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Blood, Vol. 114, Issue 2, 459-468, July 9, 2009
In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta
Blood Panaroni et al.
114: 459
Supplemental materials for: Panaroni et al
Files in this Data Supplement:
- Table S1. Comparison of donor cell engraftment in long bones and bone marrow of same mice determined by real time PCR and FACS (PDF, 18.6 KB)
- Table S2. Composition of type I collagen isolated from trabecular and cortical bones of 2 mo old recipient mice (PDF, 56.5 KB)
- Table S3. FACS analysis of Lin−/Sca1+/CD117−/CD45− cells from 2 month old treated and untreated mice (PDF, 79.4 KB)
- Figure S1. Differentiation potential of bone marrow cells from eGFP transgenic mice towards osteoblasts (A, B, C) and adipocytes (D, E, F) (JPG, 236 KB)
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Whole bone marrow from donor mice was isolated as described in methods. For osteoblasts differentiation, the cells were cultured in presence of 10−8 M dexamethasone, 10 mM 2-β-glycerophosphate and 0.2 mM ascorbic acid and the deposited minerals were stained after 20 days by Von Kossa. For adipocytes differentiation, the cells were cultured in presence of 10−8 M dexamethasone, 0.5 mM IBMX, 125 µm indomethacin and 10 µg/ml insulin and oil drops were stained by Oil Red O. Bright-field (A,D) and eGFP fluorescence (B,E) images before staining; bright-field images (C,F) after staining. Images were recorded by DMIL-Leica microscope with FC480-Leica camera and 10×/0.22 NA objective.
- Figure S2. Donor eGFP+ Colony Forming Unit-Fibroblasts (CFU-F) from 2-month-old recipient mice (JPG, 63.1 KB)
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Bone marrow stromal cells isolated from 2 mo old recipient mice were isolated, as described in methods, and cultured for 8 days. CFU-F were stained by Giemsa and observed by bright-field (A) and fluorescent microscope (B) using a DMIL-Leica microscope,with DFC480-Leica camera and 10×/0.22 NA objective.
- Figure S3. FACS analysis of Lin−/CD117−/Sca1+ bone marrow progenitor cells from in utero transplanted mice (JPG, 75.1 KB)
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Representative plots of (A) the analysis performed for the isolation of Lineage negative (Lin−) cells, (B) selection of CD117−/Sca1+ cell population among Lin− cells and (C) determination of eGFP+ cells among a single Lin−/CD117−/Sca1+ population. PerCP-A: Peridinin Chlorophyllin-a Protein, CD117-PE-A: monoclonal phycoerythrin conjugated anti CD117, Sca1-APC-A: monoclonal allophycocyanin conjugated anti Sca1, FSC: forward scatter, correlated with the cell volume.
- Figure S4. Representative FACS evaluation of peripheral blood chimerism in transplanted mice (JPG, 237 KB)
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(A) Selection of myeloid cells (CD11b) and granulocytes (Ly 6G/Gr-1) and identification of eGFP+ cells in these populations (B and C respectively); (D) selection of B lymphocytes (B220) and identification of eGFP+ cells among them (E); (F) selection of erythroid cells (Ter119) and identification of eGFP+ cells (G); (H) selection of T lymphocytes (CD3) and identification of eGFP+ cells (I).
- Figure S5. Mineralization variability of cortical bone matrix near host and donor cells measured by confocal Raman microspectroscopy (A,B) and visible images used to select sampling points (C–F) (JPG, 565 KB)
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(A) A representative profile of the ratio of mineral phosphate (ν1PO43−) to organic CH near two donor (eGFP+) cells and one host (Brtl) cell. This profile was measured along the red line shown in the bright field (D), eGFP fluorescence (E), and dark-field polarized (F) images of the same 15 µm bone section. (B) The PO43−/CH ratios measured at selected points within lamellar and fine-fibred primary bone near 62 osteocytes in 10 bone sections containing eGFP+ cells. The bone types were distinguished via the polarized images (F). The distinction is also illustrated in panel C for a 4 µm section producing a higher contrast polarized image. The images of solvated samples at room temperature were captured via the Raman microscope, Nikon D70 camera and UPlanSApo 40×/0.95 NA (A,B,D,E) and SLCPlanFl 40×/0.55 NA (C,F) objectives.
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