SEARCH

SEARCH BY CITATION

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
stem1555-sup-0001-suppfig1.tiff26330K

Figure S1. Characteristics of CD45- and CD45+ cells isolated from WT BMMSCs. Primary cultures of WT BMMSCs were passaged to disperse the colony-forming cells (passage 1). CD45- and CD45+ cells were isolated from these samples by negative or positive selection using anti-CD45 immunomagnetic microbeads. CD45- WT BMMSCs had stromal characteristics, whereas CD45+ WT BMMSCs did not. (A) Multi-differentiation analysis of CD45- and CD45+ cells isolated from WT BMMSCs. Morphology of separated CD45- (left) and CD45+ (right) WT BMMSCs prior to culture in differentiation-inducing conditions (upper panels). Alizarin Red S staining of CD45- and CD45+ WT BMMSCs cultured in osteogenesis-inducing conditions in vitro (middle panels). Oil Red O staining of CD45- and CD45+ WT BMMSCs cultured in adipogenesis-inducing conditions in vitro (lower panels). Representative images are shown. Original magnification, 40×. Bars, 50 μm. (B) Expression of CXCL12/SDF-1 mRNA in CD45- (n = 5) and CD45+ (n = 5) WT BMMSCs after 3 days of culture. (C) Concentration of CXCL12/SDF-1 protein in the culture supernatants of the same number (3×105 cells in 1.5mL per dish) of CD45- (n = 3) and CD45+ (n = 5) WT BMMSCs after 3 days of culture, as determined by ELISA. (D) Expression of IL-7 and SCF mRNA in CD45- (n = 5) and CD45+ (n = 5) WT BMMSCs after 3 days of culture. *, P < 0.05.

stem1555-sup-0002-suppfig2.tiff26330K

Figure S2. The B-cell population is not reduced in the spleen of C/EBPβ-KO mice. (A) Percentage of B220+ B-cells in the spleen (SP) of WT mice (n = 14) and C/EBPβ-KO mice (KO, n = 13), as determined by flow cytometric analysis. (B, C) Detailed flow cytometric analysis indicating the percentage of immature B-cells (Fraction E; B220+CD43-sIgM+sIgDlow) and mature B-cells (Fraction F; B220+CD43+sIgM+sIgDhigh) in the spleen of WT and C/EBPβ-KO mice. Representative counter-plots are shown (C).

stem1555-sup-0003-suppfig3.tiff26330K

Figure S3. The B220+CD43- B-cell population is not reduced in the bone marrow of C/EBPβ-KO mice. (A, B) Detailed flow cytometric analysis indicating the percentages of pre-BII cells (Fraction D; B220+CD43-sIgM-sIgD-), immature B-cells (Fraction E; B220+CD43-sIgM+sIgDlow), and mature B-cells (Fraction F; B220+CD43-sIgM+sIgDhigh) in the bone marrow (BM) of WT and C/EBPβ-KO mice. (B) Representative counter-plots are shown. Numbers in each box indicate the percentage of cells.

stem1555-sup-0004-suppfig4.tiff26330K

Figure S4. C/EBPβ-deficient and WT bone marrow hematopoietic cells generate an equivalent level of B-cells when transplanted into lethally irradiated WT mice. (A, B) Lethally irradiated WT mice received hematopoietic cells derived from WT mice (WT[RIGHTWARDS ARROW]WT, n = 5) or C/EBPβ-KO mice (KO[RIGHTWARDS ARROW]WT, n = 3). Levels of B-cells in the spleens of recipients were analyzed 14 weeks after transplantation. (A) Percentage of B220+ B-cells in the recipient spleen (SP) of the WT[RIGHTWARDS ARROW]WT (closed bar) and the KO[RIGHTWARDS ARROW]WT (open bar) groups, as determined by flow cytometric analysis. (B) Detailed flow cytometric analysis indicating the percentage of immature B-cells (Fraction E; B220+CD43-sIgM+sIgDlow) and mature B-cells (Fraction F; B220+CD43-sIgM+sIgDhigh) in the recipient spleen (SP) of the WT[RIGHTWARDS ARROW]WT (closed bar) and the KO[RIGHTWARDS ARROW]WT (open bar) groups.

stem1555-sup-0005-suppfig5.tiff26330K

Figure S5. Bone marrow transplantation experiments using C/EBPβ-KO mice as recipients. In reverse transplantation experiments, bone marrow cells (1×106 cells/mouse) from WT mice (Ly5.1) were administrated intravenously through the tail vein into C/EBPβ-KO mice (Ly5.2) that received total body irradiation at a dose of 10 Gy or lower prior to transplantation. B-cell lymphopoiesis was evaluated by flow cytometric analysis at 12 and 18 weeks after transplantation. (A) Schema of the experiments. BM hematopoietic cells (1×106 cells) from WT mice (Ly5.1) were transplanted into C/EBPβ-KO mice (Ly5.2) (WT[RIGHTWARDS ARROW]KO). As a control, WT mice (Ly5.2) were used as recipients (WT[RIGHTWARDS ARROW]WT). The recipient mice were irradiated at doses of 10, 7 or 5 Gy prior to transplantation. (B, C) Engraftment of donor cells in surviving recipient mice that received 5 Gy or 7 Gy irradiation, because all C/EBPβ-KO recipients died early after transplantation when a dose of 10 Gy was used. (B) The percentage of Ly5.1+ donor cells in peripheral blood (PB) of WT[RIGHTWARDS ARROW]KO and WT[RIGHTWARDS ARROW]WT mice 12 weeks after transplantation. (C) The percentage of Ly5.1+ donor cells in bone marrow (BM) of WT[RIGHTWARDS ARROW]KO and WT[RIGHTWARDS ARROW]WT mice 18 weeks after transplantation. (D, E) B-cell reconstitution by donor cells in surviving recipient mice that received 7 Gy irradiation. The percentage of Ly5.1+ PB cells (D) and Ly5.1+ BM cells (E) that were B220+ at 12 and 18 weeks after transplantation, respectively, in WT[RIGHTWARDS ARROW]KO and WT[RIGHTWARDS ARROW]WT mice.

stem1555-sup-0006-suppfig6.tiff26330K

Figure S6. Frequencies of B-cell subsets generated from KSL cells following addition of exogenous CXCL12/SDF-1 to the co-cultures, and the expression of B-cell lymphopoiesis-associated molecules in C/EBPβ-deficient BMMSCs. (A, B) Co-cultures of WT (Ly5.1)-KSL cells with WT (Ly5.2) or C/EBPβ-deficient (Ly5.2) BMMSCs (Fig. 3A), in the presence or absence of CXCL12/SDF-1. (A) Frequency of B220+ cells differentiated from WT-KSL cells following addition of CXCL12/SDF-1. (B) Frequencies of CD24-BP-1- (Fraction A), CD24+BP-1- (Fraction B) and CD24+BP-1+ (Fraction C/C') subsets in the B220+CD43+ precursor cell population that differentiated from WT-KSL cells following addition of CXCL12/SDF-1. Three experiments were performed in each condition (A, B). (C‒E) Expression of Flt3-L, IL-7, and SCF mRNA in WT and C/EBPβ-deficient BMMSCs. Quantitative real-time PCR analysis examining the expression of Flt3-L (C), IL-7 (D), and SCF (E) mRNA in WT BMMSCs (WT, n = 6) and C/EBPβ-deficient BMMSCs (KO, n = 6). *, P < 0.05; **, P <0.01.

stem1555-sup-0007-suppfig7.tiff26330K

Figure S7. Skeletal development is impaired in C/EBPβ-KO mice. (A, B) Crown-rump length (A) and total length (B) of WT and C/EBPβ-KO (KO) mice. Left graphs, males (9–10-weeks-old: WT, n = 15; KO, n = 15); right graphs, females (9–10- weeks-old: WT, n = 10, KO, n = 10). (C, D) Trabecular bone (TB) formation in WT and C/EBPβ-KO mice. (C) Histological analysis of the vertebrae of WT and C/EBPβ-KO mice (males, 9-weeks-old; WT, n = 5, KO, n = 5). Representative images of the vertebrae as assessed by HE staining; Original magnification, 100×. Bars, 50 μm (D). Quantification of TB area in WT and C/EBPβ-KO mice. (E, F) Immunohistochemical staining of the vertebrae of WT and C/EBPβ-KO mice for osteocalcin (OSC). For immunostaining with OSC, paraformaldehyde-fixed paraffin-embedded sections were first incubated with proteinase K (Dako) for antigen retrieval and then incubated with 3% hydrogen peroxidase to block endogenous peroxidase activity. Sections were then incubated with a primary antibody against OSC (TAKARA-BIO INC) or a negative control antibody. Signals were visualized using the horseradish peroxidase/3, 3-diaminobenzidine detection system. (E) Representative images. Yellow arrows indicate OSC+ cells. Original magnification 200×. Bars, 50 μm. (F) Quantitative measurement of OSC+ cells in the immunostained vertebrae sections of WT and C/EBPβ-KO mice (males, 9-weeks-old; WT, n = 3, KO, n = 3). OSC+ cells on the surface of TB were counted and the number of OSC+ cells per 100 μm of TB was calculated using ImageJ software. *, P < 0.05; **, P <0.01.

stem1555-sup-0008-suppfig8.tiff26330K

Figure S8. mRNA levels of CXCL12/SDF-1 and C/EBPβ in BMMSCs derived from human precursor B-ALL bone marrow samples. Quantitative real-time PCR analysis examining the mRNA expression of CXCL12/SDF-1 (A) and C/EBPβ (B) in normal human BMMSCs (n = 3) and BMMSCs derived from human precursor B-ALL bone marrow samples (#1‒#4). For normal human BMMSCs, mean ± SD is shown.

stem1555-sup-0009-supptbl1.tiff24304KTable S1. List of primer sets and universal probes for quantitative real-time PCR
stem1555-sup-0010-supptbl2.tiff24304KTable S2. Clinical features of human precursor B-ALL samples.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.