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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-
Editors: Cristina Lo Celso, Kristy Red-Horse, and Fiona M.
WattAdditional Perspectives on Stem Cells: From Biological
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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.
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(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
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(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-
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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
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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).
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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
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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
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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
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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-
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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
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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).
REFERENCES
Abud EM, Ramirez RN, Martinez ES, Healy LM, NguyenCHH, Newman
SA, Yeromin AV, Scarfone VM, MarshSE, et al. 2017. iPSC-derived
humanmicroglia-like cells tostudy neurological diseases. Neuron 94:
278–293.e9.doi:10.1016/j.neuron.2017.03.042
Almeida RG, LyonsDA. 2017.Onmyelinated axon plasticityand
neuronal circuit formation and function. J Neurosci37: 10023–10034.
doi:10.1523/jneurosci.3185-16.2017
Bagley JA, Reumann D, Bian S, Lévi-Strauss J, Knoblich JA.2017.
Fused cerebral organoids model interactions be-tween brain regions.
Nat Methods 18: 170–751.
Bershteyn M, Nowakowski TJ, Pollen AA, Di Lullo E,
NeneA,Wynshaw-Boris A, KriegsteinAR. 2017.Human iPSC-derived
cerebral organoids model cellular features of lis-sencephaly and
reveal prolonged mitosis of outer radialglia. Cell Stem Cell 20:
435–449.e4. doi:10.1016/j.stem.2016.12.007
Birey F, Andersen J, Makinson CD, Islam S, Wei W, HuberN, Fan
HC,Metzler KRC, Panagiotakos G, ThomN, et al.2017. Assembly of
functionally integrated human fore-brain spheroids. Nature 545:
54–59. doi:10.1038/nature22330
Camp JG, Badsha F, Florio M, Kanton S, Gerber T,
Wilsch-Bräuninger M, Lewitus E, Sykes A, Hevers W, LancasterM, et
al. 2015. Human cerebral organoids recapitulategene expression
programs of fetal neocortex develop-ment. Proc Natl Acad Sci 112:
15672–15677.
Cederquist GY, Asciolla JJ, Tchieu J, Walsh RM, CornacchiaD,
Resh MD, Studer L. 2019. Specification of positional
S. Benito-Kwiecinski and M.A. Lancaster
14 Advanced Online Article. Cite this article as Cold Spring
Harb Perspect Biol doi: 10.1101/cshperspect.a035709
on July 7, 2021 - Published by Cold Spring Harbor Laboratory
Press http://cshperspectives.cshlp.org/Downloaded from
http://cshperspectives.cshlp.org/
-
identity in forebrain organoids. Nat Biotechnol 37: 436–444.
doi:10.1038/s41587-019-0085-3
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M,Sadelain M,
Studer L. 2009. Highly efficient neural con-version of human ES and
iPS cells by dual inhibition ofSMAD signaling. Nat Biotechnol 27:
275–280. doi:10.1038/nbt.1529
Child C. 1928. Axial development in aggregates of dissoci-ated
cells from Corymorpha palma. Physiol Zool 1: 419–461.
doi:10.1086/physzool.1.3.30151056
Clevers H. 2016. Modeling development and disease withorganoids.
Cell 165: 1586–1597. doi:10.1016/j.cell.2016.05.082
Cunningham CL, Martínez-Cerdeño V, Noctor SC. 2013.Microglia
regulate the number of neural precursor cellsin the developing
cerebral cortex. J Neurosci 33: 4216–4233.
doi:10.1523/jneurosci.3441-12.2013
DeLong GR. 1970. Histogenesis of fetal mouse isocortex
andhippocampus in reaggregating cell cultures. Dev Biol 22:563–583.
doi:10.1016/0012-1606(70)90169-7
Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M,Yonemura S,
Matsumura M, Wataya T, Nishiyama A,Muguruma K, Sasai Y. 2008.
Self-organized formationof polarized cortical tissues from ESCs and
its active ma-nipulation by extrinsic signals. Cell Stem Cell 3:
519–532.doi:10.1016/j.stem.2008.09.002
Eiraku M, Takata N, Ishibashi H, Kawada M, Sakakura E,Okuda S,
Sekiguchi K, Adachi T, Sasai Y. 2011. Self-orga-nizing optic-cup
morphogenesis in three-dimensionalculture. Nature 472: 51–56.
doi:10.1038/nature09941
Evans MJ, Kaufman M. 1981. Establishment in culture
ofpluripotential stem cells from mouse embryos. Nature292:
151–156.
Fainsod A, Deißler K, Yelin R, Marom K, Epstein M, Pil-lemer G,
Steinbeisser H, Blum M. 1997. The dorsalizingand neural inducing
gene follistatin is an antagonist ofBMP-4. Mech Dev 63: 39–50.
doi:10.1016/S0925-4773(97)00673-4
Gaspard N, Bouschet T, Hourez R, Dimidschstein J, NaeijeG, van
den Ameele J, Espuny-Camacho I, Herpoel A,Passante L, Schiffmann
SN, et al. 2008. An intrinsicmechanism of corticogenesis from
embryonic stem cells.Nature 455: 351–357.
doi:10.1038/nature07287
Giandomenico SL, Lancaster MA. 2017. Probing humanbrain
evolution and development in organoids. CurrOpin Cell Biol 44:
36–43. doi:10.1016/j.ceb.2017.01.001
Giandomenico SL, Mierau SB, Gibbons GM, Wenger LMD,Masullo L,
Sit T, Sutcliffe M, Boulnger J, Tripodi M, De-rivery E, et al.
2019. Cerebral organoids at the air-liquidinterface generate
diverse nerve tracts with functionaloutput. Nat Neurosci 22:
669–679. doi:10.1038/s41593-019-0350-2
Halfter W, Dong S, Yip YP, Willem M, Mayer U. 2002. Acritical
function of the pial basement membrane in cor-tical histogenesis. J
Neurosci 22: 6029–6040. doi:10.1523/jneurosci.22-14-06029.2002
HomanKA,GuptaN,Kroll KT, KoleskyDB, Skylar-ScottM,Miyoshi
T,MauD, ValeriusMT, Ferrante T, Bonvenre JV,et al. 2019.
Flow-enhanced vascularization and matura-tion of kidney organoids
in vitro. Nat Methods 16: 255–262.
doi:10.1038/s41592-019-0325-y
Huttenlocher PR, Dabholkar AS. 1997. Regional differencesin
synaptogenesis in human cerebral cortex. J CompNeurol 387: 167–178.
doi:10.1002/(SICI)1096-9861(19971020)387:23.0.CO;2-Z
Ishii K. 1966. Reconstruction of dissociated chick brain cellsin
rotation-mediated culture. Cytologia 31: 89–98.
doi:10.1508/cytologia.31.89
Itskovitz-Eldor J, SchuldinerM, Karsenti D, EdenA, YanukaO, Amit
M, Soreq H, Benvenisty N. 2000. Differentiationof human embryonic
stem cells into embryoid bodiescomprising the three embryonic germ
layers. Mol Med6: 88–95. doi:10.1007/BF03401776
Jiang X, Nardelli J. 2016. Cellular and molecular introduc-tion
to brain development.Neurobiol Dis 92: 3–17.
doi:10.1016/j.nbd.2015.07.007
Jo J, Xiao Y, SunAX, Cukuroglu E, TranHD,Göke J, Tan ZY,Saw TY,
Tan CP, Lokman H, et al. 2016. Midbrain-likeorganoids from human
pluripotent stem cells containfunctional dopaminergic and
neuromelanin-producingneurons. Cell Stem Cell 19: 248–257.
doi:10.1016/j.stem.2016.07.005
Kadoshima T, Sakaguchi H, Nakano T, Soen M, Ando S,Eiraku M,
Sasai Y. 2013. Self-organization of axial polar-ity, inside-out
layer pattern, and species-specific progen-itor dynamics in human
ES cell-derived neocortex. ProcNatl Acad Sci 110: 20284–20289.
doi:10.1073/pnas.1315710110
KarusM, Blaess S, Brüstle O. 2014. Self-organization of neu-ral
tissue architectures from pluripotent stem cells.J Comp Neurol 522:
2831–2844. doi:10.1002/cne.23608
Kazanskaya O, Glinka A, Niehrs C. 2000. The role of Xen-opus
dickkopf1 in prechordal plate specification and neu-ral patterning.
Development 127: 4981–4992.
Kessaris N, Fogarty M, Iannarelli P, Grist M, Wegner
M,RichardsonWD. 2006. Competing waves of oligodendro-cytes in the
forebrain and postnatal elimination of anembryonic lineage. Nat
Neurosci 9: 173–179. doi:10.1038/nn1620
KimH, XuR, Padmashri R, DunaevskyA, Liu Y, Dreyfus CF,Jiang P.
2019. Pluripotent stem cell-derived cerebral or-ganoids reveal
human oligodendrogenesis with dorsaland ventral origins. Stem Cell
Rep 12: 890–905. doi:10.1016/j.stemcr.2019.04.011
Kirwan P, Turner-Bridger B, Peter M, Momoh A, Arambe-pola D,
Robinson HPC, Livesey FJ. 2015. Developmentand function of human
cerebral cortex neural networksfrom pluripotent stem cells in
vitro. Development 142:3178–3187. doi:10.1242/dev.123851
Kleinman HK, Martin GR. 2005. Matrigel: basement mem-brane
matrix with biological activity. Semin Cancer Biol15: 378–386.
doi:10.1016/j.semcancer.2005.05.004
LaMonica BE, Lui JH, Wang X, Kriegstein AR. 2012.
OSVZprogenitors in the human cortex: an updated perspectiveon
neurodevelopmental disease. Curr Opin Neurobiol 22:747–753.
doi:10.1016/j.conb.2012.03.006
Lancaster MA, Renner M, Martin CA, Wenzel D, BicknellLS, Hurles
ME, Homfray T, Penninger JM, Jackson AP,Knoblich JA. 2013. Cerebral
organoids model humanbrain development and microcephaly. Nature
501: 373–379. doi:10.1038/nature12517
Lancaster MA, Corsini NS, Wolfinger S, Gustafson EH,Phillips AW,
Burkard TR, Otani T, Livesey FJ, Knoblich
Brain Organoids
Advanced Online Article. Cite this article as Cold Spring Harb
Perspect Biol doi: 10.1101/cshperspect.a035709 15
on July 7, 2021 - Published by Cold Spring Harbor Laboratory
Press http://cshperspectives.cshlp.org/Downloaded from
http://cshperspectives.cshlp.org/
-
JA. 2017. Guided self-organization and cortical plate for-mation
in human brain organoids. Nat Biotechnol 11:546–666.
LeonzinoM, Busnelli M, Antonucci F, Verderio C,MazzantiM, Chini
B. 2016. The timing of the excitatory-to-inhib-itory GABA switch is
regulated by the oxytocin receptorvia KCC2. Cell Rep 15: 96–103.
doi:10.1016/j.celrep.2016.03.013
Levine AJ, Brivanlou AH. 2007. Proposal of a model ofmammalian
neural induction. Dev Biol 308:
247–256.doi:10.1016/j.ydbio.2007.05.036
Li ML, Aggeler J, Farson DA, Hatier C, Hassell J, Bissell
MJ.1987. Influence of a reconstituted basement membraneand its
components on casein gene expression and secre-tion in mouse
mammary epithelial cells. Proc Natl AcadSci 84: 136–140.
doi:10.1073/pnas.84.1.136
Li W, Sun W, Zhang Y, Wei W, Ambasudhan R, Xia P,Talantova M,
Lin T, Kim J, Wang X, et al. 2011. Rapidinduction and long-term
self-renewal of primitive neuralprecursors from human embryonic
stem cells by smallmolecule inhibitors. Proc Natl Acad Sci 108:
8299–8304.doi:10.1073/pnas.1014041108
Li Y, Muffat J, Omer A, Bosch I, Lancaster MA, Sur M,Gehrke L,
Knoblich JA, Jaenisch R. 2017. Induction ofexpansion and folding in
human cerebral organoids.Cell Stem Cell 20: 385–396.e3.
doi:10.1016/j.stem.2016.11.017
Long KR, Huttner WB. 2019. How the extracellular matrixshapes
neural development. Open Biol 9: 180216.
doi:10.1098/rsob.180216
Luo C, Lancaster MA, Castanon R, Nery JR, Knoblich JA,Ecker JR.
2016. Cerebral organoids recapitulate epige-nomic signatures of the
human fetal brain. Cell Rep 17:3369–3384.
doi:10.1016/j.celrep.2016.12.001
Madhavan M, Nevin ZS, Shick HE, Garrison E, Clarkson-Paredes C,
Karl M, Clayton BLL, Factor DC, Allan KC,Barbar L, et al. 2018.
Induction of myelinating oligoden-drocytes in human cortical
spheroids. Nat Methods 15:700–706.
doi:10.1038/s41592-018-0081-4
Mansour AA, Gonçalves JT, Bloyd CW, Li H, Fernandes S,Quang D,
Johnston S, Parylak SL, Jin X, Gage FH. 2018.An in vivo model of
functional and vascularized humanbrain organoids. Nat Biotechnol
36: 432–441. doi:10.1038/nbt.4127
Mariani J, Coppola G, Zhang P, Abyzov A, Provini L, To-masini L,
Amenduni M, Szekely A, Palejev D, Wilson M,et al. 2015.
FOXG1-dependent dysregulation of GABA/glutamate neuron
differentiation in autism spectrum dis-orders. Cell 162: 375–390.
doi:10.1016/j.cell.2015.06.034
Martin GR. 1980. Teratocarcinomas and mammalian em-bryogenesis.
Science 209: 768–776. doi:10.1126/science.6250214
Martin GR. 1981. Isolation of a pluripotent cell line
fromearlymouse embryos cultured in medium conditioned
byteratocarcinoma stem cells. Proc Natl Acad Sci 78: 7634–7638.
doi:10.1073/pnas.78.12.7634
Moscona A. 1957. Formation of lentoids by dissociated ret-inal
cells of the chick embryo. Science 125:
598–599.doi:10.1126/science.125.3248.598
Moscona A, Moscona H. 1952. The dissociation and aggre-gation of
cells from organ rudiments of the early chickembryo. J Anat 86:
287–301.
Muguruma K, Nishiyama A, Kawakami H, Hashimoto K,Sasai Y. 2015.
Self-organization of polarized cerebellartissue in 3D culture of
human pluripotent stem cells.Cell Rep 10: 537–550.
doi:10.1016/j.celrep.2014.12.051
Nasr B, Chatterton R, Yong JHM, Jamshidi P, D’Abaco GM,Bjorksten
AR, Kavehei O, Chana G, Dottori M, SkafidasE. 2018. Self-organized
nanostructure modified micro-electrode for sensitive
electrochemical glutamate detec-tion in stem cells-derived brain
organoids. Biosensors 8:E14. doi:10.3390/bios8010014
Nasu M, Takata N, Danjo T, Sakaguchi H, Kadoshima T,Futaki S,
Sekiguchi K, Eiraku M, Sasai Y. 2012. Robustformation and
maintenance of continuous stratified cor-tical neuroepithelium by
laminin-containing matrix inmouse ES cell culture. PLoS ONE 7:
e53024. doi:10.1371/journal.pone.0053024
Okabe S, Forsberg-Nilsson K, Spiro AC, Segal M, McKayRDG. 1996.
Development of neuronal precursor cellsand functional postmitotic
neurons from embryonicstem cells in vitro. Mech Dev 59: 89–102.
doi:10.1016/0925-4773(96)00572-2
Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, Sugi-hara H,
Fujimoto K, Weissman IL, Capecchi MR, et al.2009. Sustained in
vitro intestinal epithelial culturewithina Wnt-dependent stem cell
niche. Nat Med 15: 701–706.doi:10.1038/nm.1951
Ormel PR, Vieira de Sá R, van Bodegraven EJ, Karst H,Harschnitz
O, Sneeboer MAM, Johansen LE, van DijkRE, Scheefnals N, Berdenis
van Berlekom A, et al. 2018.Microglia innately developwithin
cerebral organoids.NatCommun 9: 4167.
doi:10.1038/s41467-018-06684-2
Ozone C, Suga H, Eiraku M, Kadoshima T, Yonemura S,Takata N,
Oiso Y, Tsuji T, Sasai Y. 2016. Functional an-terior pituitary
generated in self-organizing culture ofhuman embryonic stem cells.
Nat Commun 7: 10351.doi:10.1038/ncomms10351
Paşca AM, Sloan SA, Clarke LE, Tian Y, Makinson CD,Huber N, Kim
CH, Park JY, O’Rourke NA, Nguyen KD,et al. 2015. Functional
cortical neurons and astrocytesfrom human pluripotent stem cells in
3D culture. NatMethods 12: 671–678. doi:10.1038/nmeth.3415
Pollen AA, Bhaduri A, Andrews MG, Nowakowski TJ,Meyerson OS,
Mostajo-Radji MA, Di Lullo E, AlvaradoB, Bedolli M, Dougherty ML,
et al. 2019. Establishingcerebral organoids as models of
human-specific brainevolution. Cell 176: 743–756.e17.
doi:10.1016/j.cell.2019.01.017
Qian X, Nguyen HN, Song MM, Hadiono C, Ogden SC,Hammack C, Yao
B, Hamersky GR, Jacob F, Zhong C,et al. 2016. Brain-region-specific
organoids using mini-bioreactors for modeling ZIKV exposure. Cell
165: 1238–1254. doi:10.1016/j.cell.2016.04.032
Qian X, Song H, Ming GL. 2019. Brain organoids:
advances,applications and challenges. Development 146: dev166074.
doi:10.1242/dev.166074
Quadrato G, Nguyen T, Macosko EZ, Sherwood JL, MinYang S, Berger
DR, Maria N, Scholvin J, Goldman M,Kinney JP, et al. 2017. Cell
diversity and network dynam-ics in photosensitive human brain
organoids.Nature 545:48–53. doi:10.1038/nature22047
Rathjen J, Haines BP, HudsonKM,Nesci A, Dunn S, RathjenPD. 2002.
Directed differentiation of pluripotent cells to
S. Benito-Kwiecinski and M.A. Lancaster
16 Advanced Online Article. Cite this article as Cold Spring
Harb Perspect Biol doi: 10.1101/cshperspect.a035709
on July 7, 2021 - Published by Cold Spring Harbor Laboratory
Press http://cshperspectives.cshlp.org/Downloaded from
http://cshperspectives.cshlp.org/
-
neural lineages: homogeneous formation and differenti-ation of a
neurectoderm population. Development 129:2649–2661.
Renner M, Lancaster MA, Bian S, Choi H, Ku T, Peer A,Chung K,
Knoblich JA. 2017. Self-organized develop-mental patterning and
differentiation in cerebral orga-noids. EMBO J 36: 1316–1329.
doi:10.15252/embj.201694700
Rothermel A, Willbold E, Degrip WJ, Layer PG. 1997. Pig-mented
epithelium induces complete retinal reconstitu-tion from dispersed
embryonic chick retinae in reaggre-gation culture. Proc Biol Sci
264: 1293–1302. doi:10.1098/rspb.1997.0179
Sakaguchi H, Kadoshima T, Soen M, Narii N, Ishida Y, Oh-gushiM,
Takahashi J, EirakuM, Sasai Y. 2015. Generationof functional
hippocampal neurons from self-organizinghuman embryonic stem
cell-derived dorsomedial telen-cephalic tissue. Nat Commun 6: 8896.
doi:10.1038/ncomms9896
Sasai Y, Lu B, Steinbeisser H, Geissert D, Gont LK, De Ro-bertis
EM. 1994. Xenopus chordin: a novel dorsalizingfactor activated by
organizer-specific homeobox genes.Cell 79: 779–790.
doi:10.1016/0092-8674(94)90068-X
Sato T, Vries RG, Snippert HJ, van deWeteringM, Barker N,Stange
DE, van Es JH, Abo A, Kujala P, Peters PJ, et al.2009. Single Lgr5
stem cells build crypt-villus structuresin vitro without a
mesenchymal niche. Nature 459: 262–265. doi:10.1038/nature07935
Shi Y, Kirwan P, Livesey FJ. 2012. Directed differentiation
ofhuman pluripotent stem cells to cerebral cortex neuronsand neural
networks. Nat Protoc 7: 1836–1846. doi:10.1038/nprot.2012.116
Shiraishi A, Muguruma K, Sasai Y. 2017. Generation of tha-lamic
neurons from mouse embryonic stem cells. Devel-opment 144:
1211–1220. doi:10.1242/dev.144071
Sloan SA, Darmanis S, Huber N, Khan TA, Birey F, CanedaC, Reimer
R, Quake SR, Barres BA, Paşca SP. 2017. Hu-man astrocytematuration
captured in 3D cerebral corticalspheroids derived from pluripotent
stem cells.Neuron 95:779–790.e6.
doi:10.1016/j.neuron.2017.07.035
Smith WC, Harland RM. 1992. Expression cloning of nog-gin, a new
dorsalizing factor localized to the Spemannorganizer in Xenopus
embryos. Cell 70: 829–840. doi:10.1016/0092-8674(92)90316-5
Steinberg MS, Gilbert SF. 2004. Townes and Holtfreter(1955):
directed movements and selective adhesion ofembryonic amphibian
cells. J Exp Zool A Comp Exp Biol301A: 701–706.
doi:10.1002/jez.a.114
Steinberg MS, Roth SA. 1964. Phases in cell aggregation
andtissue reconstruction an approach to the kinetics of
cellaggregation. J Exp Zool 157: 327–338.
doi:10.1002/jez.1401570304
Sutcliffe M, Lancaster MA. 2017. A simple method of gen-erating
3D brain organoids using standard laboratoryequipment. Methods Mol
Biol 3: 519–512.
Tomooka Y, Kitani H, Jing N, Matsushima M, Sakakura T.1993.
Reconstruction of neural tube-like structures in vi-tro from
primary neural precursor cells. Proc Natl AcadSci 90: 9683–9687.
doi:10.1073/pnas.90.20.9683
Vollmer G, Layer PG, Gierer A. 1984. Reaggregation of em-bryonic
chick retina cells: pigment epithelial cells induce ahigh order of
stratification. Neurosci Lett 48:
191–196.doi:10.1016/0304-3940(84)90018-1
Watanabe K, Kamiya D, Nishiyama A, Katayama T, NozakiS, Kawasaki
H, Watanabe Y, Mizuseki K, Sasai Y. 2005.Directed differentiation
of telencephalic precursors fromembryonic stem cells. Nat Neurosci
8: 288–296. doi:10.1038/nn1402
Watanabe K, UenoM, Kamiya D, Nishiyama A, MatsumuraM, Wataya T,
Takahashi JB, Nishikawa S, Nishikawa S,Muguruma K, et al. 2007. A
ROCK inhibitor permitssurvival of dissociated human embryonic stem
cells. NatBiotechnol 25: 681–686. doi:10.1038/nbt1310
Weiss P, Taylor AC. 1960. Reconstitution of complete organsfrom
single-cell suspensions of chick embryos in ad-vanced stages of
differentiation. Proc Natl Acad Sci 46:1177–1185.
doi:10.1073/pnas.46.9.1177
Wilson H. 1907. On some phenomena of coalescence andregeneration
in sponges. J Exp Zool 5: 245–258. doi:10.1002/jez.1400050204
Wilson H. 1911. On the behavior of the dissociated cells
inhydroids, alcyonaria, and Asterias. J Exp Zool 11: 281–338.
doi:10.1002/jez.1400110304
Xiang Y, Tanaka Y, Patterson B, Kang YJ, Govindaiah G,Roselaar
N, Cakir B, Kim KY, Lombroso AP, HwangSM, et al. 2017. Fusion of
regionally specified hPSC-de-rived organoids models human brain
development andinterneuron migration. Cell Stem Cell 21:
383–398.e7.doi:10.1016/j.stem.2017.07.007
Xiang Y, Tanaka Y, Cakir B, Patterson B, Kim KY, Sun P,Kang YJ,
Zhong M, Liu X, Patra P, et al. 2019. hESC-derived thalamic
organoids form reciprocal projectionswhen fused with cortical
organoids. Cell Stem Cell 24:487–497.e7.
doi:10.1016/j.stem.2018.12.015
Ying QL, Stavridis M, Griffiths D, Li M, Smith A.
2003.Conversion of embryonic stem cells into neuroectoder-mal
precursors in adherent monoculture. Nat Biotechnol21: 183–186.
doi:10.1038/nbt780
Zhang SC, Wernig M, Duncan ID, Brüstle O, Thomson JA.2001. In
vitro differentiation of transplantable neural pre-cursors from
human embryonic stem cells. Nat Biotech-nol 19: 1129–1133.
doi:10.1038/nbt1201-1129
Brain Organoids
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published online November 25, 2019Cold Spring Harb Perspect Biol
Silvia Benito-Kwiecinski and Madeline A. Lancaster Brain Organoids:
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