In vivo modeling of human neuron dynamics and Down syndrome

Abstract

Harnessing the potential of human stem cells for modeling the physiology and diseases of cortical circuitry requires monitoring cellular dynamics in vivo. We show that human induced pluripotent stem cell (iPSC)-derived cortical neurons transplanted into the adult mouse cortex consistently organized into large (up to ~100 mm³) vascularized neuron-glia territories with complex cytoarchitecture. Longitudinal imaging of >4000 grafted developing human neurons revealed that neuronal arbors refined via branch-specific retraction; human synaptic networks substantially restructured over 4 months, with balanced rates of synapse formation and elimination; and oscillatory population activity mirrored the patterns of fetal neural networks. Lastly, we found increased synaptic stability and reduced oscillations in transplants from two individuals with Down syndrome, demonstrating the potential of in vivo imaging in human tissue grafts for patient-specific modeling of cortical development, physiology, and pathogenesis.

Figure 1. Single-cell-resolution in vivo imaging of human cortical tissue grafts reveals mechanisms of pruning.

(A) Schematic of experimental design (left) and 2-photon in vivo imaging time line (right). NeuRef, neurite refinement. CaDyn, calcium dynamics. SynDyn, synaptic dynamics. (B) Representative 2-photon overview of the cranial window over the injection site at 3 mpt. (C) Bright field view of a cranial window (~ 20 mm²) at 5 mpt. Arrowheads indicate blood vessels. (D) Representative immunostaining of endothelial marker CD31 in the human graft at 5 mpt. Arrowheads indicate blood vessels. (E) Representative example of axonal bundles (arrows) along blood vessels; dashed red lines represent a blood vessel. (F) Representative example of axonal layering in human grafts. Same example as movie S3. (G) Example of a human neuron migrating (*) and remodeling the leading processes (arrows) over 7h. (H) Representative example of extensive remodeling of a dendritic arbor in a human pyramidal neuron over 25h. (I) Pruning of axonal branch over 6h. Dashed red lines represent a blood vessel. (I’) Neurite degeneration over 22h. Arrows indicate axonal fragments. (J) Representative examples of axon elongation and retraction over 24h. The arrows in the inset indicate EPBs. gc, growth cone. (K) Speed of neurite elongation and retraction at 3 mpt (n = 113 neurites from 104 cells in 6 animals, average 17 cells/animal). Mann-Whitney U-test, ***P < 0.001. (L) Proportion of neurites elongating, retracting and stable in 24h intervals at 3 mpt (n = 92 neurites from 88 cells in 6 animals, average 15 cells/animal). Bonferroni’s multiple comparisons test after one-way ANOVA, F_2,15 = 43.74, P < 0.0001; *P < 0.05; ****P < 0.0001. Scale bars, 500 μm (B), 100 μm (D), 50 μm [(E) and (F)], 20 μm [(G), (H) and (J)], 10 μm (I), 2 μm (I’).

Figure 2. Developing human synaptic networks are characterized by substantial restructuring and balanced rates of gains and losses.

(A) Overview of cranial window at 136 and 138 dpt; red arrows represent examples of cells with a stable location over a 48h period. (B) Detail of a representative dendrite imaged over 24h (white box in the top panel and red box in fig. S8A); green, red and white arrowheads indicate gained, lost and stable dendritic spines, respectively. (C) Dendritic spine density over 4-6 days at 3 mpt (red: n = 8 cells, 1.40 mm total dendritic length, from 3 animals) and 4 mpt (blue: n = 6 cells, 0.93 mm total dendritic length, from 2 animals). Two-way ANOVA, interaction F_3,46 = 0.4357, P = 0.7285. ****P < 0.0001. (D) Average fraction of dendritic spines gained and lost over 48h at 3 mpt (red: n = 8 cells) and 4 mpt (blue: n = 6). Two-way ANOVA, interaction F_1,24 = 0.1894, P = 0.6673. Sidak’s multiple comparisons test, *P < 0.05 (gains); P = 0.063 (losses). (E) Dendritic spine turnover rate (TOR) over 4 days at 3 mpt (n = 8) and 4 mpt (n = 6 cells). Mann-Whitney U-test, *P < 0.05. Each data point represents a cell. (F) Dendritic spines survival fraction at 3 mpt (red: n = 7 cells) and 4 mpt (blue: n = 6 cells). Two-way ANOVA, interaction F_3,47 = 1.513, P = 0.2235; *P < 0.05. (G) Representative example of a branched human axon at 130 dpt; arrow indicates a growth cone. (H) Detail of axon in (G), imaged every 48h over 4 days; green, magenta and white arrowheads indicate gained, lost and stable EPBs, respectively. (I) EPB density over 2-4 days at 3 mpt (n = 8 cells, 1.3 mm total axonal length, from 3 animals). One-way ANOVA, F_2,17 = 0.4014; P = 0.6756. (J) Quantification of EPB TOR over 4 days at 3 mpt (n = 4 cells). Each data point represents an axon. (K) Quantification of EPB survival fraction at 3 mpt (n = 8 cells). (L) Average fraction of EPB gains and losses over 48h at 3 mpt (n = 8 cells). Wilcoxon matched-pairs signed rank t-test; ns, not significant. Dashed lines represent individual cells and full lines represent means (C, D, F, I, K, L). Scale bars, 50 μm (A), 20 μm (B, top panel), 2 μm (B, bottom panel), 10 μm (G), 5 μm (H).

Figure 3. In vivo calcium imaging shows that patterned population activity emerges early and has a defined spatiotemporal order.

(A) Example of an imaged cortical region taken from a WT-1 graft at 1 mpt in the somatosensory cortex of an adult mouse. Neurons express tdTomato (left - red) and GCaMP6 (middle - green). GCaMP positive neurons are shown as a maximum intensity projection of activity over a 4 min period of spontaneous activity. Active neurons (yellow) are shown by overlaying the images (right – merge). (B) Representative ΔF/F₀ calcium traces from 5 active neurons imaged in a WT-1 graft at 1 mpt. (C) Distribution of spontaneous calcium activity in WT-1 grafts at 1-2 mpt. Activity was measured as the integral of the average ΔF/F₀ signal over the entire region of interest (ROI), normalized to the total duration of the recording in seconds (n = 88 cells, 6 ROIs, 3 mice). Inset: percentage of ROIs in WT-1 grafts at 1-2 mpt (3 out of 16 ROIs, 18.8%; n = 4 mice) and 3 mpt (31 out of 35 ROIs, 89.0%; n = 5 mice) that exhibit bursts. Chi-square test, *P < 0.05. (D) Montage of image frames from a typical recurrent burst in a WT-1 graft. (E) Example of burst activity over two different spatial regions (gray and black) shown in left cartoon, taken from the bursts in (D). Scale bars, 10 μm (A), 20 μm (D).

Figure 4. In vivo modelling of structural and functional neuronal dynamics in tissue grafts from individuals with Down syndrome.

(A) Representative example of axon elongation in a Ts21-1 neuron, over a 24h period. The inset highlights the presence of EPBs. (B) Example of axonal branch retraction in a Ts21-1 neuron over 17h. (C) Proportion of elongating, retracting and stable neurites in 24h intervals in WT-1 (n = 96 neurites from 79 cells, 7 grafted animals, average 11 cells/animal), Ts21-1 (n = 65 neurites from 60 cells, 7 grafted animals, average 9 cells/animal), WT-2 (n = 65 neurites from 53 cells, 4 grafted animals, average 13 cells/animal) and Ts21-2 (n = 60 neurites from 51 cells, 4 grafted animals, average 13 cells/animal) grafts at 3 wpt. WT-2 is a revertant disomic cell line from Ts21-2. Unpaired two-tailed t-test; ns, not significant. Each data point represents an animal. (D) Speed of neurite elongation and retraction in WT-1 (n = 96 neurites from 73 cells, average 10 cells/animal), Ts21-1 (n = 62 neurites from 54 cells, average 8 cells/animal), WT-2 (n = 53 neurites from 47 cells, average 12 cells/animal) and Ts21-2 (n = 54 neurites from 46 cells, average 12 cells/animal) grafts at 3 wpt. Unpaired multiple t-test; ns, not significant. Each data point represents an animal. (E) Example of dendritic branches and spines on a Ts21-1 neuron, imaged at 48h intervals for 4 days; green, red and white arrowheads indicate gained, lost and stable dendritic spines, respectively. (F) 3D-rendering of the same dendritic region imaged in vivo in (E), obtained from electron microscopy reconstruction. Presynaptic terminals are shown in green. (G) Electron microscopic images of the dendritic spines marked with 1 and 2 in (E). Red arrows indicate the location of synapses. Green asterisk, presynaptic terminal. (H) Dendritic spines survival fraction over 4 days in WT-1 (n = 10 cells from 2 animals), Ts21-1 (n = 9 cells from 4 animals) and Ts21-2 (n = 7 cells from 2 animals) grafts at 3-4 mpt. Two-way ANOVA, interaction F_4,69 = 5.435, P = 0.0007; Tukey’s multiple comparisons test, ****P < 0.0001. Each data point represents a cell. (I) Quantification of dendritic spine turnover rate over 4 days in WT-1 (n = 10 cells from 2 animals), Ts21-1 (n = 9 cells from 4 animals) and Ts21-2 (n = 7 cells from 2 animals) grafts at 3-4 mpt. Sidak’s multiple comparisons test after one-way ANOVA, F_2,23 = 3.078, **P < 0.01; ***P <0.001. Each data point represents a cell. (J) Representative example of an axon on a Ts21-2 neuron imaged at 48h intervals for 4 days. The arrowheads in the insets indicate stable (white), new (green) and lost EPBs (red). (K) EPBs survival fraction over 4 days in WT-1 (n = 6 cells), TS21-1 (n = 24 cells) and TS21-2 (n = 10 cells) grafts at 3-4 mpt from 3 mice each. Two-way ANOVA, interaction F_4,111 = 0.8211, P = 0.5144; ns, not significant. Each data point represents an axon. (L) EPBs turnover rate over 4 days in WT-1 (n = 6 cells), TS21-1 (n = 24 cells) and TS21-2 (n = 10 cells) grafts at 3-4 mpt from 3 mice each. Sidak’s multiple comparison test after one-way ANOVA, F_2,37 = 5.588, **P < 0.01; ns, not significant. Each data point represents an axon. (M), (N) Left: Example of imaged cortical regions taken from Ts21-1 (M) and Ts21-2 (N) grafts in the somatosensory cortex of adult mice. Neurons express tdTomato (red) and GCaMP6s (green). Active neurons (yellow) are shown by overlaying the images. Right: Representative ΔF/F₀ calcium traces from 5 active neurons imaged in Ts21-1 (M) and Ts21-2 (N) grafts. Note weak synchronized burst activity across different neurons compared to the traces in fig. S7E. (O) Percentage of ROIs in WT-1 (50 out of 52 ROIs, 96.1 %, 6 grafted mice), Ts21-1 (10 out of 38 ROIs, 26.3 %, 3 grafted mice), WT-2 (34 out of 34 ROIs, 100 %, 3 grafted mice) or TS21-2 (11 out of 23 ROIs, 47.8 %, 3 grafted mice) grafts that exhibit bursts at 3-5 mpt. Z-test, ***P <0.001. (P) Frequency of burst events in WT-1, Ts21-1, WT-2 and Ts21-2 grafts measured at 3-5 mpt. Kruskal-Wallis test, **P < 0.01; ***P < 0.001. (Q) Global ROI activity in WT-1, Ts21-1, WT-2 and Ts21-2 grafts measured at 3-5 mpt. Kruskal-Wallis test, ***P <0.001. Scale bars, 10 μm [(A) and (B)], 5 μm [(E, left) and (J)], 2 μm (E, right), 0.2 μm (G), and 20 μm [(M), (N)].