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ORGANOIDS

Polarizing brain organoids

Human brain organoids are endowed with regional topography using engineered signaling centers.

Yuki Miura and Sergiu P. Pașca

B
rain organoids are self-organizing,
3D cellular structures generated
in vitro that recapitulate aspects of
neural development and function1. Derived
from pluripotent stem cells, they can be
guided to resemble specific brain regions,
such as the dorsal or ventral forebrain,
through the application of developmental
signaling factors or, alternatively, can be
produced by undirected differentiation to
generate various neural and non-neural cell
lineages. Organoids, however, do not have a
predictable anatomy, and they do not reflect
the asymmetries of the developing brain,
such as topography along dorsoventral,
mediolateral and anteroposterior axes.
In this issue, Cederquist et al.2 describe
a method to induce dorsoventral- and
anteroposterior-like polarization
in forebrain organoids. Mimicking
development, the authors supplied early-
stage organoids with a small cell cluster that
creates a gradient of the morphogen Sonic
Hedgehog (SHH) and subsequently specifies
cell fate in a concentration-dependent
manner. The work offers a method to derive
brain organoids with a certain level of
topographic organization.
The forebrain arises through a dramatic
expansion of the anterior part of the
neural tube and ultimately gives rise to the
cerebral cortex, basal ganglia and other
structures3. Early in development, the
forebrain divides into distinct subregions
through the action of secreted morphogens,
which form gradients that instruct cells
to acquire specific fates4. For instance,
SHH is expressed in the ventral midline
of the central nervous system (CNS)5 and
contributes, along with other factors, to
the division of the forebrain into dorsal
and ventral domains6 (Fig. 1). The dorsal
part gives rise to a progenitor-rich region
that generates the cortical plate and later
becomes the laminated cerebral cortex. The
ventral part includes several eminences,
including a medial eminence that generates
GABAergic interneurons that subsequently
migrate dorsally to integrate into cortical
circuits and a lateral eminence that generates
the basal ganglia.
Previous work had achieved the reliable
generation of brain organoids that resemble
either the dorsal forebrain, containing
cortical glutamatergic neurons, or the
ventral forebrain, containing GABAergic
interneurons. Specification into dorsal or
ventral fates was controlled by adding to
the culture medium specific amounts of
small molecules that modulate the SHH
and WNT pathways. But a key challenge
for the field is to discover methods that
model the topographical patterning and
inter-regional interactions that characterize
brain development.
In a first step toward recapitulating
greater complexity in vitro, dorsal and
ventral forebrain organoids have been
generated separately and then combined.
The resulting structures, known as
assembloids, exhibit the saltatory migration
of interneurons in the fetal forebrain,
display functional integration of GABAergic
interneurons into circuits, and have been
used to model genetic disease7. But this
approach depends on manipulating a limited
number of forebrain domains that do not
develop together and that must be fused at
specific time points.
The ingenious method of Cederquist
et al.2 takes its cue from development by
assembling early brain organoids together
with a cluster of SHH-secreting cells that
functions as a signaling or polarizing cellular
center (Fig. 1b). The authors began by
genetically engineering a human pluripotent
stem cell line to express SHH under the
control of the antibiotic doxycycline. They
mixed a small aggregate of cells from
this line with a larger aggregate of cells

a b

Dorsal

Midbrain
hPSCs

Forebrain
Genetic Aggregation Differentiation
Hind-
engineering
brain

SHH
Polarized
Anterior organoid

Ventral
hPSCs with
Dox-inducible
SHH expression

Fig. 1 | Assembly of polarized human brain organoids from human pluripotent stem cells. a, Schematic of the developing human CNS illustrating the main
developmental axes and the source of the ventralizing morphogen SHH. b, Derivation of polarized brain organoids from human pluripotent stem cells (hPSCs)
by assembly of brain organoids containing clusters of cells conditionally expressing SHH.

Nature Biotechnology | www.nature.com/naturebiotechnology

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from an unmodified pluripotent stem cell
line and started neural differentiation.
Doxycycline was added over the next
week to induce SHH expression from the
genetically modified cells. The resulting
organoids were polarized into several
topographical forebrain subregions,
including areas resembling the lateral or
medial ganglionic eminences (GSX2+ or
NKX2.1+), the hypothalamus (NKX2.2+ or
NKX2.1+FOXG1–), the thalamus (TCF7L2+)
and the dorsal forebrain (PAX6+FOXG1+).
At later stages in vitro, the organoids
expressed markers for cortical layer–
specific neurons (for example, TBR1+),
striatal neurons (DARPP32+), GABAergic
interneurons (LHX6+) and hypothalamic
neurons (for example, POMC+, OTP+).
Importantly, SHH was distributed in
the organoids as a gradient. SHH had a
longer signaling range than in 2D cultures,
as quantified by the ability to suppress
expression of the dorsal forebrain marker
PAX6. Notably, the authors also showed that
embedding the cells into an extracellular
matrix, such as Matrigel, is not necessary to
derive organoids, a modification that should
improve the reliability of differentiation.
Cholesterol can modulate the long-range
signaling of SHH and acts as an agonist
of Smoothened receptors. In an initial
application of the system, the authors
demonstrated that exposure of polarized
organoids to inhibitors of the cholesterol
synthesis pathway, including the cholesterol-
lowering medication lovastatin, reduced the
efficiency and range of SHH signaling and a
shifted cell differentiation to a more dorsal
forebrain fate.
The experiments of Cederquist et al.2
suggest several refinements that could
further improve the anatomic fidelity of
polarized organoids. Although the timing
and level of SHH expression were controlled
by doxycycline, more precise methods to
titrate SHH expression will be essential.
Implementation of in situ single-cell
transcriptomics would enable more accurate
identification of cell fates. In the developing
nervous system, organizers secrete
combinations of morphogens, such as WNT
and BMP in addition to SHH, in complex
spatiotemporal patterns, and these patterns
could be simulated in brain organoids or
assembloids using engineering approaches
to precisely position and control multiple
signaling centers.
Many interesting applications of the
approach can be envisaged. Polarized
organoids could be used to study
signaling and cell specification in the
human forebrain. They could be applied
to model complex interactions between
different regions, such as the migration
of interneurons into various parts of the
forebrain or the connectivity between the
cortex and striatum, and to investigate
the modulatory effects of hypothalamus-
derived neuropeptides on activity in the
cerebral cortex. Because they reproduce
long-range SHH signaling effects,
polarized organoids could serve as models
of diseases such as the autosomal recessive
Smith–Lemli–Opitz syndrome, which is
caused by mutations in the gene encoding
the cholesterol biosynthetic enzyme
7-dehydrocholesterol reductase8. Similar
approaches could be developed for other
CNS regions, such as the midbrain or the
spinal cord. For example, inclusion of
organizers expressing dorsalizing factors,
such as various WNTs, and techniques
to lateralize or break symmetry would
further increase complexity in these
in vitro models.
Moving toward structures with more
elaborate cytoarchitecure, organizer-like
cells that resemble, for example, the cortical
hem9 or floorplate10 could be differentiated
from human pluripotent stem cells and then
Nature Biotechnology | www.nature.com/naturebiotechnology
combined with brain organoids to instruct
the fate and topography of specific CNS
regions. Moreover, early organizer-like cells,
as recently derived11, could be fused with
3D cell aggregates to model fundamental
processes in early human CNS development,
such as neural tube formation.
The mechanisms underlying cell
fate decisions and wiring in specific
locations of the human CNS are still
poorly understood, mostly because of
limited access and an inability to precisely
manipulate neural tissues in vitro. The
approach of Cederquist et al.2 for polarizing
forebrain organoids, together with other
methods for generating assembloids,
provide powerful tools for accelerating
our understanding of human brain
development, evolution and disease. ❐
Yuki Miura and Sergiu P. Pașca   *
Department of Psychiatry and Behavioral
Sciences & Human Brain Organogenesis Program,
Stanford University, Stanford, CA, USA.
*e-mail: spasca@stanford.edu
Published: xx xx xxxx
https://doi.org/10.1038/s41587-019-0084-4
References
1. Pașca, S. P. Nature 553, 437–445 (2018).
2. Cederquist, G. Y. et al. Nat. Biotechnol.. https://doi.org/10.1038/
s41587-019-0085-3 (2019).
3. Silbereis, J. C., Pochareddy, S., Zhu, Y., Li, M. & Sestan, N. Neuron
89, 248–268 (2016).
4. Kiecker, C. & Lumsden, A. Annu. Rev. Neurosci. 35,
347–367 (2012).
5. Ericson, J. et al. Cell 81, 747–756 (1995).
6. Lupo, G., Harris, W. A. & Lewis, K. E. Nat. Rev. Neurosci. 7,
103–114 (2006).
7. Birey, F. et al. Nature 545, 54–59 (2017).
8. Blassberg, R., Macrae, J. I., Briscoe, J. & Jacob, J. Hum. Mol. Genet.
25, 693–705 (2016).
9. Sakaguchi, H. et al. Nat. Commun. 6, 8896 (2015).
10. Jo, J. et al. Cell Stem Cell 19, 248–257 (2016).
11. Martyn, I., Kanno, T. Y., Ruzo, A., Siggia, E. D. & Brivanlou, A. H.
Nature 558, 132–135 (2018).
Competing interests
The authors declare no competing interests.