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. Author manuscript; available in PMC: 2014 Mar 19.
Published in final edited form as: Nature. 2013 Aug 28;501(7467):10.1038/nature12517. doi: 10.1038/nature12517

Figure 6. Cerebral organoid modeling of microcephaly.

Figure 6

a. MRI scan from patient A3842 taken at birth (top) compared with age-matched control (bottom) showing brain and head size reduction and simplified cortical folding (arrows). Saggital T1 (left) and axial T2 (right) images. Scale bar 1cm. b. Sequencing chromatograms demonstrating compound heterozygous nonsense mutations inherited from each parent. c. CDK5RAP2 protein is undetectable on immunoblotting of patient cell lysate (A3842) compared with control skin fibroblasts. Vinculin (VCL), loading control. d. Representative bright-field images of control and patient-derived cerebral organoids (A3842 line 1M, all lines shown in Extended Data Figure 7d) at 6, 11, 15, and 22 days of differentiation. Control exhibits large fluid-filled cortical regions (arrows), while patient-derived exhibits increased outgrowth (arrowheads). e. Immunohistochemistry in control and patient-derived (10M) tissues at day 30 of differentiation revealing fewer neurons (Doublecortin, DCX, arrows) and smaller progenitor zones (Sox2, arrowheads). f. Staining at day 22 showing increased neurons (Tuj1, arrows) in patient-derived tissue (14B). g. BrdU pulse-chase in control and patient-derived organoids (14B) showing higher percentage of BrdU+ cells with neural identity and less in the VZ compared with control. Results quantified at right. Error bars are S.D. **P<0.01, Student’s t-test. n=3 organoids for each condition (300 cells total for control, 204 cells for patient). h. P-Vimentin staining in control and patient-derived tissues (14B) showing RG mitotic divisions. Control RGs at anaphase divided exclusively horizontal (0-30 degree angle, arrow) whereas patient RGs displayed many oblique and vertical orientations (arrowhead). Results quantified at right (P<0.01, 2×3 Fisher’s exact test, n=11 cells for control, n=15 cells for patient-derived, from >5 cortical regions each). i. hESC organoids co-electroporated with GFP and scrambled or CDK5RAP2 shRNAs and examined after 5 days. Electroporated regions (demarcated by arrowheads) exhibit loss of Sox2+ progenitors and increased Doublecortin (DCX) neurons. Scale bars: 200 μm (d, e, i), 50 μm (f-h).