeloid progenitor (donor pro-B/GMP and pre-B/GMP reconstitution ratios) (c), % donor B cell progenitors divided by % donor erythro-megakaryocytic progenitors (donor pro-B/MEP and pre-B/MEP reconstitution ratios) (d), and % donor B cell progenitors divided by % donor LSK-SLAM HSCs (donor pro-B/LSK-SLAM and pre-B/LSK-SLAM reconstitution ratios) (e) in the bone marrow of recipients transplanted with HSCs from Ebf3-CreERT2;CXCL12^+/+ control (n = 6) or CXCL12^ΔCAR (n = 5) mice are shown. f % donor B cells divided by % donor granulocytes (donor B/granulocyte reconstitution ratios) in the peripheral blood of recipients transplanted with HSCs from Ebf3-CreERT2;CXCL12^+/+ control (n = 6) or CXCL12^ΔCAR (n = 3) mice are shown. g Frequencies of the CD150^lo subset in the LT-HSC population in 21- to 25-week-old Ebf3-CreERT2;CXCL12^+/+ control (n = 37) and CXCL12^ΔCAR (n = 30) mice or CD229^hi subset in the LT-HSC population in 21- to 25-week-old Ebf3-CreERT2;CXCL12^+/+ control and CXCL12^ΔCAR (n = 6) mice. h Ratios of B cell progenitor numbers to myeloid progenitor numbers (pro-B/GMP and pre-B/GMP) or erythro-megakaryocytic progenitor numbers (pro-B/MEP and pre-B/MEP) and ratio of myeloid progenitor numbers to erythro-megakaryocytic progenitor numbers (GMP/MEP) in 21- to 25-week-old Ebf3-CreERT2;CXCL12^+/+ control (n = 31) and CXCL12^ΔCAR (n = 21) mice. All error bars represent SE of the mean. Statistical significances were calculated using the two-tailed unpaired Student’s t test. Source data are provided as a Source data file.

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CXCL12 enabled the maintenance of the B lineage repopulating ability of HSCs in vitro

The preferential reduction in lymphoid-biased HSCs in the absence of CAR cell-derived CXCL12 prompted us to examine direct actions of CXCL12 on maintenance of the lymphoid repopulating ability of HSCs. A serum-free culture system that supports the functional mouse HSCs ex vivo for over one month has been developed³⁰. Using this culture system, we sorted 50 cells in the LT-HSC population into fibronectin-coated plate wells containing serum-free medium and polyvinyl alcohol (PVA), cultured them for 28 days in the presence of stem cell factor (SCF) and thrombopoietin (TPO) with or without CXCL12, and transplanted cultured cells with 1 × 10⁶ CD45.1⁺ competitor bone marrow cells into wild-type mice (Fig. 5a). At 16 weeks after transplantation, although percentages of donor-derived hematopoietic cells were variable between recipients (Fig. 5b and Supplementary Fig. 13), the donor B cell progenitor/myeloid progenitor (pro-B/GMP and pre-B/GMP) reconstitution ratios in recipients of cultured cells without CXCL12 was lower than the donor B cell progenitor/myeloid progenitor reconstitution ratios in recipients of HSCs from wild-type mice (compare Fig. 4c with Fig. 4d). However, interestingly, the donor B cell progenitor/myeloid progenitor (pro-B/GMP and pre-B/GMP) reconstitution ratios in the bone marrow and B cell/myeloid cell reconstitution ratios in the peripheral blood in recipients of cultured cells with CXCL12 was higher than those in recipients of cultured cells without CXCL12 (Fig. 5c, d) and was comparable to those in recipients of HSCs from wild-type mice (compare Fig. 4c with Fig. 4d).

a Experimental design. Sorted 50 cells in the HSC population from CD45.2⁺ wild-type mice were cultured for 28 days in the presence of SCF and TPO with or without CXCL12, and 75% of cells in each well were transplanted with 1×10⁶ CD45.1⁺ competitor bone marrow cells into CD45.1⁺CD45.2⁺ wild-type mice. b Donor chimerism of LSK-SLAM HSCs, GMPs, pro-B cells, and pre-B cells in the bone marrow of recipients at 16 weeks after transplantation is shown (n = 5). All error bars represent SE of the mean. c Representative flow cytometry plots of bone marrow and peripheral blood from recipient mice at 16 weeks after transplantation. d Donor-derived B lymphoid/myeloid (pro-B/GMP and pre-B/GMP) ratios in recipients at 16 weeks after transplantation are shown (n = 5). All error bars represent SE of the mean. Statistical significances were calculated using the two-tailed unpaired Student’s t test. Source data are provided as a Source data file. e Working model for how CXCL12 regulates HSCs to produce the required number of B cell progenitors. CAR cell-derived CXCL12 plays a critical role in the maintenance of HSCs, especially lymphoid-biased HSCs.

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• Source: https://www.nature.com/articles/s41467-023-42047-2