Id1^−/− mice show decreased BM cellularity with fewer myeloid, erythroid, and lymphoid cells but increased neutrophils and monocytes in the PB. (A) PBCs obtained from Id1^−/− (n = 5) and Id1^+/+ (n = 5) mice were analyzed with lineage-specific monoclonal antibodies. The horizontal bars indicate the mean percentage (*P < .01). Data were obtained from 2 independent experiments. (B) Total BM (Id1^−/−, n = 30; Id1^+/+, n = 32) and spleen (Id1^−/−, n = 9; Id1^+/+, n = 8) cells were enumerated by the use of a hemacytometer (*P < .01). (C) Left panel shows the flow cytometric analysis for granulocytes/monocytes (Gr-1 × Mac-1) and erythroid cells (TER119 × CD71) in Id1^−/− and Id1^+/+ BMC. The numbers in each quadrant indicate the percentage of each cell type among the total analyzed cells. Right panel shows percentages of Gr-1⁺/Mac-1⁺ or TER119⁺ BMC from all Id1^−/− (n = 9) and Id1^+/+ (n = 6) mice analyzed (*P = .01). (D) The absolute number of Gr-1⁺, B220⁺, and TER119⁺ BMC was calculated by multiplying the fraction of positive cells to total BMC (*P < .01). Data were obtained from 3 independent experiments, and the horizontal bars indicate the mean cell counts.

Increased cell cycling of myeloid progenitors in Id1^−/− mice. (A) Mice received an intraperitoneal injection of 3 mg BrdU and then 1 mg/mL BrdU in the drinking water for 2 days. BMC were isolated from treated mice, depleted of lineage-positive cells, and then stained with IL7Rα, c-Kit, and Sca-1 antibodies to detect LSK and LS⁻K progenitors. BrdU incorporation was measured by use of the APC BrdU flow cytometry kit (Becton Dickinson). Data are presented as the mean ± SEM of 3 mice for each genotype (*P = .002). (B) Cell proliferation was measured by sorting the indicated number of LS⁻K or LSK cells from Id1^−/− and Id1^+/+ mice into 96-well plates (24 replicates per cell concentration) with cIMDM media containing cytokines (mouse SCF, 100 ng/mL; human IL-6, 50 ng/mL; human Flt3, 100 ng/mL; and human Tpo, 100 ng/mL for LSK and mouse SCF,100 ng/mL; mouse IL-3, 30 ng/mL; and mouse GM-CSF, 100 ng/mL for LS⁻K). Cell proliferation, including colonies (> 50 cells) and clusters (> 10 and < 50 cells), was determined after 10 days of culture, and then the log of the percentage of negative wells was plotted against the number of cells per well. The frequency of a positive colony or cluster is defined as the inverse of the number of seeded cells that corresponds to 37% negative cells.

Role of Id1 in the formation of the BM microenvironment. (A) The homing efficiency of Id1^−/− and Id1^+/+ BMC was assayed by the labeling of cells with CFSE. Staining was performed by incubating cells for 10 minutes in 37°C with 1 μmol/L concentration of CSFE. Then, 1.5 × 10⁷ cells were injected into irradiated recipient mice. At 24 hours, recipient BM and spleen cells were harvested, and CSFE-positive cells were analyzed by flow cytometry (n = 2 for each genotype, P = .56). Lineage-negative (Lin⁻) hematopoietic cell homing was determined by transplanting 8.5 × 10⁶ Lin⁻ cells per mouse (n = 3 for each genotype, P = .46). (B) LTC-IC assays of mouse hematopoietic cells using primary BM stromal cells and limiting dilution analysis was used to compare the supportive function of Id1^−/− (○—○) and Id1^+/+ (●—●) BM-derived stromal cells. Wells containing clonogenic cell colonies were scored after 10 days of culture of Id1^+/+ Lin⁻Sca-1⁺ BM progenitors with established stromal cells from Id1^−/− or Id1^+/+ and plotted as the number of wells that showed no growth for each cell dilution. The data are presented as representative data from 2 separate experiments. (C) Serum chemokine and cytokine concentrations (expressed as picograms per milliliter except SDF-1α, which is nanograms per milliliter) were measured and compared between 2 groups of age- and sex-matched mice (n = 21 for Id1^−/−, n = 18 for Id1^+/+, *P < .05). (D) Total RNA was isolated from cultured bone marrow–derived stromal cells and real-time PCR was used to measure mRNA. Relative mRNA units were normalized to glyceraldehydes-3-phosphate dehydrogenase levels. Data shown are the mean ± SEM in triplicate (*P < .05).

HSCs are maintained in Id1^−/− mice. (A) The percentages and absolute numbers of LSK, CD34loLSK, CMP, MEP, and GMP were determined by flow cytometry of BMC obtained from 12- to 14-week-old Id1^−/− (n = 5) and Id1^+/+ (n = 6) mice in 3 separate experiments (*P < .05). The percentage of each population among total Lin⁻IL-7Rα⁻ BMC (top panel), and the absolute counts to total Lin⁻IL-7Rα⁻ BMC of each group of mice (bottom panel) is shown. (B) Myeloid colony assays were performed on BM, spleen, and PB cells from Id1^−/− and Id1^+/+ mice. BMCs were cultured in methylcellulose semisolid media with cytokines (mouse GM-CSF, 100 ng/mL; mouse IL-3, 30 ng/mL; mouse SCF, 100 ng/mL). The results depict data from triplicate plates (*P ≤ .001). (C) Mice were irradiated (n = 10 per group) and then serially transplanted with 2 × 10⁶ Id1^−/− or Id1^+/+ BMC. The percentage of donor BMC were determined in recipient mice 70 days after transplantation by the use of antibodies specific for donor and host cells (Ly5.1 and Ly5.2). Survival data of recipient mice were plotted as Kaplan-Meier curves and analyzed by log rank test (P = .13). (D) The percentage of donor-derived BMC versus the serial transplant number was plotted (P = .30). Data are representative of 1 of 2 independent experiments. (E) BMC (10⁶) from Id1^−/− or Id1^+/+ mice were mixed with BM competitor cells (10⁶) and were transplanted into irradiated recipient mice. Donor cell chimerism in recipient BMs was evaluated 4 months after transplantation. Left panel graph shows competitive repopulation unit (CRU) of 10⁶ BMC of each group (*P = .03), whereas right panel graph shows CRU data that was normalized by use of the mean total BMC for each group (P = .15). The data are presented as the mean ± SEM (n = 6).

Id1 is required for proper hematopoietic microenvironment function. (A) BMCs were harvested 4 months after Id1^−/− or Id1^+/+ BMC were transplanted into irradiated Id1^+/+ recipient mice and then analyzed for donor hematopoietic reconstitution by flow cytometry. Flow cytometric data are representative data from individual mice. The left panel shows donor myeloid (Gr-1 × Mac-1) and erythroid (TER119 × CD71) reconstitution in Id1^−/− and Id1^+/+ recipient mice. The numbers indicate the percentage of donor cells in each quadrant. The right panel shows percentages of Gr-1⁺/Mac-1⁺ or TER119⁺ BMC in all the mice analyzed (each genotype, n = 3). (B) Repopulation of Id1^+/+ BMC in irradiated Id1^−/− or Id1^+/+ recipient. Total BMC of recipient mice were enumerated 4 months after transplantation (n = 6, *P = .04). (C) BMC were harvested 4 months after Id1^+/+ BMC were transplanted into irradiated Id1^+/+ and Id1^−/− recipient mice and then analyzed for donor hematopoietic reconstitution by flow cytometry. Left panel shows myeloid and erythroid repopulation of representative mice, whereas the right panel shows percentages of Gr-1⁺/Mac-1⁺ myeloid or TER119⁺ erythroid BMC in all the mice analyzed (each genotype, n = 6, *P < .001).

Summary of Id1^−/− hematopoietic phenotypes. Id1^−/− mice show decreased BM cellularity, which results in decreased absolute numbers of B cells and erythroid cells, while myeloid cell development is enhanced. Id1^−/− LSK and LS⁻K progenitor cell cycling is increased in vivo but not in vitro. Cytokine and chemokine production is dysregulated in stroma cultures in vitro and in vivo, suggesting that Id1 is required for proper microenvironment function. As a result of dysregulated growth factor production, Id1^−/− mice show enhanced myeloid proliferation and differentiation, increased egress of myeloid cells from the BM, and increased neutrophil and monocyte numbers in the PB.