Fig 1.
Longitudinal wide-area visualization of the cellular-level behavior of the transplanted BM cells.
Mosaic and magnified images longitudinally obtained from the calvarial bone marrow of the recipient mouse in vivo. (A) (upper panel) Mosaic images showing the transplanted BM cells (red, actin-DsRed in the cytoplasm and nucleus; green, H2B-GFP in the nucleus) and vascular sinus (blue). (lower panel) Red-color separated mosaic images highlighting the distribution of the transplanted BM cells on each day. (B) Magnified images of (A) at the region marked with dotted squares. The large BM cell clusters with a size larger than 5,000 μm2 were marked with an arrow. The small BM cell clusters with a size ranging from 500–5,000 μm2 were marked with an arrowhead. The asterisk indicates the BM cell cluster with a significantly increased size at day 4. Scale bars are (A) 1 mm, and (B) 100 μm, respectively.
Fig 2.
Quantitative imaging analysis of transplanted BM cells.
Quantitative imaging analysis was performed by using the large mosaic images of calvarial BM of three BMT mouse models. (A) The ratio of the total area occupied by actin-DsRed expressing cells in the calvalrial BM. (n = 3). (B) The ratio of the total cell area and the fractional ratio occupied by three groups comprising single BM cells or their clusters categorized by their size: < 500 μm2; 500–5,000 μm2; > 5,000 μm2. (C) The number of cell clusters in each group. (n = 3).
Fig 3.
Nuclear-cytoplasmic visualization of the transplanted BM cell.
(A) Differentiation of the transplanted bone marrow cells into megakaryocytes (spot 1) and cluster formation (spot 2) were longitudinally observed at sequential time-points in vivo. (B) Various shapes of nucleus visualized by nuclear-cytoplasmic dual labeling. (C, D) Fractional ratio of nuclear morphology in (C) the normal BM cells isolated from the BM of H2B-GFP/β-actin-DsRed double-transgenic mouse, (D) the transplanted BM cells expressing H2B-GFP/β-actin-DsRed isolated from the BM of the BMT recipient wildtype mouse (n≥3). Scale bars are (A) 50 μm, and (B) 10 μm, respectively.
Fig 4.
Dynamic time-lapse nuclear-cytoplasmic visualization of the transplanted BM cell.
Multi-position time-lapse imaging of the calvarial bone marrow at 6 minute intervals was performed 3 days after the BMT. (A) Proliferation events observed during the 5 hour time-lapse imaging (S1 and S2 Movies). (B, C) Representative time-lapse image sequences showing (B) proliferation (S3 and S4 Movies) or (C) migration of the transplanted BM cell (S5 Movie). Scale bar is 10 μm.