Brain Organoids: Human Neurodevelopment in a Dish...2019/11/25 · into various retinal cells...

Brain Organoids: Human Neurodevelopmentin a Dish

Silvia Benito-Kwiecinski and Madeline A. Lancaster

MRCLaboratory ofMolecular Biology, Cambridge Biomedical Campus, CambridgeCB2 0QH,United Kingdom

Correspondence: [email protected]

The human brain is often described as the most complex organ in our body. Because of thelimited accessibility of living brain tissue, human-specific features of neurodevelopment anddisease remain largely unknown. The ability of induced pluripotent stemcells to self-organizeinto 3D brain organoids has revolutionized approaches to studying brain development invitro. This reviewwill first look at the historyof studyingneural development in a dish andhoworganoids came to be. We evaluate the ability of brain organoids to recapitulate key devel-opmental events, focusing on the generation of various regional identities, cytoarchitecture,cell diversity, features of neuronal maturation, and circuit formation. We also consider thelimitations of the model and review recent approaches to improve reproducibility and thehealthy maturation of brain organoids.

Central to developmental biology is the re-markable ability of one fertilized egg to re-liably generate a whole complex body throughcarefully orchestrated spatial and temporal pat-terns that generate an immense diversity of cellfates. All living multicellular organisms rely onthe ability of a single cell to self-organize into 3Dtissues with many specialized cell types, func-tions, and architectures. Because the instruc-tions to carry out these complex morphogeneticevents are contained within each cell, these pro-cesses can be recapitulated in vitrowith the rightstarting cell type and culture conditions. Stemcells, or organ progenitors, provide the startingpoint, while recent improvements in tissue en-gineering and 3D culture techniques enable theformation of macrostructures reminiscent ofhuman organs. These so-called organoids are3D tissues that self-organize into a spatially or-

ganized structure consisting of multiple organ-specific cell types in a manner highly reminis-cent to the actual organ. Furthermore, becauseof their embryonic identity and fate potential,pluripotent stem cell (PSC)-derived organoidsmodel developmental trajectories with surpris-ingly minimal extrinsic guidance leading tospontaneous self-organization into specific ear-ly organ structures.

Because PSC-derived organoids follow pri-marily intrinsic developmental programs, thesemethods are less like tissue engineering andmore akin to gardening. The PSC can bethought of as the seed, while culture conditionsresemble the sunlight, soil, fertilizer, and waterthat nurture the seed to take root and sprout.Thus, these in vitro models are grown ratherthan built. Because of their intrinsic ontogeny,organoids offer the unprecedented ability to ob-

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serve, analyze, and manipulate human develop-ment in a dish.

In this context, the human brain in particu-lar is of great interest because of the widespreadhealth burden of neurodevelopmental and neu-rological disorders, the uniqueness of our excep-tionally large primate brains, and the ethical andtechnical inaccessibility of functionally testingfeatures of human brain development in vivo.Although not the focus of this review, brain or-ganoids have already been used to study variousaspects of neurodevelopmental diseases andevolution, and we direct the reader to other re-views covering such work (Clevers 2016; Gian-domenico and Lancaster 2017; Qian et al. 2019).This review will focus on neural organoids, cov-ering the history and development of methodsfor studying in vitro self-organization of neuraltissues. We will compare various stages of invitro development to in vivo development,mainly focusing on methods for generating or-ganoids with cortical identities and furtheringtheir developmental potential.

HISTORY

Reaggregates

The remarkable ability of cells to self-organizeinto 3D tissues in vitrowas alreadyobserved overa century ago. In 1907, Wilson demonstratedthat dissociated sea sponges would reaggregateinto complete organisms. This regeneration of awhole animal from its dissociated cells was sub-sequently shown in other simple organisms(Wilson 1907, 1911; Child 1928). These findingsled to a surge of in vitro experiments in the 1950sand 1960s looking at how distinct combinationsof dissociated cells from more complex organ-isms would reaggregate into organized struc-tures. In 1955, Townes andHoltfreter (Steinbergand Gilbert 2004) showed that dissociated cellsfrom the three germ layers of early amphibianembryos would recombine into spatially segre-gated germ layers, reproducing their proper em-bryonic positions. This reaggregation of cellsinto organized structures was also shown usingcells from later vertebrate embryos, in particular,the chick. Dispersed cells from an already func-

tioning vertebrate organ, such as skin or kidney,can under the right conditions self-assemble toreconstitute the tissue (Moscona and Moscona1952; Weiss and Taylor 1960).

These dissociation–reaggregation experi-ments showed that tissue formation can occurindependent of a prepattern and a sequence ofpreceding developmental events. Despite initial-ly being randomly clustered, reaggregated cellswill self-assemble into distinct regions of a tissuethrough a process of a cell “sorting out.” Thisprocess is explained by the differential adhesionhypothesis (Steinberg and Roth 1964), wherebycells will rearrange their positions to bind to cellsexpressing similar cell surface adhesive mole-cules, resulting in a more thermodynamicallystable structure, segregated into domains of cellswith differing adhesive strengths. Sorting out ofcells is also observable in aggregates of corticalneurons where early postmitotic neurons havebeen shown to associate with one another (De-Long 1970).

Reaggregates of tissues from the embryonicnervous system, in particular neural retina cells,were shown to result in neuroepithelial cells self-organizing into radial rosette structures reminis-cent of the embryonic neural tube (Moscona1957; Ishii 1966). Reaggregates derived fromearly avian retinal cells were not only shown toform rosettes, but these retinal stem cells werealso capable of proliferating and differentiatinginto various retinal cells types, resulting in reti-nal layers (Vollmer et al. 1984; Rothermel et al.1997). This demonstrated another level of self-organization observable in vitro as not onlycould reaggregates sort out cell types into orga-nized structures, but these cells could thenmain-tain their lineage commitment and reproducefeatures of a developmental program. Similarly,aggregates from embryonic neural precursorcells were also shown to reproduce some basicfeatures of developmental neurogenesis as, afterforming neural tube-like structures, neural pre-cursors would proliferate at the lumen and dif-ferentiate into neurons destined to an outer layer(Tomooka et al. 1993). Although cells withinthese aggregates were capable of self-renewingand generating neurons, in terms of modelingbrain development, the end organization was

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very primitive and did not recapitulate corticaltissue architecture and layering. One key factorin the failure of these 3D aggregates tomodel theintricate architecture of the developing brain islikely the developmental stage at which theywere taken, which was too late to initiate com-plex temporal and spatial patterning (Karus et al.2014), suggesting that more naive cell typesmight make a better starting point.

Embryonic Stem Cells and Embryoid Bodies

Embryogenesis can be viewed as a gradual loss ofdevelopmental capacity. Soon after fertilization,a blastocyst forms, containing an inner cell masswith PSCs, from which the whole embryo willform through progressive differentiation andspecialization into all cell types of the organism.Embryonic stem cells (ESCs) are the pluripotentcells derived from the inner cell mass of a blas-tocyst (Fig. 1). The ability to culture ESCs hasopened many doors to furthering our under-standing of developmental biology since, beyondstudying specific cells isolated from developingtissue, these PSCs actually provide a tool to watchfeatures of early development and observe dif-ferentiation and self-organization into differentidentities in vitro. When grown in suspension,the ability of ESCs to follow developmental trajec-tories becomes evident as they aggregate to formsmall spheres called embryoidbodies (EBs) (Mar-tin 1980, 1981; Evans and Kaufman 1981). EBsessentiallymimic the very early events of embryo-genesis as, following the blastocyst stage, an em-bryo will undergo gastrulation and formation ofthe three germ layers. EBs are capable of mimick-ing these very early events of embryogenesis asthey spontaneously differentiate into the threegerm layers—ectoderm, mesoderm, and endo-derm (Itskovitz-Eldor et al. 2000)—and thus pro-vide an appealing system for directing early devel-opmental processes in vitro and deriving manytissueandcell typesbypromotingspecific lineages.

Promoting Neural Fates/Neural Rosettes

The formation of organized neural tissue fromEBs was first shown using mouse ESCs (Okabeet al. 1996) and later from human ESCs (hESCs)

(Zhang et al. 2001). These studies showed thatwhen EBs are spread onto an adhesive substrateand directed toward a neural lineage in the pres-ence of bFGF, initially tightly packed epithelialcells change their morphology into elongatedneural stem cells that self-organize into 2D neu-ral rosettes, reminiscent of the embryonic neuraltube, radially organized around a lumen.

Insights into key signaling pathways under-lying early neural differentiation allowed for thedirect differentiation of hESCs and induced plu-ripotent stem cells (iPSCs) into primitive neuralstem cells in 2D monolayer culture, bypassingthe EB step by providing a neural-inducing en-vironment. In the absence of caudalizing signals,the default differentiation trajectory for plurip-otent cells in the early epiblast is toward anteriorneural fates (Levine and Brivanlou 2007). Un-less other signaling factors are provided, cellswill progress through ectodermal to neuroecto-dermal, then neuroepithelial and anterior neuralstates. Signals that induce nonneural identitiesin the early embryo include Wnts, BMPs, andNodal and neural differentiation of the ecto-derm is achieved by locally suppressing thesesignals through secretion of inhibitors such asDKK1, Noggin, chordin, and follistatin (Smithand Harland 1992; Sasai et al. 1994; Fainsodet al. 1997; Kazanskaya et al. 2000). In vitro,however, it was found that simply providing ad-herent mouse ESCs with serum-free minimalmedia (N2B27 supplements) and eliminatinginductive signals for other identities was suffi-cient to trigger significant differentiation of EScells into neural precursors, highlighting anteri-or neural as the default state (Ying et al. 2003). Inadherent human ESCs and iPSCs, it was foundthat inhibition of BMP and TGF-β/NODAL sig-naling, known as “dual SMAD inhibition” be-cause both pathways use SMADs downstream,destabilizes pluripotency and suppresses non-neural fates, resulting in a more efficient neuralinduction and the rapid differentiation of cellsinto early anterior neuroectoderm (Chamberset al. 2009). Neural induction by dual SMADinhibition resulted in primitive neural stem cellsthat, despite expressing apical markers at themembrane, lack a polarized expression of theseproteins. The addition of bFGF, however, allows

Brain Organoids

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these primitive neuroepithelial cells to organizeinto rosette structures (Li et al. 2011).

Similar to the developing neural tube, pro-liferative neuroepithelial cells of the neural ro-sette are apicobasally polarized, with apicallylocalized proteins such as ZO-1 and aPKC de-tected at the luminal surface (Shi et al. 2012).Furthermore, these radial glia-like stem cells are

pseudostratified and show evidence of interki-netic nuclear migration, with nuclei migratingapically to undergo mitosis. Later, they generatebasal TBR2+ intermediate progenitors and somemitotic cells are observed more basally alongwith basal radial glia, suggesting a rough segre-gation of progenitor zones reminiscent of theventricular zone (VZ) and subventricular zone

Neural plate Neuroectoderm

+ bFGFNeural induction

Polarized neuroepithelium(neural tube-like “buds”)

Neurulation

Neural tube

Dorsal telencephalon

Ventral telencephalonDiencephalonMidbrainHindbrain

Gastrulation

ShhFgf

Wnt/Bmp

Cerebral organoid Region-specific brain organoids

ECM components

BlastocystICM ESC/iPSC Reprogramming

Somatic cells

In vivo In vitro

Trilaminar disc

Embryoidbody

EctodermMesoderm

EndodermNotochord

Sel

f-or

gani

zatio

nPatterningmolecules

Figure 1.Acomparisonbetween invivoand invitrobraindevelopment. In vivo, thebraindevelops fromtheneuralplate that folds in on itself to form a neural tube. In vitro, aggregates of embryonic stem (ESCs) or inducedpluripotent stem cells (iPSCs) are guided toward a neuroectodermal fate and form neural tube-like buds upon theaddition of extracellular matrix (ECM) components. In vivo, the brain is patterned into different regional iden-tities bymultiplemorphogen gradients (e.g., Fgf, Bmp/Wnt, Shh) along the body axis. In vitro, cerebral organoidswill self-organize and self-pattern into various brain regional identities in a heterogeneousmanner. Alternatively,signalingmolecules can also be added to pattern organoids into specific regional identities. (ICM) inner cellmass.





(SVZ). Rosette neural stem cells are kept in aproliferative state in the presence of mitogenicbFGF, and the withdrawal of bFGF stimulatesthe onset of neurogenesis in rosette protocols.Remarkably, the temporal order of cortical neu-rogenesis is maintained in neural rosettes withthe sequential generation of neurons in an in-side-out pattern, whereby deep layer neuronsare generated first, followed by outer layer neu-rons (Gaspard et al. 2008). These layer-specificneurons were shown to be capable of formingaction potentials and excitatory synapses (Shiet al. 2012; Kirwan et al. 2015).

ORGANOID HISTORY

Moving toward 3D Brain Protocols

Although neural rosette structures exhibit a highdegree of self-organization and recapitulatemany important features of early brain develop-ment, such as the temporal generation of neu-rons, these neurons lacked the ability to spatiallyorganize into distinct neuronal layers. It wasclear that more accurate modeling of the com-plex architecture of the developing brain wouldrequire 3D growth. One importantmovement inthis direction was the discovery that EBs couldbe directed to form primitive neuroectoderm insuspension. This was first shown by culturingmouse EBs in MEDII, a media conditionedfrom a human hepatocellular carcinoma cellline, resulting in the organization of cells onthe surface into a stratified primitive neuroepi-thelium (Rathjen et al. 2002). In vitro neuraldifferentiation was further improved usingmore reproducible serum-free methods, knownas SFEB (serum-free, floating culture of EB-like aggregates). Aggregates plated on coateddishes would efficiently differentiate into telen-cephalic progenitors, and subsequent additionof patterning signaling molecules Wnt3a andShh allowed for directed differentiation intosubregional dorsal and ventral telencephalicidentities, respectively (Watanabe et al. 2005,2007). Adapting this method and adding theROCK inhibitor Y-27632 to promote survivalof dissociated human ESCs (Watanabe et al.2007) as well as allowing for a quicker reaggre-

gation of ESCs into EBs using 96-well U-bottomplates (SFEBq) (Eiraku et al. 2008), resulted inthe more effective formation of cortical tissue.During the first week of culture, a continuouspolarized neuroepithelial sheet would form onthe surface of floating EBs, which would even-tually self-organize into multiple small rosettesof neural precursors surrounding and growingaround apical lumens (Eiraku et al. 2008). Plat-ing of these aggregates onto adherent dishescoated with poly-D-lysine, laminin and fibro-nectin allowed for telencephalic differentiationand SFEBq rosettes also mimic the developmentof neural tube-like progenitor zones, generatingneurons in a temporally defined manner. Whenapplied to human ESCs, plated SFEBq aggre-gates were not entirely flattened and appeared“dome-like.” Unlike mouse-derived tissues andprevious human 2D rosette protocols, these ro-settes for the first time produced much largerand continuous apical lumens, perhaps a reflec-tion of the greatly expanded cortex of humansrelative tomice.While recapitulatingmany earlyspatial and temporal features of corticogenesis,this semi-3D culture system was, however, stillnot sufficient to observe the spatial organizationof neuronal subtypes into discrete layers.

ECM Gels and the Formation of the First 3DNeural Organoids

Pivotal to the progression of the wider organoidfield was the addition of an extracellular matrix(ECM) hydrogel, for exampleMatrigel, a solublebasement membrane-rich extract that forms a3D gel at 37°C, derived from a mouse tumorthat produces abundant ECM (Li et al. 1987;Kleinman and Martin 2005). Matrigel or moredefined collagen gels were shown in 2009 tosupport the formation of 3D intestinal orga-noids from intestinal stem cells or small explantsgrown in the gel (Ootani et al. 2009; Sato et al.2009). The combination of physical propertiessuch as rigidity of the gel along with additionalsignaling cues present in basement membraneligands of Matrigel means that it can supportorganoid formation by providing both a scaffoldand influencing various biological functionssuch as tissue polarity and cell migration

Brain Organoids




(Long and Huttner 2019). Indeed, the additionof Matrigel at various stages of 3D brain orga-noid protocols appears to have the effect ofrapidly promoting the formation of polarizedneural tube-like buds from neuroepithelial tissue.

Adding dissolved Matrigel to EBs guidedtoward retinal identity was successful in sup-porting the morphogenesis of an ordered opticcup, the first example of an entirely 3D self-or-ganizing neural tissue (Eiraku et al. 2011). Dis-solved Matrigel was subsequently successful insupporting the growth of 3D cortical forebraintissues from floating SFEBq aggregates (Nasuet al. 2012; Kadoshima et al. 2013). Alternative-ly, 3D brain architecture could also be supportedby embedding EBs in pure droplets of Matrigel(Lancaster et al. 2013). The addition of Matrigelto neural-induced EBs supports the formationof a polarized neuroepithelium with the basalside facing the external ECM-like environment,and also provides the epithelium with the sup-port to undergo subsequent morphogeneticchanges. In vivo, the polarized neuroepitheliumof the neural plate will fold in on itself to form arounded neural tube, a pseudostratified neuro-epithelium surrounding an apical fluid-filled lu-men. Although this folding of the neural platehas not yet been replicated in organoids, Matri-gel supports the generation of multiple neuraltube–like “buds” in which neuroepithelial cellsorganize in 3D around large apical lumens.When comparing cortical regions of organoidsto 2D rosettes, 3D culture systems show a higherlevel of spatial organization of proliferative pro-genitors into a VZ, SVZ, and intermediate zone,followed by neurons with primitive inside-outlayering into deep early-born neurons followedby later-born neurons that migrate more super-ficially (Qian et al. 2016). In vivo, neurons in thedeveloping cortex align radially into a denseband called the cortical plate. Despite the abilityof organoids to generate basally migrating cor-tical neurons capable of primitive layering, theaddition of dissolved Matrigel to neurogenicstages of organoid development was found tobe crucial for generating a cortical plate (Fig. 2;Kadoshima et al. 2013; Lancaster et al. 2017).This is likely a result of the fact that the ECMof the pial basement membrane, generated by

overlying nonneural mesenchyme and thusnot present in organoids, has been found tobe critical for proper neuronal migration andlocalization within the neural plate (Halfteret al. 2002).

PATTERNING

Self-Patterned/Cerebral Organoids

In the absence of external factors, differentiationinto neural fates occurs by default. Building onthis, Lancaster et al. (2013) used a relatively sim-ple media for culturing organoids, without theaddition of any signaling molecules to the cul-ture. By not directing a specific identity thatwould restrict the developmental landscape,these organoids spontaneously self-pattern andself-organize into distinct brain regions withinthe same organoid. Because of the presence ofbroad regional identities, this method wasnamed cerebral organoids (Fig. 1).

Interestingly, adjacent brain identities with-in cerebral organoids were not entirely random-ly interspersed; there were some neighboring re-gions separated by clear boundaries mimickingborders found in vivo. The early brain developsfrom a neuroepithelial sheet that is flanked bymultiple organizing centers, responsible for pat-terning the brain through the secretion of vari-ousmorphogen gradients. Cells will acquire spe-cific regional identities as a result of theirposition and the combination of various levelsof signaling factors (Fig. 1). Two important tel-encephalic signaling centers are the hem, whichis found at the midline adjacent to the choroidplexus and dorsal telencephalon and promotesdorsal identities through the secretion of BMPsand Wnts, and the antihem, which sits oppositethe hem and separates dorsal and ventral telen-cephalic regions through the expression of var-ious morphogens including Wnt antagonists.Tissue reminiscent of these organizing centerswas found in cerebral organoids with ventricularzone–like regions showing abrupt bordersbetween dorsal (TBR2+) and ventral (GSX2+)forebrain identities as would be found at theantihem, and tissue-positive for Wnt2b andBMP6 (secreted from the hem in vivo) was ob-





served adjacent to choroid plexus (TTR+ cuboi-dal morphology), which was immediately fol-lowed by the presence of dorsal telencephalictissue (TBR2+) (Renner et al. 2017). This dem-onstrated another level of in vitro self-organiza-tion; not only could cerebral organoids developinto complex brain architectures, but withoutany cues or a body axis for reference, neuroepi-thelial tissue was also capable of spontaneouslysetting up signaling centers and developing localtissue patterning.

Patterning Organoids with Small Molecules

As remarkable as it is to observe the generationof broad regional identities in vitro, it is oftendesirable to reproducibly and efficiently gener-ate organoids consisting of specific brain areasof interest. To overcome regional heterogeneityand restrict identity to a single brain region, themajority of protocols described to date alter me-dia composition to guide organoid developmenttoward a specific fate. Defined developmentalpatterning factors, many of which had previous-ly been successfully used in 2D differentiationprotocols, are used to promote specific neuralfates generating organoids of various identitiesfrom forebrain tomidbrain to hindbrain (Fig. 1).“Dual SMAD inhibition” is frequently used toprepattern cells to a neuroectodermal fatethrough treatment with various combinationsof inhibitors of the SMADpathway downstreamfrom BMP and Nodal signaling. Maintainingthese inhibitors of the SMAD pathway on grow-ing EBs results in production of organoids witha higher yield of dorsal forebrain identity (Ka-doshima et al. 2013; Paşca et al. 2015; Qian et al.2016). To promote ventral forebrain identities,initial dual SMAD inhibition may be followedby exposure to SHH agonists (Bagley et al.2017; Birey et al. 2017; Xiang et al. 2017), mim-icking the gradient observed in vivo of Shhactivity from the ventral to dorsal neural tubeinitially produced by underlying notochordthat acts as an organizing center on overlyingneural tissue.

In vivo, the neuroepithelium of the dorso-medial telencephalon develops into the hippo-campus and choroid plexus under inductive

signals from BMP andWnt. Following the orig-inal protocol for generating SFEBq cortical tis-sue followed by a transient exposure toWnt andBMP resulted in the development of hippocam-pal organoids, whereas prolonged exposure in-duced choroid plexus identity (Sakaguchi et al.2015).

In addition to optic cup organoids, orga-noids with other diencephalic identities havealso been produced in vitro. Following initialdual SMAD treatment to prepattern EBs to neu-roectodermal fates, treatment with caudalizinginsulin along with a MAPK/ERK inhibitor toprevent overcaudalization to midbrain fatesand BMP7, results in the development of tha-lamic tissue (Shiraishi et al. 2017; Xiang et al.2019). Treating EBs with SHH and Wnt3a pro-duced organoids with hypothalamic identity(Qian et al. 2016). By inducing both hypotha-lamic and nonneural oral ectoderm identitiesfrom the same EB, Ozone et al. (2016) wereable to generate hormone-producing anteriorpituitary tissue, which in vivo emerges fromthe oral ectoderm through interactions withthe overlying hypothalamic epithelium.

To generate midbrain organoids, Qian et al.built on 2D protocols for generating midbraindopaminergic neurons. In addition to earlytreatment with SMAD inhibitors, SHH, FGF8,and a Wnt activator were added to the mediaresulting in neuroepithelial cells expressing thefloorplate precursor marker, FOXA2, which atlater stages went on to produce TH+ dopami-nergic neurons (Qian et al. 2016). Furthermore,Jo et al. (2016) found that these dopaminergicneurons secrete brown-colored granules of neu-romelanin. In vivo, these neuromelanin gran-ules have been observed in primates but notmice and similarly they were found to be absentfrommouse-derivedmidbrain organoids (Fig. 2;Jo et al. 2016).

Moving more caudally, cerebellar organoidshave also been successfully generated. In addi-tion to SMAD inhibition, treatment with cau-dalizing FGF2 and insulin was sufficient toinduce differentiation into cerebellar plate neu-roepithelium that initially formed small rosettes.Subsequent treatment with FGF19, expressed atthemidbrain–hindbrain boundary and involved

Brain Organoids




in the development of dorsal hindbrain progen-itors, resulted in the remarkable transformationof cerebellar plate rosettes into large oval-shaped structures, dorsoventrally patterned ina manner reminiscent of the polarized tubestructure of early developing human cerebellum.Dorsal region-specific markers were observedon the side of the oval neuroepithelium facingthe outside of the whole aggregate, and thusexposed to higher levels of FGF19, whereas ven-tral markers were expressed on the inner side

facing the center of the organoid (Mugurumaet al. 2015).

Tradeoff between Heterogeneity versusUniformity of Organoids

Something that should be consideredmore care-fully within the organoid field is the tradeoffbetween heterogeneity versus uniformity of thetissue generated. Although the addition of smallmolecules and patterning signals to the culture

VZ

SVZ/IZ

CP

Subcortical projection neurons

Radial glia

Dividing radial glia and intermediate progenitors

Intermediate progenitorsand immature callosal neurons

Maturing neurons

Interneurons

Figure 2. Brain organoids produce various neural cell types that mimic the architecture of the developing brain.(Top row) An example of a tSNE plot generated from scRNA-seq data to visualize various cell identities present inan organoid. (Bottom left) A cartoon image color-coded to represent the various cell types detected by scRNA-seqand their location within the ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), andcortical plate (CP) of the developing cortical organoid. (Bottom right) A representative image of an organoidcontaining these architectural and identity divisions stained for DAPI (blue), CTIP2 (magenta), and DCX(yellow).





directs organoids to desired and reproduciblebrain identities, several of these small moleculesmay play roles on developing cells beyondsimple patterning and generate desired tissueidentities in a manner that does not reflectfetal organogenesis. The addition of excessiveexternal signals runs the risk of flattening thedevelopmental landscape and the intrinsic de-velopmental program of organoids, which couldpotentially reduce organoid complexity or maskimportant features of development that mightbe relevant in the context of a disease. Con-versely, organoids that are generated in the ab-sence of patterning molecules, spontaneouslyself-organizing and self-patterning, suffer fromincreased “batch-effect” and inconsistent gener-ation of desired tissues, meaning their heteroge-neity could potentially conceal subtle diseasephenotypes.

Some methods have been able to producemore reproducible brain identities without theaddition of external signaling molecules. Grow-ing EBs on floating scaffolds made of fiber mi-crofilaments results in elongated EBs with alarger surface area exposed to neural inductionmedia, resulting in increased neuroectodermformation and subsequently increased corticaldifferentiation (Lancaster et al. 2017). Anotherrecent strategy, discussed later in this review, isto generate intrinsic signaling gradients withinthe organoid, resulting in spatially encoded or-ganoids (Cederquist et al. 2019).

DIFFERENTIATION/MATURATIONOF NEURAL CELL TYPES

3D Architecture and Cell Diversityin Organoids

Brain organoids have the ability to mimic thearchitecture of the developing brain and formvarious neural cell types in a spatiotemporalmanner. To determine whether these simi-larities with in vivo brain development oc-cur through the reactivation of developmentalgene expression programs, several studies havecompared organoid gene expression to primaryfetal tissue using microarrays (Paşca et al. 2015),RNA-seq (Mariani et al. 2015; Luo et al. 2016;

Qian et al. 2016), and single-cell RNA-seq(Camp et al. 2015; Quadrato et al. 2017; Sloanet al. 2017; Pollen et al. 2019). These gene ex-pression analyses have shown that organoid pro-tocols replicate early brain development partic-ularly well, generating a wide diversity of cellsthat share transcriptomic profiles with the earlyfetal neocortex (Fig. 2). We will focus on howorganoids with specifically cortical identitymimic early brain architecture and gene expres-sion programs.

Organoids containmultiple neural tube–likeregions that exhibit VZ-like regions populatedby proliferative apical progenitors that expresstypical radial glial marker genes (SOX2, NES-TIN, PAX6) and make up the majority of cellsin the organoid prior to neurogenesis. Theseneural stem cells are pseudostratified, displaythe typical elongated morphology of radial glialcells and undergo mitosis at the apical surface,via interkinetic nuclear migration (Bershteynet al. 2017). After the onset of neurogenesis,populations of intermediate progenitor cells(TBR2+) begin to appear in an SVZ-like regionbasal to the VZ-like region (Fig. 2). Cells ex-pressing basal radial glial markers (HOPX,PTPRZ1) have also been observed (Qian et al.2016; Li et al. 2017), which are present exclu-sively in the outer SVZ (oSVZ) in vivo, anadditional germinal zone thought to be absentin rodent neocortices (LaMonica et al. 2012).However, although cells expressing oSVZmarkers are being generated in organoid proto-cols, an oSVZ-like region has yet to be observedreliably.

In vivo, neurons generated from VZ andSVZ regions migrate basally along radial glialprocesses to the cortical plate and form a six-layered structure, each composed of neuronswithdifferent properties.Neurogenesis occurs ina spatiotemporal manner with deep-layer neu-rons generated earlier followed by later genera-tions of upper-layer neurons. Although orga-noid protocols to date only show a restrictedspatial layering of neurons, they do generatethe various classes of neurons following the tem-poral trajectory of initially making deep-layer(CTIP2+), followed by upper-layer (SATB2+)neurons (Renner et al. 2017).

Brain Organoids




Neuronal Activity and Maturation

Several studies have now investigated the phys-iological properties of neurons generated in or-ganoid protocols and shown their functionalmaturation over time. Over time, neurons gen-erated in brain organoid protocols begin tofunctionally mature and exhibit spontaneousfiring, shown by Ca2+ surges (Eiraku et al.2008; Lancaster et al. 2013) and electrophysio-logical recordings (Qian et al. 2016), with firingfrequency sensitive to the application of gluta-mate and glutamate receptor antagonists, indi-cating the presence of glutamatergic neurons(Lancaster et al. 2013). Intrinsic glutamate re-lease and levels within cortical organoids havealso been measured using enzyme-modified mi-croelectrodes (Nasr et al. 2018).

GABA is known as the major inhibitoryneurotransmitter in the brain; however, duringdevelopment, it exerts a depolarizing effect onimmature neurons (Leonzino et al. 2016). Onekey feature of neuronal maturation is the switchfrom a depolarizing to a hyperpolarizing re-sponse toGABA,mediated in vivo by decreasingintracellular Cl− concentrations as neurons ma-ture because of the down-regulation and up-reg-ulation of NKCC1 and KCC2 membrane trans-port proteins, respectively. Qian et al. (2016)found that organoids also appear to replicatethis neuronal maturation by showing increasedexpression of KCC2 and a reduction in neuronsresponsive to GABA-induced depolarization asorganoids mature.

Circuit Formation

Single labelingof neurons generatedbyorganoidsdemonstrates the ability of these neurons to gen-erate complex morphologies and synapse ontoeach other, with dendritic spines in close prox-imity to presynaptic terminals (Qian et al. 2016).

Serial electron microscopy of older orga-noids (Quadrato et al. 2017) further showedthe ability of neurons to form synapses, reveal-ing a density of synapses within the range ofdensities observed in human fetal brains (Hut-tenlocher and Dabholkar 1997). Also observedwere single dendrites making synapses withmultiple axons, suggesting the formation of

complex networks. In addition to confirmingspontaneous firing activity in individual neu-rons, Quadrato et al. measured population firingactivity and found some organoids that dis-played clear bursts of coordinated activity, indi-cating that organoids are capable of generatingneuronal networks that form self-organized fir-ing patterns.

Additionally, they found that organoids cul-tured long-term generated a population of light-sensing retinal cells, photoreceptors. These pho-toreceptors were functional as a subpopulationof neurons showed attenuation in firing rates inresponse to light exposure. The capability of or-ganoids to respond to physiological sensory in-put suggests that organoids may be used in thefuture to study how circuit formation and net-work activity is regulated by sensory stimuli.

Recently, organoids cultured at the air–liq-uid interface (ALI-CO) (Giandomenico et al.2019) were able to show a great improvementin survival and maturation of neurons that werecapable of forming long, dense bundles of axonswith specific orientations, reminiscent of nervetracts. In vivo, deep-layer neurons of the cortexproject axons subcortically to other regions ofthe brain, whereas upper-layer neurons projectintracortically and form callosal tracts. Twomain morphologies of axon tracts reminiscentof callosal and subcortical projections were ob-served in ALI-COs: ones that projected internal-ly within the organoid and others that projectedoutwardly and away from the organoid altogeth-er, formed by neurons with a primarily upper-layer (CUX2+) and deep-layer (CTIP2+) identi-ty, respectively. Intracortical-like tracts oftenmade sharp turns along their path and stainingfor known developmental guidance cues, suchas WNT5a, revealed its presence surroundingaxon tracts, demonstrating the ability of orga-noids to self-organize axonal pathways.Measur-ing neuronal activity across the ALI-COsshowed correlated firing activity between re-gions at various distances, demonstrating thatfunctional intracortical-like connections pro-duce short-, medium-, and long-range connec-tivity within the organoid. To test the functionaloutput of escaping subcortical-like tracts, ALI-COs were cocultured with dissected embryonic





mouse spinal cord still attached to peripheralnerves and perispinal muscles. Escaping axontracts were able to innervate the spinal cordand trigger coordinated contractions of themuscles. Furthermore, muscle contractionscould be controlled by extracellular stimulationof axon tracts and the latency of response be-tween stimulation and contraction was similarto latencies recorded in developing human de-scending motor pathways.

The experiments performed by Quadratoet al. and Giandomenico et al. show functionalsensory input and motor output of organoids,meaning that organoids could be used to studyneural connectivity, potentially between differ-ent brain region-specific organoids assembledtogether, as discussed further below.

Other Neural Cell Types

Neurons are not the only cell types necessary tomake a brain. Glial cells are essential for brainfunctioning in late development and the adultbrain, making up at least half of human braincells. The two main glial cells, astrocytes andoligodendrocytes, are essential for supportingsynaptic function and rapid transmission ofnerve impulses, respectively. These cell typesarise in late embryonic development, beginningin the middle of the second trimester and con-tinuing after birth, when the same neural pro-genitors that were undergoing neurogenesisswitch from a primarily neurogenic to gliogenicfate (Jiang and Nardelli 2016).

Immunostaining of organoids culturedlong-term reveals GFAP-expressing cells withthe typical stellar-like morphologies of astro-cytes (Paşca et al. 2015; Renner et al. 2017). As-trocyte-like cells isolated from cortical orga-noids were capable of recapitulating severalkey functions of astrocytes, with several in vitroassays showing their ability to uptake glutamate,induce synapse formation, phagocytose synap-tosomes, and modulate neuronal calcium sig-naling (Sloan et al. 2017). As astrocytes maturefrom fetal to postnatal states, they undergo var-ious transcriptomic changes, including changessuch as increasedmorphological complexity, re-duced proliferative capacity, and reduced func-

tional ability to phagocytose synaptosomes.Sloan et al. showed, by purifying astrocytesfrom organoids in a time series ranging from100 to 590 days of culture and performing sin-gle-cell RNA sequencing and various functionalassays, that these features were replicated in vitrowith earlier time points correlating with fetalastrocytes and showing increased proliferativecapacities and ability to phagocytose synapto-somes. Later time points (around 400 days ofculture) correlated with mature astrocytes andshowed more complex morphologies and in-creased ability to augment calcium signaling inneurons.

Single-cell RNA sequencing has detected asmall proportion of cells that express oligoden-drocyte precursor cell (OPC) markers afterlong-term culture (Quadrato et al. 2017; Gian-domenico et al. 2019). This makes sense as theformation of myelin sheaths around axons bymature oligodendrocytes only begins aroundbirth in vivo. To study oligodendrogenesis invitro, Madhavan et al. (2018) exposed corticalspheroids to known oligodendrocyte lineagegrowth factors and hormones to promote OPCproliferation and further maturation into myeli-nating oligodendrocytes that were capable offorming myelin sheaths wrapped around axonswithin the neurosphere. Kim et al. (2019) alsoused a protocol to accelerate oligodendrocytematuration and demonstrated differences inthe timing of oligodendrogenesis and matura-tion when the protocol was applied to ventralversus dorsal patterned forebrain organoids.These differences mimicked in vivo observa-tions in mice, where ventral neural precursorsundergo a wave of oligodendrogenesis prior tothe dorsal wave (Kessaris et al. 2006). Prolongedculture past initial oligodendrocyte maturationin both these studies, however, did not lead tocontinued structural organization of myelinsheaths such as myelin compaction and forma-tion of nodes of Ranvier, potentially caused bythe lack of mature neurons and network activity,necessary to signal and drive myelination (Al-meida and Lyons 2017), or caused by the lack ofnutrients exposed to oligodendrocytes in these3Dmodels. Perhaps promoting oligodendrocytelineages in slice cultured organoids grown at the

Brain Organoids




air–liquid interfacewould allow for further mat-uration of myelin sheaths in a system with in-creased nutrient exposure and cell survivability,in addition to more mature network formation.

Microglia are the resident innate immunecells of the brain that have also been shown tohave roles in fine-tuning neuronal circuits andregulating neural progenitor cell number (Cun-ningham et al. 2013). Microglia arise from ex-traembryonic and mesoderm lineages and aretherefore usually absent from brain organoidprotocols that primarily or exclusively containneuroectodermal lineage. However, a complete-ly undirected cerebral organoid protocol, whichproduces less consistent brain regions by notmanipulating molecular patterning pathways,does produce a proportion of mesoderm pro-genitor cells that develop into microglia-likecells (Ormel et al. 2018). Another study foundthat microglia-like cells differentiated fromiPSCs could enter and integrate in brain orga-noids simply by adding them to the culturingmedia (Abud et al. 2017). Furthermore, uponinjury to the organoid these microglia-like cellschanged theirmorphology to resemble activatedmicroglia found in diseased or injured brains.

IMPROVEMENTS/ENGINEERING

Cell Survival and Maturation

In early brain development, neural tube forma-tion, proliferation, and neurogenesis occur inthe absence of vascularization. Eventually, asthe tissue grows and thickens, vascularizationbecomes essential. In vivo organs consist ofvast branched vascular networks, and cells arewithin a few hundred micrometers of a capillaryallowing for diffusion. Vascular networks, beingof nonneural origin, are not formed in orga-noids, impairing cell survivability and architec-ture in older, larger organoids. Culturing orga-noids in agitation on a shaker or in spinningbioreactors (Lancaster et al. 2013; Qian et al.2016; Sutcliffe and Lancaster 2017) can increasetheir diffusion limit; however, organoids will usu-ally reach a maximum size of ∼5–6 mm andconsist of a necrotic core because of the lack ofvascularization, limiting the supply of oxygen

and nutrients to the core of the organoid. Asdescribed above, slice cultures of organoidscan overcome issues involving lack of nutrientsupply, massively improving cell survival andmaturation (Fig. 3; Giandomenico et al. 2019).

An alternative approach to address this issueof vascularization was tested by Mansour et al.(2018) through transplanting brain organoidsinto the adult mouse brain. The host vascularsystem was capable of invading and nourishingthe organoid with active blood flow. This result-ed in a replacement of the necrotic core withhealthy mature neurons (Fig. 3). Furthermore,these organoids also integrated host microglia.This transplantation approach enables vascular-izationmore similar towhat would occur in vivoand opens avenues to studying the interaction oforganoids with microglia and immune cells;however, it does involve a surgical procedureand damage to the host, and the growth of theorganoid is limited by the size of the cavity in themouse cortex. It will be interesting to accom-plish in vitro vascularization in a manner thatdoes not disrupt the self-organizing architectureof brain organoids. This may be achieved bycoculturing organoids with endothelial pre-cursors. Perhaps the brain organoid fieldmay borrow vascularization approaches fromother organoid systems, for example, the vascu-larized kidney organoids developed by Homanet al. (2019), by culturing them under flowon microfluidic chips with endothelial cells,which resulted in the generation of vascular net-works.

Assembled Organoids

The fact that we are capable of directing andgenerating a multitude of brain regions in vitrobrings the exciting prospect of combining themto form more complex structures to studyconnectivity and interactions between differentbrain regions.

In cerebral organoids, it was shown that dif-ferent brain regions within the same organoidmight be interacting in a manner similar to invivo as GABAergic interneurons originatingfrom ventral forebrain were frequently detectedin dorsal forebrain regions of organoids con-





taining both tissues. This suggests a ventral-to-dorsal migration of these neurons, similar to thedeveloping forebrain where interneurons are in-tegrated into cortical circuits (Lancaster et al.2013). Building on these observations, severalstudies have fused region-specific dorsal andventral forebrain organoids to each other tostudy the saltatory migration of interneuronsfrom the ventral organoid into the dorsal orga-noid (Fig. 3; Bagley et al. 2017; Birey et al. 2017;Xiang et al. 2017). Furthermore, it was shownthat interneurons that hadmigrated altered theirmorphology, made synapses with dorsal gluta-matergic neurons, and formed electrically activefunctional cortical microcircuits (Birey et al.2017). These fusions are achieved by close posi-tioning of the two organoids in a microwell ortube for a few days (Birey et al. 2017; Xiang et al.2017) or by embedding two EBs in the samedroplet ofMatrigel after patterned neural induc-tion (Bagley et al. 2017).

Cortical organoids have also been fused tothalamic organoids (Xiang et al. 2019). Thesefused organoids form reciprocal thalamo-cortical and corticothalamic projections, withthalamocortical projections found to innervateregions of cortical organoids containing differ-entiated neurons. Electrophysiological record-ing of thalamic neurons revealed an increasedfiring frequency of neurons in fused organoidsversus thalamic organoids alone.

Assembling Signaling Centers

Recently, Cederquist et al. (2019) developed asystem to induce a morphogen gradient fromone pole of a developing forebrain organoid,mimicking developmental organizing centers,and resulting in organoids with spatially encod-ed forebrain subdivisions similar to fetal pat-terning (Fig. 3). First, a small aggregate ofESCs expressing inducible SHH is allowed to

Necroticcore

Vascularization Slice culture

Engineering brainorganoids

Patterning

Assembled organoids

Assembled signaling center

iSHH PSCs

Cell survival and maturation

WT PSCs + Doxycycline

Ventral forebrain

Dorsal forebrainFused

Transplant intomouse cortex

Axon tracts/long-rangeconnectivity

Organoid grownin 3D agitation

Interneurons migratingdorsally

Figure 3.Methods in use for engineering brain organoids. (Left panel) Patterned organoids can bemade by fusingorganoids of different regional identities to one another or by generating organoids containing a region oftransgenic cells expressing an inducible signaling molecule that produces spatially patterned organoids. (Rightpanel) Organoids grown in vitro in 3D suffer from a necrotic core that impairs their maturation as a result ofdiffusion limits and a lack of vasculature. Transplanting organoids into the adult mouse cortex allows healthyneural tissue to form in the core of the organoid. Growing organoids as slice cultures improves neuronal survivaland maturation and allows for the formation of long axon tracts connecting different regions or tissues. (WT)wild type, (PSC) pluripotent stem cell.

Brain Organoids




form, and then a larger amount of ESCs is seed-ed on top, resulting in an EBwith an aggregate oforganizer cells at one pole of the developingorganoid. This asymmetric SHH cue mimickedventral-high and dorsal-low SHH gradients, re-sulting in the self-organization of a dorsoventralforebrain axis with ventral identities appearingnearer to and dorsal identities further from theorganizer cells.

CONCLUSION AND FUTURE PROSPECTS

The ability to generate various ordered brainregions with remarkably little external inputdemonstrates the outstanding capacity of stemcells to carry out an intrinsic program involvingmultiple stages of complex tissue movements,differentiation, and cell interactions. Despiteundergoing development in a dish, outside oftheir natural environment, brain organoid pro-tocols are capable of recapitulating many fea-tures of early brain development in a spatiotem-poral manner, both in terms of gene expressionand cytoarchitecture.

The past few years have witnessed majorbreakthroughs in brain organoid developmentand tissue engineering, allowing for the genera-tion of a multitude of regional identities and themodeling of later stages of brain developmentsuch as neuronal maturation, connectivity, andthe formation of functional circuits. The mod-eling of these later, more complex stages of braindevelopment still has various limitations that arebeginning to be addressed, as it involves the in-terplay of several cell types of nonneural originsand interactions between various brain regionswith a predictable topographic organizationalong the developing body axis. We predictthat the coming years will attempt to more re-producibly model these later stages of brain de-velopment by optimizing culture conditions toallow for in vitro vascularization and the incor-poration of microglia and immune cells in brainorganoids. We also predict that additional andmore complex types of brain circuits will bemodeled by assembling multiple organoids ofspecific regional identities to one another in adefined orientation. The fact that organoids canself-organize circuits that respond to physiolog-

ical stimuli and are capable of innervatingmouse spinal cords to evoke functional muscu-lar output, brings the exciting prospect of gen-erating full circuits where a sensory input wouldtrigger a physical output. We think it is impor-tant to exercise a certain level of caution whenattempting to generate complex brain structuresmore reproducibly, as overly engineering thedevelopment of brain tissue in vitro runs therisk of skipping over key steps in tissue morpho-genesis and not faithfully recapitulating the de-velopmental program of brain formation.

ACKNOWLEDGMENTS

We thank members of the Lancaster laboratoryfor helpful insight. We also apologize to thosecolleagues whose work could not be coveredbecause of space constraints. Work in the Lan-caster laboratory is supported by the MedicalResearch Council (MC_UP_1201/9) and theEuropean Research Council (ERC STG 757710).

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Brain Organoids




published online November 25, 2019Cold Spring Harb Perspect Biol Silvia Benito-Kwiecinski and Madeline A. Lancaster Brain Organoids: Human Neurodevelopment in a Dish

Subject Collection Stem Cells: From Biological Principles to Regenerative Medicine

A Stem Cell Approach to Cure Type 1 DiabetesAharon Helman and Douglas A. Melton Diversity of Tissue Stem Cell Niche Systems

Mouse Spermatogenesis Reflects the Unity and

Shosei Yoshida

Alveolar Stem Cells In VivoNiche Cells and Signals that Regulate Lung

Tushar J. DesaiNicholas H. Juul, Courtney A. Stockman and

Divergence of Cell Types across EpitheliaA Synthesis Concerning Conservation and

Rood, et al.Daniel T. Montoro, Adam L. Haber, Jennifer E.

the Context of Aging and Cancer InitiationStem Cell DNA Damage and Genome Mutation in

Lara Al zouabi and Allison J. Bardinthrough Lineage Tracing and In Vivo ImagingDiscovering New Progenitor Cell Populations

Rudra Nayan Das and Karina Yaniv

Live Cell MicroscopyCapturing Stem Cell Behavior Using Intravital and

Scheele, et al.Arianna Fumagalli, Lotte Bruens, Colinda L.G.J.

Manipulation of Stem CellsNext-Generation Biomaterials for Culture and

DeForestKoichiro Uto, Christopher K. Arakawa and Cole A.

DishBrain Organoids: Human Neurodevelopment in a

Silvia Benito-Kwiecinski and Madeline A. LancasterDegenerationDevelopment of Stem Cell Therapies for Retinal

Emma L. West, Joana Ribeiro and Robin R. Ali

Multimodal Single-Cell AnalysisAdvancing Stem Cell Research through

Iwo Kucinski and Berthold GottgensPlasticity between Liver and PancreasDirect Lineage Reprogramming: Harnessing Cell

SpagnoliSilvia Ruzittu, David Willnow and Francesca M.

Tracing the Dynamics of Stem Cell FateLemonia Chatzeli and Benjamin D. Simons Pluripotent Stem Cells

Modeling Brain Disorders Using Induced

Linker, et al.Krishna C. Vadodaria, Jeffrey R. Jones, Sara

GenodermatosesToward Combined Cell and Gene Therapy for

Secone Seconetti, et al.Laura De Rosa, Maria Carmela Latella, Alessia

Lineages In Vivo and In VitroSpecification of the First Mammalian Cell

Melanie D. White and Nicolas Plachta

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved


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