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Osteolineage niche cells initiate hematopoietic stem cell mobilization
Blood Mayack and Wagers
10.1182/blood-2008-01-133710
Supplemental materials for: Mayack and Wagers
Files in this Data Supplement:
- Figure S1. Overlapping expression of ALP and PTHR1 on Opn+ osteoblast cells (JPG, 78.2 KB)
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(A) Flow cytometric analysis of ALP activity, determined using a modified version of the fluorogenic ELF-97 assay (Molecular Probes). Histograms show ALP activity in purified Opn+CD45−Ter119 PTHR1lo cells (black line) and Opn+CD45−Ter119−PTHR1hi cells (grey line, see gating of PTHR1lo and PTHR1hi subsets shown in B), and are compared to ALP activity in a control cell population of total BM cells (grey filled). (B) Flow cytometric analysis of CD45−Ter119− bone-associated cells. FACS plots depict Opn vs. alkaline phosphatase (ALP, left) or Opn vs. PTHR1 (right) for cells in the CD45−Ter119− gate. The numbers in the upper corner of each quadrant indicate the frequency of cells in that quadrant. The majority of OPN+CD45−Ter119− cells are ALP+ and PTHR1+.
- Figure S2. OPN+CD45−Ter119− cells are phenotypically homogenous based on expression of many cell surface markers (JPG, 175 KB)
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OPN+ cells (shown gated on CD45−Ter119−) exhibited largely homogeneous expression of RANKL, CD44, CD51, and VCAM-1, and lacked expression of CD43, Sca-1, CD31, and c-Kit. The frequency of double positive cells (expressing Opn+ and the indicated cell surface marker) is shown in the upper right corner of each plot, and the frequency of OPN+ cells negative for the indicated markers is shown in the bottom right corner. FACS plots are representative of 2 independent experiments.
- Figure S3. G-CSF treatment alone does not promote changes in the osteoblastic niche (JPG, 84.2 KB)
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(A) Osteoblast frequency is increased following Cy/G treatment (D2) but is not increased in bone-assoicated cells of mice treated with G-CSF alone for 2 days (G2). The frequency of Opn+ cells is shown in lower right corner of each plot. FACS plots are representative of 3 independent experiments. (B) ALP activity is altered in Cy/G mobilized osteoblasts. ALP activity was determined using the fluorogenic ELF-97 assay (Molecular Probes), modified for FACS. Representative histograms show the ALP activity in osteoblasts isolated from UNT (light gray filled), D2 (dark gray line), or G2 (black line) mice and as compared to the ALP activity in a control cell population of total BM cells (light gray line) (n=4).
- Figure S4. Cy/G modified osteoblasts promote HSC expansion (JPG, 78.1 KB)
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(A) Frequency of ckit+Thy1.1loLin−Sca1+ (KTLS) HSC among Lin-depleted BM cells (HPC) cultured alone or with UNT or Cy/G-treated (D2) osteoblasts (OSBs) for 12 hrs. Contour plots are representative of 6 independent experiments and indicate c-kit and Sca-1 staining of Lin-Thy1.1lo gated cells. The frequency of KTLS cells is shown on each plot. (B) Osteoblast mediated HSC proliferation (HPC:D2), indicated by CFSE dilution, is not dependent on cell contact. HSC proliferation is maintained in transwell cultures (HPC:D2, panel 2, compare gray filled T = Transwell to black dotted NT = non-transwell); however the decrease in HSC proliferation mediated by UNT osteoblasts is dependent on cell contact, as indicated by the increase in HSC proliferation rates in a transwell assay (HPC:UNT, panel 1). Histograms shown are representative of 3 independent transwell experiments.
- Figure S5. Non-osteoblastic stromal cells (Opn−CD45−TER119−) do not expand HSC in culture (JPG, 45.3 KB)
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Frequency of ckit+Thy1.1loLin−Sca1+ (KTLS) HSC among Lin-depleted BM cells (HPC) cultured alone or with UNT or cytokine-modified (D2) OPN+ osteoblastic cells or OPN− non-osteoblastic stromal cells for 12 hrs. Differences in HSC frequency among HPC exposed to OPN+ D2 cells or to any other stromal population are statistically significant (P<0.05). No significant increase in HSC frequency was detected in cultures containing Opn− stromal cells harvested from untreated (UNT) or Cy/G-mobilized (D2) mice, as compared to HPC cultured alone. Data are combined from 2 independent experiments.
- Figure 6. Primer sequences used for either standard quantitative RT-PCR or single cell RT-PCR (JPG, 58.6 KB)
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Primer sequences used for either standard RT-PCR or single cell real time PCR analysis, as shown in table for Alkaline Phosphatase (ALP), Osteoactivin (OA), Runx2 (RX2), Osterix (OX), Osteocalcin (OC). All PCR amplification was performed in a 10 µl final volume containing 1 × SYBR Green PCR buffer, 2 mM MgCl2, 0.5mM dNTPs, 10ng of each primer as listed in the table, 0.25 U AmpliTaq Gold, and 1 µl of cDNA templates.  -actin gene expression was used to normalize the amount of each investigated transcript.
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