In vivo modeling of human neuron dynamics and Down …...Human neuron dynamics imaged in vivo.We combined a human-specific genetic background with live imaging in cortical tissue grafts

RESEARCH ARTICLE SUMMARY◥

NEURODEVELOPMENT

In vivo modeling of human neurondynamics and Down syndromeRaquel Real*, Manuel Peter*, Antonio Trabalza*, Shabana Khan, Mark A. Smith,Joana Dopp, Samuel J. Barnes, Ayiba Momoh, Alessio Strano, Emanuela Volpi,Graham Knott, Frederick J. Livesey†, Vincenzo De Paola†

INTRODUCTION: Scientists are building de-tailed maps of the cellular composition in thehuman brain to learn about its development.In the human cortex, the largest area of themammalian brain, neural circuits are formedthroughanatomical refinement, including axonand synaptic pruning, and the emergence ofcomplex patterns of network activity duringearly fetal development. Cellular analyses inthe human brain are restricted to postmortem

material, which cannot reveal the process ofdevelopment. Model organisms are, therefore,commonly used for studies of brain physiol-ogy, development, and pathogenesis, but theresults from model organisms do not alwaystranslate to humans.

RATIONALE: Systems to model human neu-ron dynamics and their dysfunction in vivo areneeded. While biopsy specimens and the gen-

eration of neurons from induced pluripotentstem cells (iPSCs) could provide the neces-sary human genetic background, two- andthree-dimensional cultures lack factors thatnormally support neuronal development, in-cluding blood vessels, immune cells, and in-teractionwith innervating neurons fromotherbrain areas. On the basis of previous stem cell

transplantation studies inmice, we reasoned thatthe physiological micro-environment of the adultmouse brain could sup-port the growth of humancortical tissue grafts that

had been generated from iPSC-derived neu-ronal progenitors. With human neurons im-planted into the mouse brain, high-resolution,real-time in vivomonitoring of human neurondynamics for periods of time spanning therange from subseconds to several months be-comes feasible.

RESULTS:We found that transplanted humaniPSC–derived neuronal progenitors consist-ently assembled into vascularized territorieswith complex cytoarchitecture, mimicking keyfeatures of the human fetal cortex, such as itslarge size and cell diversification. Single-cell-resolution intravital microscopy showed thathumanneuronal arborswere refined via branch-specific retraction, rather than degeneration.Human synaptic networks restructured overthe course of 4 months, while maintaining bal-anced rates of synapse formation and elimina-tion. Human functional neurons rapidly andconsistently acquired oscillatory populationactivity, which persisted over the 5-month ob-servation period. Lastly, we used cortical tis-sue grafts derived from the fibroblasts of twoindividuals with Down syndrome, caused bysupernumerary chromosome 21. We foundthat neuronal synapses in cells derived fromthese individuals were overly stable and thatoscillatory neural activity was reduced in thesegrafts, revealing in vivo cellular phenotypes nototherwise apparent.

CONCLUSION: By combining live imaging inamultistructured tissue environment inmicewith a human-specific genetic background,we provide insights into the earliest stages ofhuman axon, synaptic, and network activitydevelopment and uncover cellular phenotypesin Down syndrome. Our work provides an al-ternative experimental system that can be usedto study other disorders affecting the develop-ing human cortex. ▪

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Real et al., Science 362, 793 (2018) 16 November 2018 1 of 1

The list of author affiliations is available in the full article online.*These authors contributed equally to this work.†Corresponding author. Email: [email protected](V.D.P.); [email protected] (F.J.L.)Cite this article as R. Real et al., Science 362, eaau1810 (2018).DOI: 10.1126/science.aau1810

Human neuron dynamics imaged in vivo.We combined a human-specific geneticbackground with live imaging in cortical tissue grafts to investigate the earliest stages ofhuman axon, synaptic, and network activity development and model Down syndrome.

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RESEARCH ARTICLE◥

NEURODEVELOPMENT

In vivo modeling of human neurondynamics and Down syndromeRaquel Real1,2,3*, Manuel Peter4*, Antonio Trabalza1,3*, Shabana Khan1,3,Mark A. Smith1,3, Joana Dopp1, Samuel J. Barnes5, Ayiba Momoh4, Alessio Strano4,Emanuela Volpi6, Graham Knott7, Frederick J. Livesey4,8†, Vincenzo De Paola1,3†

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

Cellular analyses in the human brain arerestricted mainly to postmortem material,which cannot provide direct observationof dynamic events, such as anatomical re-finement (1) and the emergence of com-

plex patterns of network activity. This limitationraises the question of how to model human neu-ron dynamics and their dysfunction in the manyincurable disorders that affect the developingcortex (2).Rodent models have been valuable for un-

derstanding the pathophysiology of complex ge-netic disorders, such as Down syndrome (DS)(3–5), which is associated with neurodevelop-mental alterations and is caused by trisomyof chromosome 21 (Ts21), but certain pheno-types are better captured in the context of ahuman genetic background (6).Human induced pluripotent stem cell (iPSC)–

derived neurons can be used in patient-specificstudies to model human cortical development(7), but in vitro two-dimensional (2D) and 3Dcultures (8, 9) lack key interactions with neuro-

glia and vasculature (10). Therefore, systems thatmore closely recapitulate the complex cellulardynamics of the living brain by using patient-specific cells are urgently needed.Building on previous transplantation work

(11), we hypothesized that the existing physio-logical microenvironment in the adult mousebrain could support the expansion of humancortical tissue grafts from iPSC-derived neu-rons, thus allowing high-resolution, real-timein vivo monitoring of human neuron dynam-ics for extended periods of time.In this study, we used single-cell-resolution

intravital microscopy (12) in human tissue graftsto gain insights into the dynamics of pruning,synaptogenesis, and network activity during theearliest stages of cortical neuron developmentand demonstrated this approach by modelinghuman neuron structural and functional dynam-ics in DS. This research was approved by theU.K. Stem Cell Bank Steering Committee andthe U.K. Home Office, in accordance with theU.K. Code of Practice for the Use of HumanStem Cell Lines and the U.K. Animals (ScientificProcedures) Act 1986, respectively.

Complex cytoarchitecture in humancortical tissue grafts

To study the dynamics of human axon and syn-aptic development and population activity invivo, we generated cortical excitatory neuronsfrom a control human iPSC line (13) (fig. S1) andtransplanted them into the adult mouse soma-tosensory cortex (SCx1) for chronic multiphotonimaging (Fig. 1A). Cells were transplanted after36 to 38 days of differentiation, a stage at whichcultures contained ~50% neural progenitor cellsand ~50% deep-layer cortical neurons [of which

~15% expressed T-box, brain 1 (TBR1+), and~85% expressed COUP transcription factor–interacting protein 2 (CTIP2+)] (fig. S2, A andB). As expected, and consistent with ongoingneurogenesis after engraftment, upper-layer cor-tical excitatory neurons and a small proportionof astrocytes and oligodendrocytes could also befound at both 3 and 5 months posttransplanta-tion (mpt) (fig. S2, C and D). Electron microscopy(EM) confirmed that human grafts resembled im-mature cortical tissue at 130 days posttransplan-tation (dpt) (fig. S3, A to C), with few synapsesand few myelinated axons, and showed no de-tectable boundary with the mouse brain (fig. S3C),suggestive of structural integration (14). The graftscontained proliferating cells (fig. S3, C and D),enlarged with time (movie S1), and consisted ofmultiple human- and host-derived cell types (figs.S2 and S3). The cell types from the host includedmicroglial cells, oligodendrocytes, astrocytes,and both excitatory neurons and inhibitory in-terneurons (fig. S3, D to F), whereas no inter-neurons of human origin were found (n = 3transplants). Microglia recruitment in the graftwas minimal (fig. S4). Postmortem analysis re-vealed that the human tissue grafts developedorganizational features resembling the structuralarrangement of the early fetal cortex (fig. S5)(15, 16).At earlier stages (

Real et al., Science 362, eaau1810 (2018) 16 November 2018 2 of 9

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Fig. 1. Single-cell-resolution in vivo imaging of human cortical tissuegrafts reveals mechanisms of pruning. (A) Schematic of experimentaldesign (left) and two-photon in vivo imaging time line (right). NeurRef,neurite refinement; CaDyn, calcium dynamics; SynDyn, synaptic dynamics.(B) Representative two-photon overview of the cranial window over theinjection site at 3 mpt. (C) Bright-field view of a cranial window (~15 mm2) at5 mpt. Arrowheads indicate blood vessels. (D) Representative immuno-staining of endothelial marker CD31 in the human graft at 5mpt. Arrowheadsindicate blood vessels. hNu, human nucleus marker. (E) Representativeexample of axonal bundles (arrows) along blood vessels. Dashed red linesrepresent a blood vessel. (F) Representative example of axonal layering inhuman grafts.The example shown is the sameas that inmovie S3. (G) Exampleof a human neuron migrating (*) and remodeling the leading processes(arrows) over 7 hours. (H) Representative example of extensive remodeling

of a dendritic arbor in a human pyramidal neuron over 25 hours. (I) Pruningof axonal branch over 6 hours. Dashed red lines represent a blood vessel.(I′) Neurite degeneration over 22 hours. Arrows indicate axonal fragments.(J) Representative examples of axon elongation and retraction over 24 hours.The boxed area in the right panel is magnified in the inset. The arrows inthe inset indicate EPBs. gc, growth cone. (K) Speed of neurite elongation andretraction at 3 mpt (n = 113 neurites from 104 cells in six animals, average17 cells per animal). Means and SEM are indicated. Mann-Whitney U test,***P < 0.001. (L) Proportion of neurites elongating, retracting, and stable in24-hour intervals at 3 mpt (n = 92 neurites from 88 cells in six animals,average 15 cells per animal). Error bars indicate SEM. Bonferroni’s multiplecomparisons test after one-way analysis of variance (ANOVA), F2,15 = 43.74,P < 0.0001; *P < 0.05; ****P < 0.0001. Scale bars, 500 mm (B), 100 mm (D),50 mm [(E) and (F)], 20 mm [(G), (H), and (J)], 10 mm (I), and 2 mm (I′).

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which spanned up to 6 months, and spread awayfrom the injection site (Fig. 1B) [on average, upto 1.2 ± 0.6 mm (mean ± SD) from the bregmain the rostral direction over the first 3 mpt (n =4 mice)]. Consistent with the immature braincell-cell interactions (10), human axons grewalong blood vessels and as fiber bundles (Fig. 1Eand movie S2), and parallel and radially orientedaxonal layers could be detected below the duramater (Fig. 1F and movie S3), similar to the onesfound in the human cortex (18).Given the widespread axonal extension out-

side the graft area, we asked which brain regionshuman neurons target 5 mpt. Main SCx1 targetareas showed a higher number of human fibersthan in areas known to receive fewer projectionsfrom SCx1 (fig. S6), suggesting that the directionof axon elongation is targeted. For example, theipsilateral motor cortex, striatum, thalamus, andcontralateral SCx1 received more fibers than thecerebellum and substantia nigra, and the corpuscallosum had more axonal tracts than the in-ternal capsule and cerebral peduncle (fig. S6),as expected from rodent tracing experiments(19). These data provide evidence for long-range(over centimeters) axon growth of grafted humanneurons through the mouse adult brain andindicate that, although human axons are eithernot responsive to or can overcome the inhib-itory signals present in the adult mouse brain,they may be directed by existing guidance cuesor paths.After an initial phase of growth (20), the

selective pruning of axons and dendrites isthought to occur normally via retraction anddegeneration during early development (2, 21).We explored the mechanisms of human neuritepruning up to 3 mpt (Fig. 1, G to L, and fig. S7,A and B). At this stage, neurons were still migrat-ing (Fig. 1G) and developing neural processes ina highly dynamic mode (Fig. 1, G to L). Wetracked the fate of 92 human neurites from 88cells in six mice at 3 mpt (Fig. 1, G to L). Whereasmost neurites (58.4% ± 5.5%) elongated in24 hours, neurite refinement was dynamic, andinterchanging retraction and elongation of indi-vidual neurites (31.0% ± 2.1%) over 24 hourswere observed (Fig. 1, I to L). Developmentalneurite degeneration involves cytoskeletal de-struction with widespread fragmentation overa time scale of 12 to 48 hours (22), whereas re-tracting axons do not leave fluorescent fragmentsbehind (23). Reducing the imaging interval from24 hours to 8 hours showed that branch pruning(Fig. 1I) occurred mainly by retraction (91%),rather than degeneration (Fig. 1I′) (9%). Axonalen passant boutons (EPBs), one of the two typesof presynaptic specialization on cortical axons(24), could be observed in branches with agrowth cone elongating (Fig. 1J). Neural pro-cesses extended long distances (maximum neu-rite extension = 462.769 mm in 24 hours) at aspeed of 10.29 ± 0.73 mm/hour (Fig. 1K), com-parable to that observed in the neonatal mousebrain (23). Results were validated with tissuegrafts from an independent control line (fig. S7,A and B).

Human synaptic developmentimaged in vivoNext, we studied the dynamics of synaptogen-esis up to 4 mpt. Hallmarks of developing syn-aptic networks are an increase in synaptic densityover time, followed by pruning, and the acquisi-tion of a steady state with balanced rates of syn-aptic gain and loss (25). However, when and howhuman synaptic networks acquire these proper-ties is unclear. We first considered dendriticspine formation and elimination (Fig. 2, A to F).After the initial phase of cell migration and

neurite remodeling (Fig. 1, G and H), neuronsstabilized, allowing us to track the same cellsover time (Fig. 2A and fig. S8). Dendritic spines,the structural correlates of mammalian excitatorysynapses (26), were seen as early as 20 dpt (32.8 ±5.5 dpt for either dendritic filopodia, consideredto be the precursors of dendritic spines, orspines; n = 3 mice) (27, 28). We monitored >500dendritic segments from six mice over days.However, for most dendrites, the density ofsynapses was too low to quantitatively studythe dynamics of dendritic spines before 3 mpt,as expected from previous human fetal cerebralcortex postmortem work (29) and the early de-velopmental stage modeled in this study. Eightneurons had sufficient dendritic spine numbersat 3 mpt to calculate spine density and turnoverduring three to four consecutive sessions of48-hour intervals (up to 6 days). The averagespine density was similar to that in the humanearly fetal cerebral cortex (29) and constant overthe imaging period (Fig. 2C) (0.043 ± 0.006spines/mm; n = 70 spines present in the firstsession, 176 in total; Kruskal-Wallis test, P >0.05). Synaptic structures were added and elim-inated at equal rates, even at these early develop-mental stages (Fig. 2D) (Wilcoxon matched-pairssigned-rank test, P > 0.05). The turnover ratio(TOR), a function of both spine gain and loss(30), was 46.9% ± 5.3% over 4 days (Fig. 2E),indicating synaptic reorganization.To investigate the development of synaptic

remodeling over time, we repeated the sameexperiment after 1 month. Again, spine densitywas constant over time (Fig. 2C) (0.112 ± 0.024spines/mm; n = 171 spines present in the firstsession, 291 in total; Kruskal-Wallis test, P >0.05). However, the average spine density wasincreased at 4 mpt. The majority of dendriteshad balanced rates of dendritic spine gain andloss (Fig. 2D) (paired two-tailed t test, P > 0.05),and only in one cell were we able to capture netsynaptic pruning over 2 days (Fig. 2C, thickdashed line), consistent with the idea that amajor phase of synaptic pruning occurs only atlater developmental stages (28).The TOR over 4 days was 27.6% ± 3.7%, which

was lower than at 3 mpt (Fig. 2E). Consistently,the survival fraction, defined as the fraction ofspines surviving as a function of time, was higherat 4 mpt (Fig. 2F), suggesting stabilization ofdendritic spine dynamics over time.To more thoroughly assess synaptic dynamics,

we also studied presynaptic terminals alonghuman cortical axons (Fig. 2, G to L). The density

of boutons remained stable over time (Fig. 2I)(0.051 ± 0.0075 EPBs/mm; n = 69 EPBs in thefirst session, 145 in total), indicating that axo-nal boutons were also added and eliminated atequal rates (Fig. 2L). The TOR over 4 days was45.1% ± 3.6% (Fig. 2, J and K), denoting com-parable dynamics between dendritic spines andaxonal boutons (at 3 mpt, Mann-Whitney U test,P = 0.34).In summary, we were able to study early

events of human cortical neuron synaptogenesisover the first 4 mpt. Despite the low synapticdensity, consistent with the primordial stagemodeled in this study (29), we can draw a num-ber of conclusions about early in vivo humansynaptic network development. First, trans-planted human neurons initially formed synap-tic structures within 4 to 12 weeks of in vivodevelopment, similar to the human fetal cere-bral cortex (29). Second, they underwent synapticreorganization. Third, they progressively increaseddendritic spine density over 1 month. Finally,human neurons balanced the rates of synapticgain and loss over a time scale of a few days.

Functional human cortical networksimaged in vivo

Patterned neural activity is thought to be fun-damental to neural circuit development in theimmature brain (31, 32). Although spontaneousand sparse activity can be detected in humancortical network preparations in vitro, recapit-ulating patterns typical of early human corticalpopulation activity, such as recurrent oscillatorybursts (32), remains challenging (33, 34).We first investigated the electrophysiological

properties of transplanted cells. We performedex vivo whole-cell recordings in coronal brainslices containing the grafts (fig. S9). Current-clamp recordings were made from 18 pyrami-dal neurons (n = 4 mice), as identified by usingdifferential interference contrast microscopyand expression of either GFP or tdTomato andby filling neurons with Lucifer yellow dye be-fore post hoc anatomical inspection (fig. S9A).Patched grafted pyramidal neurons were at dif-ferent stages of biophysical maturation and de-velopment, with an average resting membranepotential of −53.8 ± 1.7 mV, average capacitanceof 19.4 ± 2.2 pF, and average input resistanceof 1.4 ± 0.1 gigaohms. Although cells were qui-escent at resting membrane potentials, depola-rizing current steps evoked action potential firingin all pyramidal neurons tested (fig. S9B), withaverage action potential amplitudes of 91.3 ±2.6 mV and half-widths of 2.2 ± 0.2 ms.Immunohistochemistry showed glutamater-

gic and GABAergic terminals within the humangraft (fig. S10, A and B). To confirm that humanneurons received both excitatory and inhibitoryinput, pyramidal neurons were voltage clamped(−70 mV) and spontaneous miniature excitatorypostsynaptic currents (mEPSCs) were observedat a frequency of 0.30 ± 0.05 Hz (5 of 18 neurons)with an amplitude of 20.1 ± 3.2 pA, which werecompletely blocked by the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor




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Fig. 2. Developing human synaptic networks are characterized bysubstantial restructuring and balanced rates of gains and losses.(A) Overview of cranial window at 136 and 138 dpt. Red arrows point toexamples of cells with a stable location over a 48-hour period. (B) Detail of arepresentative dendrite imaged over 24 hours (white box in the top paneland red box in fig. S8A). Green, red, and white arrowheads indicate gained,lost, and stable dendritic spines, respectively. (C) Dendritic spine density over4 to 6 days at 3 mpt (n = 8 cells, 1.40 mm of total dendritic length, fromthree animals) and 4 mpt (n = 6 cells, 0.93 mm of total dendritic length,from two animals). Two-way ANOVA, interaction F3,46 = 0.4357, P = 0.73.****P < 0.0001. (D) Average fractions of dendritic spines gained and lost over48 hours at 3 mpt (red, n = 8 cells) and 4 mpt (blue, n = 6). Two-wayANOVA, interaction F1,24 = 0.1894, P = 0.67. Sidak’s multiple comparisonstest, *P < 0.05 (gains); P = 0.063 (losses). ns, not significant. (E) Dendriticspine TOR over 4 days at 3 mpt (n = 8 cells) and 4 mpt (n = 6 cells).Mann-Whitney U test, *P < 0.05. Each data point represents a cell.

(F) Dendritic spine survival fraction at 3 mpt (red, n = 7 cells) and 4 mpt(blue, n = 6 cells). Two-way ANOVA, interaction F3,47 = 1.513, P = 0.22;*P < 0.05. (G) Representative example of a branched human axon at 130 dpt.The arrow indicates a growth cone. The boxed area is magnified insubsequent panels. (H) Detail of the axon shown in the boxed area in (G),imaged every 48 hours over 4 days. Green, red, and white arrowheadsindicate gained, lost, and stable EPBs, respectively. (I) EPB density over 2 to4 days at 3 mpt (n = 8 cells, 1.3 mm of total axonal length, from threeanimals). One-way ANOVA, F2,17 = 0.4014; P = 0.68. (J) Quantification ofEPB TOR over 4 days at 3 mpt (n = 4 cells). Each data point represents anaxon. (K) Quantification of EPB survival fraction at 3 mpt (n = 8 cells).(L) Average fractions of EPB gains and losses over 48 hours at 3 mpt(n = 8 cells). Wilcoxon matched-pairs signed-rank t test; ns, not significant.[(C), (D), (F), (I), (K), and (L)] Dashed lines represent individual cells,and solid lines represent means. Scale bars, 50 mm (A), 20 mm [(B), toppanel], 2 mm [(B), bottom panel], 10 mm (G), and 5 mm (H).



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antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-quinoxaline-2,3-dione (NBQX) (n = 4). Al-though synaptic events were observed in theremaining neurons, spontaneous frequency wasinsufficient to acquire enough events for sta-tistical analysis (figs. S7, C and D, and S9C). Byusing a high-chloride (130 mM) internal solu-tion and in the presence of NBQX, spontaneousminiature inhibitory postsynaptic currents wereobserved at a frequency of 0.24 ± 0.12 Hz (threeof six neurons) with an amplitude of −73.3 ±21.0 pA, which were fully inhibited by bicucul-line (fig. S9C). Similar to mEPSCs, inhibitorysynaptic events were observed in the remainingneurons, but insufficient events were acquiredfor detailed kinetic analysis. In summary, graftedneurons are excitable and fire action potentials.In addition, they receive both excitatory and in-hibitory input, suggesting functional networkconnectivity.

To determine the origin of the afferent syn-aptic input to the functionally active neurons,we performed monosynaptic retrograde tracingby using a modified rabies virus. This virus lacksa glycoprotein needed for replication and caninfect only cells expressing the avian tumorvirus receptor A (TVA) (fig. S11). Human iPSC–derived cortical progenitors and neurons weretransduced with a lentiviral vector containingthe TVA, nuclear GFP, and glycoprotein underthe control of the human synapsin promoter(fig. S11A). Five months after the transplanta-tion, the modified mCherry expressing–rabiesvirus was injected in the same location, whereonly grafted cells expressing the TVA are sus-ceptible to infection. Cells that are mono-synaptically connected to the infected humancells also become infected and express mCherry,allowing for accurate tracing of the neural inputto the cells in the human grafts (fig. S11B). We

observed that whereas most of the input to thetransplanted human neurons comes from otherhuman neurons (92.5% ± 1.5%, n = 4333 cells intwo brains), host neurons also innervate thehuman graft (7.5% ± 1.5%, n = 397 cells in twobrains) (fig. S11C). The traced host neurons werelocated within the graft, in the cortical areasadjacent to the graft, in the contralateral cortex,and in the ipsilateral CA1 hippocampal region(fig. S11B). Although no traced neurons werefound in other subcortical regions, thalamocor-tical terminals were present in the graft (fig. S10;see also fig. S12) (20). These results provide evi-dence that most synaptic input to the grafts comesfrom other human neurons. Furthermore, as nointerneurons of human origin were found, thesedata, together with the demonstration that humanneurons in the graft receive inhibitory input (fig.S9C, bottom), suggest that inhibition in the hu-man grafts comes from the host.


Fig. 3. In vivo calcium imaging shows that patterned populationactivity emerges early and has a defined spatiotemporal order.(A) Example of an imaged cortical region taken from a WT-1 graft at 1 mptin the somatosensory cortex of an adult mouse. Neurons expresstdTomato (red) and GCaMP6 (green). GCaMP-positive neurons are shownas a maximum-intensity projection of activity over a 4-min period ofspontaneous activity. Active neurons (yellow) are shown by overlaying theimages (merge). (B) Representative DF/F0 calcium traces (where DF/F0is the ratio of the change in fluorescence to the baseline fluorescence)from five active neurons imaged in a WT-1 graft at 1 mpt. (C) Distribution of

spontaneous calcium activity in WT-1 grafts at 1 to 2 mpt. Activity wasmeasured as the integral of the average DF/F0 signal over the entire regionof interest (ROI), normalized to the total duration of the recording inseconds (n = 88 cells, six ROIs, three mice). (Inset) Percentage of ROIs inWT-1 grafts at 1 to 2 mpt (3 of 16 ROIs, 18.8%; n = 4 mice) and 3 mpt(31 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 ina WT-1 graft. (E) Example of burst activity over two different spatialregions (gray and black) shown on the left, taken from the bursts in (D).Scale bars, 10 mm (A) and 20 mm (D).



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Fig. 4. In vivo modeling of structuraland functional neuronal dynamics intissue grafts from individuals withDS. (A) Representative example of axonelongation in a Ts21-1 neuron over a24-hour period.The inset corresponds tothe boxed area and highlights the pres-ence of EPBs.The red line indicatesalignment between the top and bottomimages. (B) Example of axonal branchretraction (arrows) in a Ts21-1 neuron over17 hours. (C) Proportion of elongating,retracting, and stable neurites in 24-hourintervals in WT-1 (n = 96 neurites from79 cells, seven grafted animals, average11 cells per animal),Ts21-1 (n = 65 neuritesfrom 60 cells, seven grafted animals,average 9 cells per animal),WT-2(n=65 neurites from53 cells, four graftedanimals, average 13 cells per animal),and Ts21-2 (n = 60 neurites from 51 cells,four grafted animals, average 13 cells peranimal) grafts at 3wpt.WT-2 is a revertantdisomic cell line from Ts21-2. Unpairedtwo-tailed t test; ns, not significant.Each data point represents an animal.(D) Speed of neurite elongation andretraction in WT-1 (n = 96 neurites from73 cells, average 10 cells per animal),Ts21-1 (n = 62 neurites from 54 cells,average 8 cells per animal),WT-2(n = 53 neurites from 47 cells, average12 cells per animal), and Ts21-2(n = 54 neurites from 46 cells, average12 cells per animal) grafts at 3 wpt.Unpairedmultiple t test; ns, not significant.Each data point represents an animal.(E) Example of dendritic branches andspines on a Ts21-1 neuron, imagedat 48-hour intervals for 4 days.The boxedregion in the left panel is magnified insubsequent panels. Green, red, and whitearrowheads indicate gained, lost, andstable dendritic spines, respectively.(F) 3D rendering of the same dendriticregion imaged in vivo in (E), obtained fromEM reconstruction. Presynaptic terminalsare shown in green. (G) EM images of thedendritic spines marked with 1 and2 in (E). Arrowheads indicate the loca-tion of synapses. Asterisk, presynapticterminal. (H) Dendritic spine survival fraction over 4 days inWT-1 (n= 10 cells fromtwo animals),Ts21-1 (n = 9 cells from four animals), and Ts21-2 (n = 7 cells fromtwo animals) grafts at 3 to 4 mpt.Two-way ANOVA, interaction F4,69 = 5.435,P = 0.0007;Tukey’s multiple comparisons test, ****P < 0.0001. Each data pointrepresents a cell. (I) Quantification of dendritic spine TOR over 4 days in WT-1(n = 10 cells from two animals),Ts21-1 (n = 9 cells from four animals), and Ts21-2(n = 7 cells from two animals) grafts at 3 to 4 mpt. Sidak’s multiple comparisonstest after one-way ANOVA, F2,23 = 3.078, **P < 0.01; ***P < 0.001. Each datapoint represents a cell. (J) Representative example of an axon on a Ts21-2 neuronimaged at 48-hour intervals for 4 days.The arrowheads in the insets indicatestable (white), new (green), and lost (red) EPBs. (K) EPB survival fraction over4 days in WT-1 (n = 6 cells),Ts21-1 (n = 24 cells), and Ts21-2 (n = 10 cells) graftsfrom three mice each at 3 to 4 mpt.Two-way ANOVA, interaction F4,111 = 0.8211,P = 0.51; ns, not significant. Each data point represents an axon. (L) EPB TORover 4 days in WT-1 (n = 6 cells),Ts21-1 (n = 24 cells), and Ts21-2 (n = 10 cells)grafts from threemice each at 3 to 4mpt. Sidak’smultiple comparisons test after

one-way ANOVA, F2,37 = 5.588, **P < 0.01; ns, not significant. Each data pointrepresents an axon. (M andN) (Left) Example of imaged cortical regions takenfrom Ts21-1 (M) and Ts21-2 (N) grafts in the somatosensory cortices of adultmice. Neurons express tdTomato (red) and GCaMP6s (green). Active neurons(yellow) are shown by overlaying the images. (Right) Representative DF/F0calcium traces from five active neurons imaged in Ts21-1 (M) and Ts21-2 (N)grafts. Noteweak synchronized burst activity across different neurons comparedwith the traces in fig. S7E. (O) Percentage of ROIs in WT-1 (50 of 52 ROIs,96.1%, six grafted mice),Ts21-1 (10 of 38 ROIs, 26.3%, three grafted mice),WT-2(34 of 34 ROIs, 100%, three grafted mice), or Ts21-2 (11 of 23 ROIs, 47.8%,three grafted mice) grafts that exhibit bursts at 3 to 5 mpt. Z test, ***P < 0.001.(P) Frequency of burst events in WT-1,Ts21-1,WT-2, and Ts21-2 grafts measuredat 3 to 5mpt. Kruskal-Wallis test, **P

To assess the functional development of cor-tical networks in vivo, we engineered neurons toexpress the genetically encoded calcium indi-cator GCaMP6s (35) before grafting and studiedcalcium-mediated neuronal activity in vivo (Fig. 3)(n = 8 mice). Spontaneous, sparse activity (Fig. 3,A to C) was detected as early as 2 weeks post-transplantation (wpt) and persisted up to 3 mpt(Fig. 3C). In addition, bursts of activity syn-chronized across the neuropil and multiplecells (31) were also detected at 1 mpt (Fig. 3C,inset) and persisted in all grafts tested up to5 mpt (fig. S7, E to H, and movies S4 and S5).Many of these bursts had a defined spatio-

temporal order (Fig. 3, D and E), as well as re-current oscillatory behavior (

reduced in tissue grafts from two individualswith DS, suggesting a possible role for patternedactivity in regulating synaptic lifetimes in theearly stages of human cortical circuit develop-ment (32). These deficits were evident even afterTs21 cells were exposed to the in vivo physio-logical microenvironment of the mouse brainfor several months, indicating cell-intrinsic de-ficits. By using a revertant disomic iPSC line,we showed that the population activity deficitswere rescued by the loss of an extra copy ofHsa21, indicating that heightened expressionof Hsa21 genes is both necessary and sufficientto disrupt oscillatory burst activity in developingcortical DS networks in vivo.In most previous work, human ESC– or iPSC–

derived neurons have been transplanted intothe damaged cortex (38, 47), spinal cord (48),striatum (49, 50), or retina (51), with the aimof cell replacement (11) rather than for diseasemodeling (6, 52), as demonstrated in our study.Transplantation and in vivo imaging for diseasemodeling in mice is advantageous over that inhigher species such as primates, as larger num-bers of animals can be used to track cells in thegrafts over long periods of time and the modelprovides a microenvironment containing ves-sels, immune cells, and innervation, not presentin common in vitro preparations.In summary, we established a new in vivo

experimental model of DS to study how thechromosomal abnormality affects the earlieststages of human axon, synaptic, and functionalneural network development. We expect thatthis single-cell-resolution intravital microscopyapproach will advance the knowledge of cel-lular pathophysiology in this and other neuro-developmental disorders, particularly valuablein light of the scarcity of early human fetalbrain tissue material.

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ACKNOWLEDGMENTS

We thank K. Alvian, R. Festenstein, T. Keck, and M. Lancaster forcomments on the manuscript; S. Papadoupoulou for help withimmunohistochemistry; C. Bass and A. A. Bharath (ImperialCollege London) for help with the calcium imaging analysis;C. Whilding for developing a customized FIJI script for imageanalysis; M. Tortora for help with the immunohistochemistryexperiments and analysis and C. Pernaci for help with the analysis;M. Lavrov and M. Rakowska for help with synaptic dynamicsanalysis; E. Mustafa, A. Czerniak, K. Horan, A. Matthews, andE. Rowley for assistance with animal care and monitoring;M. Tripodi (LMB, Cambridge) for the kind gift of the modifiedrabies transynaptic tracer; and G. Stamp for the analysis ofhematoxylin and eosin–stained material. Funding: This work wassupported by the Medical Research Council (V.D.P.); the GABBAPh.D. program (FCT fellowship PD/BD/52198/2013), the

Rosetrees Trust, and ARUK (R.R.); the UK Dementia ResearchInstitute (grant code DRIImp17/18 Q3 to S.J.B.); a Wellcome SeniorInvestigator award (F.J.L.); and the Alborada Trust of the ARUKStem Cell Research Centre (M.P. and F.J.L.). Author contributions:V.D.P. conceived and planned the live imaging, characterization,and analysis of transplanted patient-derived neurons and broughtF.J.L. into the project; F.J.L. independently generated the iPSC-derivedneurons, conceived the in vitro aspects of the project, and contributedto the study design; R.R., A.T., and V.D.P. performed the graftingand the two-photon imaging experiments, analyzed the data, andprepared the related figures and text; M.P. performed the humaniPSC–derived neuron differentiation and in vitro characterization, thecopy number assay, and the lentiviral vector transductions andprovided input on the design of the experiments; R.R. and S.K.performed cell marker immunohistochemistry, imaging, and analysisfor the characterization of cell identity after transplantation andprepared the relevant figures with input from V.D.P.; R.R. conductedand analyzed the whole-brain rabies tracing reconstructions; M.A.S.performed the electrophysiology recordings in acute brain slicescontaining the human grafts and prepared the relevant figure andtext; S.J.B. analyzed the in vivo calcium imaging data and preparedthe relevant figures and text with input from R.R. and V.D.P.; A.M.generated and characterized the Ts21-2, WT-2, and WT-2′ lines with

input from F.J.L.; A.S. analyzed the gene expression, copy numbervariation, and STR data and prepared the relevant figures; J.D.and S.K. characterized the graft size; J.D. characterized the axonprojections from the grafts; E.V. provided input on the Hsa21 FISHexperiment and analyzed the FISH data with input from V.D.P.;G.K. performed and analyzed the EM reconstructions and preparedthe relevant figures; V.D.P. led the project; and V.D.P and R.R.wrote the paper with contributions from all authors. Competinginterests: None. Data and materials availability: All data neededto evaluate the conclusions in the paper are present in the paperor the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/362/6416/eaau1810/suppl/DC1Materials and MethodsFigs. S1 to S21Table S1References (53–60)Movies S1 to S6

24 May 2018; accepted 26 September 2018Published online 11 October 201810.1126/science.aau1810




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In vivo modeling of human neuron dynamics and Down syndrome

Alessio Strano, Emanuela Volpi, Graham Knott, Frederick J. Livesey and Vincenzo De PaolaRaquel Real, Manuel Peter, Antonio Trabalza, Shabana Khan, Mark A. Smith, Joana Dopp, Samuel J. Barnes, Ayiba Momoh,

originally published online October 11, 2018DOI: 10.1126/science.aau1810 (6416), eaau1810.362Science

, this issue p. eaau1810Sciencedendritic spine turnover and less network activity.Down syndrome, upon transplantation into the mouse brain, produced neurons that grew normally but showed reducedpruning was observed and involved a process of branch retraction, not degeneration. Cells derived from individuals with human cells produced neurons that integrated and developed synaptic networks with oscillatory activity. Dendriticinto the brains of adult mice. They used intravital imaging to visualize how resulting neurons grew and connected. The

transplanted neural progenitors derived from human iPSCset al.cells (iPSCs) can help to elucidate the process. Real The earliest stages of human brain development are very difficult to monitor, but using induced pluripotent stem

Development of human brain neurons

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MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/10/10/science.aau1810.DC1

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REFERENCES

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In vivo modeling of human neuron dynamics and Down …...Human neuron dynamics imaged in vivo.We combined a human-specific genetic background with live imaging in cortical tissue grafts