La Petite presse : journal quotidien... / [rédacteur en chef : Balathier Bragelonne]

04 juillet 18701870/07/04 (A5,N1537)

Visualization 5: Optical clearing based cellular-level 3D visualization of intact lymph node cortex

Z-stack movie of metastasized LN at 10 days after the tumor inoculation Originally published in Biomedical Optics Express on 01 October 2015 (boe-6-10-4154

Intravital longitudinal wide-area imaging of dynamic bone marrow engraftment and multilineage differentiation through nuclear-cytoplasmic labeling

<div><p>Bone marrow is a vital tissue that produces the majority of erythrocytes, thrombocytes, and immune cells. Bone marrow transplantation (BMT) has been widely performed in patients with blood disorders and cancers. However, the cellular-level behaviors of the transplanted bone marrow cells over wide-areas of the host bone marrow after the BMT are not fully understood yet. In this work, we performed a longitudinal wide-area cellular-level observation of the calvarial bone marrow after the BMT <i>in vivo</i>. Using a H2B-GFP/β-actin-DsRed double-transgenic mouse model as a donor, a subcellular-level nuclear-cytoplasmic visualization of the transplanted bone marrow cells was achieved, which enabled a direct <i>in vivo</i> dynamic monitoring of the distribution and proliferation of the transplanted bone marrow cells. The same spots in the wide-area of the calvarial bone marrow were repeatedly identified using fluorescently labeled vasculature as a distinct landmark. It revealed various dynamic cellular-level behaviors of the transplanted BM cells in early stage such as cluster formation, migration, and active proliferation <i>in vivo</i>.</p></div

Dynamic time-lapse nuclear-cytoplasmic visualization of the transplanted BM cell.

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187660#pone.0187660.s004" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187660#pone.0187660.s005" target="_blank">S2</a> Movies). (B, C) Representative time-lapse image sequences showing (B) proliferation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187660#pone.0187660.s006" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187660#pone.0187660.s007" target="_blank">S4</a> Movies) or (C) migration of the transplanted BM cell (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187660#pone.0187660.s008" target="_blank">S5 Movie</a>). Scale bar is 10 μm.</p

Quantitative imaging analysis of transplanted BM cells.

<p>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).</p

Longitudinal wide-area visualization of the cellular-level behavior of the transplanted BM cells.

<p>Mosaic and magnified images longitudinally obtained from the calvarial bone marrow of the recipient mouse <i>in vivo</i>. (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.</p

Nuclear-cytoplasmic visualization of the transplanted BM cell.

<p>(A) Differentiation of the transplanted bone marrow cells into megakaryocytes (spot 1) and cluster formation (spot 2) were longitudinally observed at sequential time-points <i>in vivo</i>. (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.</p