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.
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).
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.
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.
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.
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.tiff||24304K||Table S1. List of primer sets and universal probes for quantitative real-time PCR|
|stem1555-sup-0010-supptbl2.tiff||24304K||Table S2. Clinical features of human precursor B-ALL samples.|