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, 14 (5), 515-522

Human Brain Organoids on a Chip Reveal the Physics of Folding

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Human Brain Organoids on a Chip Reveal the Physics of Folding

Eyal Karzbrun et al. Nat Phys.

Abstract

Human brain wrinkling has been implicated in neurodevelopmental disorders and yet its origins remain unknown. Polymer gel models suggest that wrinkling emerges spontaneously due to compression forces arising during differential swelling, but these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during the in vitro development and self-organization of human brain organoids in a micro-fabricated compartment that supports in situ imaging over a timescale of weeks. We observe the emergence of convolutions at a critical cell density and maximal nuclear strain, which are indicative of a mechanical instability. We identify two opposing forces contributing to differential growth: cytoskeletal contraction at the organoid core and cell-cycle-dependent nuclear expansion at the organoid perimeter. The wrinkling wavelength exhibits linear scaling with tissue thickness, consistent with balanced bending and stretching energies. Lissencephalic (smooth brain) organoids display reduced convolutions, modified scaling and a reduced elastic modulus. Although the mechanism here does not include the neuronal migration seen in in vivo, it models the physics of the folding brain remarkably well. Our on-chip approach offers a means for studying the emergent properties of organoid development, with implications for the embryonic human brain.

Figures

Fig. 1
Fig. 1. Brain Organoid development and wrinkling
(a) Illustration of the two-dimensional compartment, h = 150μm. The top membrane is coupled to a media reservoir, and the bottom coverslip enables in situ imaging. (b) Z-stack image of the organoid showing actin using Lifeact-GFP (green) and cell nuclei using H2B-mCherry (red). (c) Illustration of an organoid optical section. (d) Fluorescence image and illustration showing cell organization in the organoid. Dashed white line marks inner organoid surface surrounding a lumen. Cells exhibit a bi-polar morphology, stretched between the outer surface (r = t) and the inner surface (r = 0). Nuclei are distributed along the radial coordinate, and cell division occurs at the inner surface (r = 0). (e) Fluorescence images showing the development of the organoid during days 3-11 after Matrigel embedment, and the emergence of wrinkles. Arrows indicate initial wrinkling instability. (f) RNA sequencing expression data in the organoids for three developmental time points: embryonic stem cell culture, organoids 5 and 12 days after Matrigel embedment. Six samples are shown for each time point (3 wild-type, 3 LIS1 +/− mutants). Color bar indicates the row z-score. The list contains genes that are typical for telencephalon and cortical brain regions. Scale bars are 100μm (e), 50μm (b), 20μm (d). Error bars represent s.e.m.
Fig. 2
Fig. 2. Organoid wrinkling occurs at a critical nuclear density and maximal strain
(a) A low magnification image of the wrinkled organoid. Dotted line marks the organoid contour of length LG. Solid line marks a convex contour of length LF. (b) Fluorescence images of H2B-mCherry showing nuclear aspect ratio and density in days 3,6. (c) Wrinkling index W = LG/LF as a function of time averaged over 14 organoids. (d) Nuclear density ρ and (e) aspect ratio R1/R2 as a function of time averaged over 250 nuclei sampled from five organoids, with 50 nuclei each. (f) Wrinkling index as a function of nuclear density ρ (Nuclei per area). Nuclear density was measured at the tissue outer region, r/t > 0.5, and normalized by the average nuclear area < a >= 89 ± 14μm2. A sharp wrinkling transition is observed at a critical density ρc = 0.85 ± 0.1 < a >. (g) Illustration showing organoid wrinkling at a critical density ρc. (h) Wrinkling wavelength λ as a function of thickness t exhibiting a linear scaling regime. Scale bars are 100μm (a), 10μm (b). Error bars represent s.e.m.
Fig. 3
Fig. 3. Nuclear motion and swelling during cell cycle lead to differential growth
(a) Time-lapse fluorescence images of nuclear motion in the organoid during cell-cycle. H2B-mCherry (red) and Lifeact-GFP (green). (b) High-resolution images during cell mitosis at the inner surface. (c) nuclear velocities ν and (d) nuclear density ρ as a function of radial position r/t. Velocities were measured for both inward (red) and outward (blue) motions. (e) Two-dimensional phase-space diagram of nuclear area A and radial-position r/t. Red color intensity indicates the percentage of nuclei at each point. The nuclei distribution is limited to closed path, reflecting the periodic cell-cycle: (1 → 2) Nuclear swelling: two-fold increase in nuclear area, coupled with an inward nuclei motion, (2 → 3) DNA condensation: motion towards the mitosis region at the inner surface, and decrease in area during DNA condensation prior to cell division. (3 → 1) Upward motion of “newly-born” nuclei. Data was taken from analysis of 13 nuclei over 48 hours with a time step of 3 minutes. Black line and arrows were drawn to indicate progression with time. (f) Nuclear area growth, during a cell cycle period, in the outer half of the organoid (r/t > 0.5) compared to the inner half of the organoid (r/t < 0.5). (g) Illustration of a physical mechanism for wrinkling. Nuclear motion and position dependent nuclear swelling create density and growth gradients, which are maximal at the organoid outer regions. The differential growth leads to residual stress and wrinkling. Scale bars are 20μm (b) and 10μm (c). Error bars represent s.e.m.
Fig. 4
Fig. 4. Cytoskeletal forces maintain organoid core contraction and stiffness
(a) Images of organoids treated with blebbistatin (+Blebbistatin) during days 6-10 and control. Dashed lines mark inner and outer surfaces. (b) Average curvature < |∂rθ(r)| > of outer (basal, blue) and inner (apical, black) organoid surfaces for control (CTRL) and treated (Bleb.) organoids (N=7-10. (c-d) Images of organoids before and after acute treatment with cytoskeleton disturbing drugs including (c,c’) blebbistatin and (d,d’) laser micro-dissection treatment of the organoid core (marked with green line and blue dots). (e) Organoid thickness before (t0) and after (t) cytoskeleton disturbing treatments including blebbistatin, ROCK inhibitor, nocodazole and laser microdissection. (f,f’) Magnified view of (c,c’) reveals that the inner surface area of cells is increased following drug treatment. (g) Illustration of the organoid during cytoskeleton inhibition, showing the reduction in thickness and increase in inner surface area. (h) Lifeact fluorescence profile along the radial coordinate r/t before (solid line) and after (dashed line) treatment with ROCK inhibitor. Blue area marks the difference in fluorescence intensity at the inner surface (0 < r/t < 0.1). (i) Reduction in inner surface fluorescence and (j) thickness for drug and microdissection treatments. Averages (squares) taken over 4-11 repeats (circles) for each condition. (k) Nuclear aspect ratio in control organoids (Ctrl) and after treatments. Data includes 40-70 nuclei from 3 organoids. (l,l’) H2B-mCherry fluorescence images before and after treatment. Dashed lines indicate inner and outer surfaces. Scale bars are 100μm (a,d), 50μm (c), 20μm (l) and 10μm (f). Error bars represent s.e.m.
Fig. 5
Fig. 5. LIS1+/- mutation results in lissencephalic organoids, modified ECM and cytoskeleton, and reduced cell elasticity
(a) Images of wild-type (WT) and (b) LIS1 mutant organoids (LIS1+/-). (c) Expression data of extra cellular matrix (ECM) and cytoskeleton related genes (N=3). (d) Distribution of organoid thickness and (e) wrinkling wavelength for WT and (f, g) LIS1+/- (N=5). (h) Wrinkle wavelength as a function of thickness. (i) Tangent correlation function, < Cos(θ(Δr)) >, between two points at a distance r along the organoid surface (N=9 WT, 10 LIS1+/-). At short distances the correlation decreases linearly, 1 − r/lp. WT organdies exhibit correlation peaks (arrowheads). Inset: correlation functions of healthy (blue) and lissencephalic (orange) human brains. (j) Average curvature < |∂rθ(r)| > of WT and three different and isogenic LIS1+/- clones (N=9 – 11). (k) Elastic modulus of WT and LIS1+/- embryonic stem cells (ES) and neuronal progenitors (NP) from several thousand force curves (N=7-10). (l) Nuclear velocities during apical (inward) and basal (outward) motion for WT and LIS1+/- (N=15). (m) Two-dimensional diagram of nuclear area A and radial-position r/t of LIS1+/- nuclei. Orange color intensity indicates the percentage of nuclei at each point. Black line and arrows indicate progression with time. (n) Nuclear area growth, over a cell-cycle period, in the outer (Out, r/t > 0.5) and inner (In, r/t < 0.5) parts of the organoid for WT (same as Fig. 2f) and LIS1+/−. Kolmogorov-Smirnov test was used to compare the WT and LIS1+/- distributions. Scale bars are 200μm (a,b), 50μm (c) and 5μm (n). Error bars represent s.e.m. Asterisks represent statistical significance (* p < 0.05, ** p < 0.01,***p < 0.001).

Comment in

  • Crinkle-Cut Brain Organoids
    MA Lancaster. Cell Stem Cell 22 (5), 616-618. PMID 29727676.
    In large mammalian brains, including those of humans, the surface of the cortex is highly folded. How these convolutions form is still unclear, but recent work in Nature …

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