Mice
B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)/Hze/J (iTdTomato) (007914), C57BL/6J (CD45.2) (000664) and B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) (002014) mice were purchased from The Jackson Laboratory. Nestin–GFP mice48 were bred in our facility. Cdh5-creER, Cdh2-creER, Cxcl12fl/fl, Kitlfl/fl, Tpo−/− and Tg(Alb-Tpo) mice were provided by R. H. Adams, L. Li, T. Nagasawa, S. J. Morrison, F. J. de Sauvage and W. S. Alexander, respectively. Unless indicated otherwise, 8–10-week-old mice of both sexes were used for experiments. All these mice were backcrossed with C57BL/6J mice for more than ten generations and maintained in pathogen-free conditions under a 12 h–12 h light–dark cycle, at a temperature of 21 ± 1 °C and humidity of 40–70%, and were fed with autoclaved food and water. This study complied with all ethical regulations involving experiments with mice, and all experimental procedures performed on mice were approved by the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine. No randomization or blinding was used to allocate experimental groups.
Femoral bone transplantation
Femurs with intact periosteum were isolated from 8–10-week-old donor mice and preserved on ice in PBS (21-040-CV, Corning) until they were implanted in recipient mice. For transplantation of a single femur, non-conditioned recipient mice that were age and sex-matched with donor mice were anaesthetized with ketamine and xylazine, and a small incision was made at their unilateral thoracic region. Subsequently, the preserved femur was implanted subcutaneously, and the wound was closed. For transplantation of six femurs, small incisions were made at the bilateral cervical, thoracic and pelvic regions of recipient mice, and then one femur was implanted in each area, followed by wound closure. A sham operation was performed by making small incisions on the same area of skin as the control bone transplantation group and closing them.
Parabiosis
Parabionts were generated by making an incision in the skin from the elbow to the knee of mice on opposite sides of each mouse. The elbows and knees were paired together by s.c. suturing. The skin was then matched from one mouse to the other, sutured together and secured with wound clips.
Splenectomy
After mice were anaesthetized with ketamine and xylazine, a longitudinal incision was made in the skin and peritoneum on the left dorsolateral side of the abdomen, caudal to the last rib. The splenic artery was ligated and the spleen was removed. The abdominal wall was then closed, and the skin was sutured. A sham operation was performed by exteriorizing the spleen and then reinserting it into the abdominal cavity.
In vivo treatment
For G-CSF treatment, G-CSF (NEUPOGEN/Filgrastim; 300 µg ml−1, purchased from Jack D. Weiler Hospital of Albert Einstein College of Medicine) was injected s.c. at a dose of 125 μg kg−1 twice a day (eight divided doses) beginning in the evening of the first day. When used in bone transplantation experiments, G-CSF was administered to all groups at 1 month after the femurs were implanted or a sham operation was performed unless otherwise indicated. When HSC mobilization was checked, blood was collected at 3 h or 7 days after the final morning dose. For induction of CreER-mediated recombination, 8–10-week-old Cdh5-creER;iTdTomato mice were injected intraperitoneally with 2 mg tamoxifen (T5648, Sigma-Aldrich) dissolved in corn oil (C8267, Sigma-Aldrich) for five consecutive days (10 mg in total per mouse). Then, 4 weeks after the injection, these mice were used as hosts, or their femurs were isolated for transplantation. In experiments examining the overlap of Cdh2+ cells and MSCs, 8–10-week-old Cdh2-creER;iTdTomato or Cdh2-creER;iTdTomato;Nestin-GFP mice were injected with tamoxifen and subjected to analyses 4 weeks after the injection. In experiments using Cdh2-creER;Cxcl12fl/fl or Cxcl12fl/fl mice as hosts, tamoxifen was administered at 2 months after the femurs were implanted or a sham operation was performed in these mice. In parabiosis experiments, each mouse of the parabionts was injected with 2 mg tamoxifen for five consecutive days (20 mg in total per parabiont) 3 weeks after the surgery. Then, 4 weeks after the injection, the parabionts were subjected to analyses. In experiments using Cdh2-creER;Kitlfl/fl or Kitlfl/fl mice as hosts, tamoxifen was administered to four to five-week-old mice before femurs were implanted or a sham operation was performed.
Whole-mount imaging of host femurs and femoral grafts
Antibodies used for immunofluorescence staining of femoral grafts and host femurs are CD31 (PECAM1) Alexa Fluor 647 (MEC13.3, 102516) and CD144 (VE-cadherin) Alexa Fluor 647 (BV13, 138006) from BioLegend. For all imaging experiments, these antibodies (5 μg each) were injected into mice through the retro-orbital plexus for the vasculature staining, and mice were euthanized 10 min after injection. Femoral grafts and host femurs were then isolated and fixed in 4% paraformaldehyde (PFA; 15710, Electron Microscopy Sciences) overnight at 4 °C. For cryopreservation, the bones were incubated sequentially in 10%, 20% and 30% sucrose/PBS at 4 °C for 1 h each, embedded and flash-frozen in SCEM embedding medium (C-EM002, SECTION-LAB) and stored at −80 °C. For whole-mount imaging, bones were placed at −20 °C overnight and shaved with a Cryostat (CM3050, Leica) until the BM cavity was fully exposed. The sections were carefully collected from the melting embedding medium, rinsed with PBS and post-fixed with 4% cold PFA for 10 min followed by permeabilization in 0.5% Triton X-100/PBS for 3 h at room temperature (20–25 °C) and incubation with 2 µg ml−1 4′,6-diamino-2-phenylindole (DAPI; D9542, Sigma-Aldrich) for 30 min. Images were acquired at room temperature using the Zeiss Axio examiner D1 microscope (Zeiss) with a confocal scanner unit (Yokogawa), and reconstructed in three dimensions with SlideBook 6 (Intelligent Imaging Innovations), Photoshop 26 (Adobe) and Fiji build of ImageJ 2 (National Institute of Health, NIH) software.
Cell preparation
For analyses of haematopoietic cells in host femurs and femoral grafts, BM cells in these bones were flushed and dissociated using a 1 ml syringe with PBS through a 21-gauge needle. For analyses of haematopoietic cells throughout the mouse body, BM cells in endogenous and grafted femurs, tibias, humeri and pelvis were collected by flushing and dissociating, and radii, skull, spine, sternum and ribs were minced into small pieces with scissors, crushed with a mortar and pestle and filtered through a 70 µm cell strainer. Splenic cells were obtained by gentle grinding with slide glasses and passing through a 70 µm cell strainer. Cells in the liver were obtained by gentle grinding with slide glasses followed by digestion at 37 °C for 30 min in 1 mg ml−1 collagenase type IV (17104019, Gibco), 2 mg ml−1 dispase (17105041, Gibco) and 50 μg ml−1 DNase I (DN25, Sigma-Aldrich). Peripheral blood was collected by retro-orbital bleeding of mice anaesthetized with isoflurane and mixed with EDTA to prevent clotting. The data from the bones above, spleen, liver and blood (assumed to be 2 ml per animal) were summed to determine the total HSC numbers in the mouse body. For analyses of BM stromal cells, intact flushed BM plugs were digested at 37 °C for 30 min in 1 mg ml−1 collagenase type IV, 2 mg ml−1 dispase and 50 μg ml−1 DNase I in Hank’s balanced salt solution with calcium and magnesium (21-023-CV, Gibco). These single-cell suspensions were then subjected to red blood cell lysis with ammonium chloride and washed in ice-cold PEB (PBS containing 0.5% BSA and 2 mM EDTA).
Flow cytometry analysis and cell sorting
Cells were surface-stained in PEB for 30–60 min at 4 °C. Antibodies used for flow cytometry analyses and sorting were as follows: anti-CD45 APC-eFluor 780 (30-F11, 47-0451-82), anti-TER-119 APC-eFluor 780 (TER-119, 45-5921-82), anti-CD31 PE-Cyanine7 (390, 25-0311-82), anti-CD51 biotin (RMV-7, 13-0512-85), anti-CD140a (PDGFRA) PE (APA5, 12-1401-81), anti-CD140a PE-Cyanine7 (APA5, 25-1401-81), anti-Ly6A/E (SCA-1) FITC (D7, 11-5981-82), anti-Ly6G/Ly6C (GR-1) FITC (RB6-8C5, 11-5931-85), anti-Ly6G/Ly6C APC-eFluor 780 (RB6-8C5, 47-5931-82), anti-CD11b PE (M1/70, 12-0112-83), anti-CD11b PE-Cyanine7 (M1/70, 25-0112-82), anti-CD11b APC-eFluor 780 (M1/70, 47-0112-82), anti-B220 APC-eFluor 780 (RA3-6B2, 47-0452-82), anti-CD3e APC-eFluor 780 (145-2C11, 47-0031-82), anti-CD48 PerCP-eFluor 710 (HM48-1, 46-0481-85), anti-CD48 PE-Cyanine7 (HM48-1, 25-0481-80), anti-CD41 PerCP-eFluor 710 (MWReg30, 46-0411-82), anti-CD34 eFluor 660 (RAM34, 50-0341-82, 1:50 dilution), anti-CD135 (FLT3) PerCP-eFluor 710 (A2F10, 46-1351-82), anti-CD115 APC (AFS98, 17-1152-82) and anti-CD45.1 PE-Cyanine7 (A20, 25-0453-82) from eBioscience; anti-CD62E PE (10E9.6, 553751) from BD Biosciences; anti-KIT PE-Cyanine7 (2B8, 105814), anti-CD117 Brilliant Violet 421 (2B8, 105828), anti-CD150 PE (TC15-12F12.2, 115904), F4/80 PE (BM8, 123110) and anti-CD45.2 APC (104, 109814) from BioLegend; and anti-CD3e PerCP-Cyanine5.5 (145-2C11, 65-0031-U100) from Tonbo Biosciences. Streptavidin FITC (11-4317-87) and Streptavidin PerCP-eFluor 710 (46-4317-82) were purchased from eBioscience. Unless otherwise specified, all antibodies, Streptavidin FITC and Streptavidin PerCP-eFluor 710 were used at a 1:100 dilution. Flow cytometry analyses were carried out on the BD LSRII (BD Biosciences) system, and cell sorting experiments were performed using BD FACSAria (BD Biosciences). Dead cells and debris were excluded by forward scatter, side scatter and DAPI staining (1 µg ml−1) profiles. Data were analysed using FACS Diva 6.1 (BD Biosciences) and FlowJo 10 software. Gating strategies are shown in Supplementary Fig. 1.
Cell cycle analysis
Single-cell suspensions were stained for cell surface markers, and subsequently fixed and permeabilized with BD Cytofix/Cytoperm solution (554714, BD Biosciences) according to the manufacturer’s instructions. The cells were then stained with DAPI (Sigma-Aldrich) at 5 μg ml−1 and anti-Ki-67 PerCP eFluor 710 antibody (SolA15, 46-5698-80, eBioscience) or anti-Ki-67 eFluor 660 antibody (SolA15, 50-5698-82, eBioscience) at 1:100 dilution for 30 min at 4 °C. After washing, the cells were analysed on the BD LSRII (BD Biosciences) system. A DAPIlowKi-67low fraction was designated as the G0 phase of the cell cycle.
Blood cell analysis
Peripheral blood was diluted in PBS, and blood parameters were determined with the Advia120 Hematology System (Siemens).
Competitive BM and HSC transplantation
Competitive repopulation assays were performed using the CD45.1/CD45.2 congenic system. CD45.1 recipient mice were lethally irradiated (12 Gy, two split doses at least three hours apart) in a caesium mark 1 irradiator (JL Shepherd & Associates). For BM repopulation assays, 1 × 106 CD45.2 donor-nucleated BM cells were transplanted into irradiated recipients together with 1 × 106 CD45.1 BM cells. For HSC repopulating assays, 200 HSCs (CD45.2) were sorted from BM cells and transplanted into irradiated CD45.1 recipients together with CD45.1 competitor BM cells calculated to contain 200 HSCs (1:1 HSC ratio). For secondary BMT, 3 × 106 BM cells from primary recipient mice were transplanted into newly irradiated (12 Gy) CD45.1 recipients. CD45.1/CD45.2 chimerism of the myeloid (CD11b+), B (B220+) and T (CD3ε+) lineages in recipient blood was analysed up to 5 months after BM or HSC transplantation using a flow cytometer, and that of BM cells was checked at 5 months after BM or HSC transplantation, at which the mice were euthanized.
Ex vivo HSC culture
Ex vivo HSC cultures were performed using F12-PVA-based culture conditions as previously described40. In brief, HSCs were sorted into 96-well flat-bottom plates containing 200 µl HSC medium and expanded at 37 °C with 5% CO2 for up to 28 days. Medium changes were made every 2–3 days. Cells were split at a 1:3 ratio into new plates when reaching 80–90% confluency. After expansion, the cells were used for non-conditioned transplantation.
Non-conditioned HSPC transplantation
HSCs were purified from CD45.2 mice and expanded, as described above. Expanded HSPCs (106 LSK cells per recipient mouse) were then transferred into non-irradiated tamoxifen-administered Cdh2-creER;Kitlfl/fl mice (backcrossed with CD45.2 mice for more than 10 generations) after the transplantation of one or six WT femurs, split into three doses over consecutive days.
Targeted limb irradiation
Animals were anaesthetized by isoflurane before irradiation using the Small Animal Radiation Research Platform, SARRP (XStrahl). The orthovoltage X-ray unit operates at 220 kVp and 13 mA. Before irradiation, a static X-ray scan was acquired using 50 kVp and 0.7 mA tube current with Al filtration. Mice were maintained in a circular lucite jig with whole-body lead shielding (to protect the individualized compartments from unwanted irradiation) and ports through which secured four limbs protruded and were irradiated to 20 Gy in a single fraction.
RNA extraction and RT–qPCR analysis
A total of 2 × 103 MSCs or HSCs were sorted directly into lysis buffer and stored at −80 °C. mRNA was extracted using the Dynabeads mRNA DIRECT Purification Kit (61012, Invitrogen) according to the manufacturer’s protocols. Conventional reverse transcription (RT) with random hexanucleotide primers was then performed using the RNA to cDNA EcoDry Premix (639549, TaKaRa) in accordance with the manufacturer’s instructions. Quantitative PCR (qPCR) was performed in 384-well plates with FastStart Universal SYBR Green Master Mix (04913914001, Roche) on the QuantStudio 6 Flex Real-Time PCR System v.1.7.2 (Applied Biosystems). The PCR protocol started with one cycle at 95 °C (10 min) and continued with 40 cycles at 95 °C (15 s) and 60 °C (1 min). All mRNA abundance was calculated relative to the corresponding amount of Actb (encoding β-actin) using the ΔCt method. A list of the primer sequences is provided in Supplementary Table 1.
ELISA
For analysis of BMEF, the BM of one femur or pelvis was flushed out using 1 ml of PBS, and the cells were subsequently pelleted by centrifugation. The resulting supernatant was transferred to another tube and stored at −80 °C until analysis. For analysis of serum, blood was allowed to clot at room temperature, and serum was separated by centrifugation and stored at −80 °C until analysis. Cytokine levels in BMEF and serum were then measured using mouse IL-1β (BMS6002), IL-6 (KMC0061) ELISA kits (Thermo Fisher Scientific) and TNF (MTA00B-1), CXCL12/SDF-1α (MCX120), SCF (MCK00) and TPO (MTP00) Quantikine ELISA kits (R&D Systems) according to the manufacturer’s protocols.
Statistics and reproducibility
All data are presented as mean ± s.e.m. n represents the number of mice in each experiment, as detailed in the figure legends, and experiments presented were successfully reproduced in at least three biological replicates. No statistical method was used to predetermine sample sizes, and sample sizes were determined by previous experience with similar models of haematopoiesis, as shown in previous experiments performed in our laboratory13,14,16,19,20,47. Sample exclusion was only done as a result of premature mouse death. Statistical significance was determined by an unpaired, two-tailed Student’s t-test to compare two groups or a one-way ANOVA with Tukey’s multiple-comparison tests for multiple group comparisons. Data presentation and statistical analyses were performed using Prism 10 (GraphPad), Excel 16 (Microsoft), SlideBook 6 (Intelligent Imaging Innovations), Photoshop 26 (Adobe) and FlowJo 10 software.
The data in Fig. 2j,k were obtained in the same experiments, and data from the sham-operated mice were reused in each of these figure panels. The data in Extended Data Fig. 7b,c were obtained in the same experiments, and data from the sham-operated mice were reused in each of these figure panels. The data in Extended Data Fig. 8i,j were obtained in the same experiments, and data from the sham-operated Cxcl12fl/fl and Cdh2-creER;Cxcl12fl/fl mice were reused in each of these figure panels. The data in Fig. 3h,i were obtained in the same experiments, and data from the sham-operated Cxcl12fl/fl and Cdh2-creER;Cxcl12fl/fl mice were reused in each of these figure panels. The data in Fig. 4c,d were obtained in the same experiments, and data from the sham-operated Kitlfl/fl and Cdh2-creER;Kitlfl/fl mice were reused in each of these figure panels. The data in Fig. 5c,d were obtained in the same experiments, and data from the sham-operated Tpo+/+, Tpo+/− and Tpo−/− mice were reused in each of these figure panels. The data in Fig. 5h,i were obtained in the same experiments, and data from the sham-operated WT and Tpo-Tg mice were reused in each of these figure panels.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.