Fig 1.
Engineered human marrow VME in vitro.
A. Model schematic of marrow cells surrounding the vessel network in a collagen matrix, shown in cross section. B. Schematic of close-up interactions of marrow cells with vessels. C-D. Overlay of bright field and immunofluorescence images of a vessel surrounded by marrow cells after two weeks of culture. Green: CD31, blue: nuclei. Arrowheads in D: openings on the endothelium. E-H. Scanning electron microscopy shows ultrastructure of the VME. E. Ultrastructure of the abluminal and luminal endothelium in relation to the collagen matrix and surrounding cells. F. Close-up view of the vessel lumen shows pores on the endothelium. G. Pro-platelet-like territories and H. pro-platelet structures on the luminal endothelium during culture.
Fig 2.
Marrow VME for the study of thrombopoiesis in vitro.
A. Z-stack projection of confocal fluorescence imaging of megakaryocytes co-cultured within a 3D microvascular system. Green: CD41, red: CD31, blue: nuclei. B. Enlarged view, z-projection of confocal fluorescence images (left panel) and orthogonal views (right two panels) of locations at dotted lines 1 and 2, showing megakaryocytes interacting with the vessel wall (stars) and in the lumen and on the abluminal vessel wall (arrowheads). Green: CD41, red: CD31, blue: nuclei. C. (i) Zoomed view of megakaryocyte indicated in A (arrowhead) showing CD41a+ (green) and nucleus staining (blue) (ii) 3D reconstruction of the nucleus lobes from the megakaryocyte in i. D. A TEM image showing a megakaryocyte with four nucleus lobes close to a vessel. E. Megakaryocyte lobe counts near and far from the vessel wall shows more mature megakaryocytes are located closer to the vessel wall.
Fig 3.
CXCR4 mediates MK migration and penetration through the endothelium.
A. Z-stack projection of confocal fluorescence image of co-cultured thrombopoietic VME in control media (i) and media supplemented with anti-CXCR4 (ii) after three days. Green: CD41, red: CD31, and blue: nuclei. B. Cell density of megakaryocytes and HS5 stromal cells in collagen with respect to distance to microvessel walls after three days of culture. C. Permeability coefficient (mean ± S.D., n = 3) of megakaryocytes co-cultured microvessels in control and anti-CXCR4 treated conditions. D. SEM of microvessel lumen showing holes in the endothelium of MK vessels in control (i), but not in anti-CXCR4 supplemented conditions (ii).
Fig 4.
Megakaryocytes transmigrate through the endothelium and release platelet-like particles.
Megakaryocytes are present in three locations relative to the microvessel wall during the process of transmigration: A. abluminal, B. transmigrating and C. luminal. Column i.: Schematics of the relative position of MKs and microvessel. Column ii.: z-stack projections and cross-sectional views of MK transmigration acquired through confocal fluorescence imaging. Red: CD31, green: CD41, blue: nuclei. Columns iii-iv.: SEM imaging of MKs interacting with the luminal endothelium at different stages, and pro-platelet fragments shedding platelet-like particles along the direction of flow (yellow arrowheads).
Fig 5.
Characterization of generated platelet-like particles.
A. Flow cytometry of whole blood, washed platelets, and collected particles from MK vessels at day five of culture showing granularity (SSC-A) and size (FSC-A). Gating was based on the platelet population of the whole blood sample. B. CD41a and CD42b expression of gated population from whole blood, washed platelets, and collected particles at day five. C-D. Immunofluorescence images of un-activated and thrombin-activated particles stained for CD41a and β-tubulin.