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. Author manuscript; available in PMC: 2017 Nov 10.
Published in final edited form as: Nat Methods. 2017 May 10;14(7):743–751. doi: 10.1038/nmeth.4304

Figure 6. Migrating cells in cerebral organoid fusions exhibit the migratory dynamics of tangentially migrating interneurons and are sensitive to CXCR4 activity.

Figure 6

(A) A schematic representation of the organoid fusion slice culture assay for either short-term time-lapse imaging of migratory dynamics, or long-term drug-treatments. A representative tile-scan image of an entire ventral::dorsalCycA organoid fusion slice is shown with the ventral GFP+ regions labeled in green and the unlabeled or tdTomato dorsal regions outlined in red. A region containing the migrating cell shown in (B) is noted by a yellow box. (B) Still images from a 3-day time-lapse experiment showing a migrating GFP+ cell. The branched leading process exhibits both extending (closed arrowheads) and retracting (open arrowheads) branches as the cell body follows one of the leading processes. (C) An extending neurite (closed arrowhead) with a tuft that appears to be an axon growth cone travels in one direction across the field of view. (D) A widefield image of GFP+ cells that migrated into the tdTomato+ dorsal region (red outline) from long-term organoid fusion slice cultures that were either untreated (control) or treated with a the CXCR4 inhibitor (AMD3100). (E) Quantification of the migrated GFP+ cell density with data represented as mean±SD with statistical significance tested using the student’s t-test (df=4) comparing control (n=3 organoids) to AMD3100 (n=3) treatment. Slices from the same organoid were split between control and AMD3100-treatments, and 3 different organoids were used from 2 different independent differentiations. Each data point represents cell density counts from one slice. Fewer migrating cells are observed in AMD3100-treated slice cultures. Scale bars are (A) 500µm, (B-C) 50µm, and (D) 500µm. The images in (A-C) are from an organoid fusion created by fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycA iPSC-derived organoid. The images and data in (D-E) are from iPSC-derived organoid fusions.