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Interfacing brain organoids with precision
medicine and machine learning
Honghui Zheng,1,2,4 Yilin Feng,1,2,4 Jiyuan Tang,1,2 and Shaohua Ma1,2,3,*

SUMMARY
The brain is a complex organ that plays a central role in human
activities. Brain organoid modeling is an advanced technique for
studying brain development, physiology, function, and disease
occurrence; the technique may provide opportunities for therapy
and healthcare management and establish the crosstalk between
human brains and engineered computing machinery. Brain organo-
ids are expected to contain both histological and physiological
information and possess the responsive characteristics toward
perturbation of their brain counterparts. Though it is in early stages
of development, the convergence of machine learning and brain or-
ganoid modeling holds enormous potential to broaden the capacity
of either technique; enables information acquisition, processing,
and optimizing for organoid establishment and maintenance
in vitro; and may open new facets of implementation of precision
medicine and healthcare engineering for brain health and diseases.

INTRODUCTION
The brain is an essential organ of the human body that coordinates systemic organ
interaction, controls body movement and emotion, and regulates immune activities
and organ metabolism, etc.1–3 Neurological disorders are among major causes of
deaths worldwide, including Alzheimer’s disease (AD), glioma, stroke, and so on.
Yet they remain short of effective therapies on account of the lack of understanding
about human brain and most neurological disorders. It is important to note that hu-
man brain development is an extremely intricate and complex process that involves
highly orchestrated neural stem cells proliferation, specification, and differentiation,
surrounded by dynamic morphogens. It has been challenging to model and manip-
ulate brain development, because of its biological complexity and limited access to
developing brains under evident ethical regulations. In addition, culturing and ex-
panding brain tissues in vitro are extremely difficult.4

Nevertheless, recent advances in stem cell technology shed light into human plurip-
otent stem cells (hPSCs)-derived mini-brain tissues, or so-called brain organoids, to
study brain development. Brain organoids are self-organized 3D cell aggregates 1Tsinghua Shenzhen International Graduate
capable of recapitulating the key features of developing human brain; those other- School (SIGS), Tsinghua University, Shenzhen
wise are lost in 2D cultures or animal models. For example, the progenitors of an 518055, China
2Tsinghua-BerkeleyShenzhen Institute (TBSI),
outer subventricular zone (oSVZ) that play an important role in determining human
Shenzhen 518055, China
cortex size and complexity are completely absent in rodent models.5,6 The high
3Institute
for Brain and Cognitive Sciences,
feasibility to track and modulate brain organoids holds great promise for elucidating Tsinghua University, Beijing 100084, China
the mechanisms during neurodevelopment and, prospectively, disease occurrence. 4These authors contributed equally
*Correspondence:
Machine learning is a computational route toward understanding and building effi- ma.shaohua@sz.tsinghua.edu.cn
cient mapping algorithms between information input (the training data) and output https://doi.org/10.1016/j.xcrp.2022.100974

Cell Reports Physical Science 3, 100974, July 20, 2022 ª 2022 The Author(s). 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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(the prediction or decision) without explicit programming. The mapping function re-
flects the relationship between input and output.

Here, we briefly summarize current protocols of human brain organoids and discuss
about their advantages, limitations, and prospects. Next, we elaborate how to ac-
quire and manipulate information from brain organoid. Then we illustrate how brain
organoids function as disease and interference models. Finally, we combine ma-
chine learning with brain organoid modeling to envision the great promise of this
cross-disciplinary field.

3D SELF-ASSEMBLED BRAIN ORGANOIDS IN A DISH

Brain organoids provide an innovative experimental paradigm for neuroscientists to
investigate brain development, which were pioneered by the group of Yoshiki Sasai
at RIKEN, Japan. They established different protocols for serum-free embryoid
bodies (EBs).7–9 These protocols combined EB-derived neural rosettes culture10
with serum-free neural induction,11 thus generating dorsal forebrain organoids
with apicobasal architecture and organized cortical plate-like structure with inside-
out layer patterns of neurogenesis.9 Further protocols have advanced rapidly in
the past decade,12–14 and an in-depth historical overview about development of
these methods has been provided elsewhere.5,15,16 In brief, these protocols begin
with dissociating PSCs, including human embryonic stem cells (hESCs) and human
induced pluripotent stem cells (hiPSCs), into 3D aggregates, i.e., EBs, and then
coaxing EBs into various brain organoids by supplementing signaling molecules,
recapitulating the microenvironment of in vivo brain development, like dorsomor-
phin, SB-431542, CHIR99021, and so on.

Based on whether using patterning factors or not, the modern methods of devel-
oping brain organoids can be divided into two categories: guided and unguided
protocols (Figure 1). Comparisons among these protocols based on guided and un-
guided methods are illustrated in Table 1.

The unguided way utilizes intrinsic self-organizing capacity of the brain and the
default stem-cell fate while only providing minimal differentiation medium, without
the use of patterning growth factors, to establish whole-brain organoids with
several discrete brain regions.17 Although unguided organoids can obtain sufficient
regional and cellular complexity, it also leads to high heterogeneity among organo-
ids and low reproducibility among batches.

As for increasing complexity and control, guided protocols are brought into being,
where a particular set of patterning signals are provided to induce EBs into specific
brain structures based on the anatomy of the brain, including forebrain that processes
information connected to complex cognitive activities,28 midbrain that plays impor-
tant functions in motor movement,35 thalamus that serves as the body’s information
relay station,37 and hypothalamus that maintains the homeostasis of human body.36
Creating on-demand, region-specific brain organoid has been proven improved in
controlling brain regionalization and gaining higher reproducibility,38 though it limits
the capacity to resemble complex connectivity and architecture of brain.

Under these circumstances, significant progress has been achieved by fusing

different brain-regional organoids into assembloids that model neural circuits for-
mation and regional interconnectivity of human brain.16,39–41 For example, subdo-
main-specific forebrain spheroids were established and assembled together to

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Figure 1. Comparisons of brain organoids generated with guided and unguided protocols
Brain organoid generation is started with formation of EB, an aggregate of hESCs derived from blastocysts or hiPSCs derived from fibroblasts. Guided
protocols with signaling molecules produce more reproducible and better regionalized organoids, reminiscent of a specific brain region. Unguided
protocols generate organoids reminiscent of the whole brain with diverse regions (left panels). Assembloids, fusion of region-specific brain organoids,
muscle organoids, or polarized organizers, are developed to study the spatiotemporal control of morphogens and assembly of neural circuits (bottom
panels). With respect to non-ectoderm cells, microglia and vasculature are omitted but can be incorporated in guided brain organoids by co-culture of
microglial and endothelial cells and organoids. However, microglia can be innately generated in unguided organoids (middle panels). With the
integration of vasculature and microglia, the neuroimmune and neurovascular interaction of the brain can be partially mimicked by brain organoids.
Figure created with BioRender.com.

study the migration of interneurons.34,42,43 A similar strategy was adopted to model

the interregional long-range axonal connectivity40 between thalamus and cortex,37
striatum and cortex,44 and hypothalamus and pituitary.45 Apart from assembling
organoids from diverse brain regions in the central nervous system (CNS), muscle or-
ganoids were also integrated to form cortico-motor assembloids that controlled
muscle contractions by functional synaptic contacts.46 Moving forward, external
growth factors were added via bath application to realize spatial feature patterning
in these extrinsic protocols, whereas in vivo, there existed signaling factor gradients
along the anteroposterior and dorsoventral axes to accomplish the correct brain
regionalization. Recently, an elegant study employed the gene-targeting strategy
to enable hPSCs to express Sonic Hedgehog (SHH) protein, which was localized at
one pole of an organoid. The SHH cells functioned as polarized organizers in devel-
oping forebrain organoids, eventually generating forebrain organoids with major
dorsoventral and anteroposterior positional identity.47 The axis patterning protocol
demonstrated better spatiotemporal control of developing brain organoids in vitro.

In the past, brain organoids derived from both methods contained mostly ecto-
dermal cells, such as neurons and astrocytes. However, oligodendrocytes were
not present. Recently, oligodendrocytes and myelin were found in patterned

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Table 1. Comparisons among selected protocols based on guided and unguided methods
Method Brain region Stem cells used Success Implements Reference
Unguided whole brain hESCs and hiPSCs  first protocol generating whole-  Matrigel as ECM Lancaster et al.17
method brain organoids  spinning bioreactor
hESCs and hiPSCs  better reproducibility and  microfilaments as a floating scaf- Lancaster et al.18
improved tissue architecture fold
hESCs and hiPSCs  better reproducibility and long-  reduced number of hPSCs Quadrato et al.19
term survival  optimized neural induction
 adding BDNF into maturation
medium
hESCs  improved nutrients diffusion and  slice culture at the air-liquid inter- Giandomenico
oxygenation face et al.20
 improved neuronal survival and
maturation
hiPSCs  correct gliogenesis: microglia  reduced levels of the neuroecto- Ormel et al.21
innately develop within organoid derm stimulant heparin
 delayed Matrigel embedment
hiPSCs  successfully vascularized brain or-  incorporated endothelial cells Pham et al.22
ganoids with organoids
Guided dorsal hiPSCs  simple and reproducible approach  better control of the neural induc- Pasxca et al.23 and
method forebrain  exhibited extensive astrogenesis tion and differentiation through Sloan et al.24
combination of small molecules
hESCs and hiPSCs  correct gliogenesis: brain orga-  supplementing medium with Marton et al.25
noids contained oligodendro- growth factors like PDGF-AA, and Madhavan
cytes as well as neurons and as- T3, and IGF-1 that promote et al.26
trocytes oligodendrocyte differentiation
in specific time points
hiPSCs  observed nested oscillatory  long-term culture of organoids on Trujillo et al.27
network neuronal activity in microelectrodes
organoids
hiPSCs  reduced heterogeneity in orga-  treatment with three factors: Qian et al.28
noid shape and size CHIR99021, WNT3A, and SB-
 better cytoarchitecture: distinct 431542
outer radial glia cell layer and  use of mini-bioreactors
diverse neuronal subtypes of six
cortical layers
hiPSCs  improved nutrients diffusion and  orbital shaker and routine slice Qian et al.29
oxygenation organoids every 4 weeks
 accomplished long-term survival
 better cytoarchitecture: distinct up-
per and deep cortical layers for
neurons and astrocytes
hiPSCs  established neuroimmune orga-  co-culture brain organoids with Popova et al.30
noid model of human brain iPSCs-derived and human pri-
development mary microglia
hESCs  successfully vascularized brain or-  transplant human brain organo- Mansour et al.31
ganoids ids into the adult mouse brain
hESCs and hiPSCs  co-culture PSCs with human um- Shi et al.32
bilical vein endothelial cells
hESCs  engineer hESCs to express ETV2 Cakir et al.33
into organoids
Ventral hESCs and hiPSCs  generated spheroids resembling  use of IWP-2 and SAG Birey et al.34
forebrain the ventral forebrain  assemble subdomain-specific
 recapitulated the migration of forebrain spheroids in vitro
interneurons between regions
Midbrain hESCs and hiPSCs  established midbrain organoid  use of Noggin, SB431542, Jo et al.35
model containing functional CHIR99021, FGF8, etc.
dopaminergic neurons
Hypothalamus hiPSCs  generated arcuate organoids  combination of patterning fac- Huang et al.36
resembling human hypothalamic tors, hypothalamus astrocyte
arcuate nucleus conditioned medium, and trophic
factors

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organoids while preserving neurons and astrocytes via supplementing certain

signaling factors to promote oligodendrocytes lineage commitment.25,30 However,
non-ectodermal lineage cells, like yolk-sac-derived microglia cells and mesoderm-
derived endothelial cells, remained missing in brain organoids. Notably, microglia
play a fundamental role in neuroimmune and neurodevelopment. They intimately
involve in brain homeostasis and neuronal interactions, as well as synaptic activities.
To tackle this issue, microglia were innately grown inside whole-brain organoids at
the late stage of neural differentiation by tuning the composition of medium,
when part of mesodermal and endodermal lineages cells were preserved in the un-
guided protocol.17,21,48 Unfortunately, in guided protocols, PSCs are inhibited to
differentiate toward mesoderm and endoderm fates by strong patterning factors
that determine the neuroectoderm fate.34 In this case, microglia were generated
separately by inducing hPSCs or dissociating from brain tissues and incorporated
into region-specific organoids via co-culture to study brain-immune response and
neurodegenerative diseases.49–52

Hypoxia and diffusion barrier exist 300–400 mm away from the outer surface of large
brain organoids29 and ultimately result in neuro-necrosis. Efforts have been made
recently to vascularize brain organoids to mimic the neurovascular interaction in vivo
by co-culturing organoids with mesodermal progenitor cells,53 engineering hPSCs
to overexpress endothelial-cell-fate-related transcriptional factors,33 or incorpo-
rating endothelial cells22 for improved oxygen and nutrient diffusion.54 Furthermore,
xeno-transplantation of human brain organoids into mouse brains achieved en-
riched vascularization in organoids.31

INFORMATION ACQUISITION AND MANIPULATION OF BRAIN

ORGANOIDS
Brain organoids are capable of modeling both physiological and pathological fea-
tures of human brains in either dynamic or static manners. However, stochastic dif-
ferentiation process occurring in various protocols introduces instability and reduces
reproducibility in organoid fabrication and culture. Different transformative analytic
techniques are geared toward obtaining reliable outputs to investigate the repro-
ducibility, accuracy, and scalability of brain organoids.16 Multimodal approaches,
including cytoarchitectural, functional, and transcriptional assessment, have been
employed for characterizing brain organoids to produce informative and unbiased
cellular and molecular descriptions along the long-term culture (Figure 2).55

To assess the cytoarchitectural arrangement and cellular diversity, organoids are

sectioned into 2D tissue slices and the neurodevelopmental stages are character-
ized by traditional immunohistochemical staining against diverse markers and opti-
cal microscopic imaging. For example, SOX2- and PAX6-positive cells reveal radial
glial cells in the ventricular zone, and TBR2+ stands for the intermediate progenitor
cells in the subventricular zone.17 By combining TBR1 and CTIP2 with other markers
in the cortical plate, it can test whether the cortical layering and the inside-out neuro-
genesis model are correct or not.18 Alternatively, unorthodox approaches like tissue
clearing and multi-photon fluorescence imaging are used in whole-mount organoids
to extract 3D spatial information in organoids.56 The volume information is usually
reconstructed by computation of multi-layered information with spatial coordinate
embeddings.

Bulk sequencing and single-cell profiling are both powerful and widely used tools to
carry out the cellular and molecular analysis. While classical bulk RNA sequencing

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Figure 2. Use manipulation as input to act on brain organoids and get output by assessment
Diverse manipulations, including electrical, genetic, thermal, optical, mechanical, and chemical manipulation, can be executed (input) along the
development of brain organoids to generate functional, transcriptional, and cytoarchitectural readouts (output) correspondingly. Figure created with
BioRender.com.

and qPCR provide simple transcriptomic readouts with limited resolution, single-cell
RNA sequencing (scRNA-seq) distinguishes itself, particularly in determining cellular
identities in brain organoids.57 scRNA-seq has been applied to compare reproduc-
ibility of brain organoids from cell diversity, differentiation trajectories, and
transcriptional biases across different protocols and brain regions.38,58 Apart from
transcriptional profiling, epigenetic analysis at single-cell resolution has also been
incorporated to probe the epigenetic landscape of brain organoids.59

Though cytoarchitectural and single-cell profiling can provide structural and composi-
tion information on organoids, they provide limited insights about functions of neurons
or wired neural networks. Specialized experimental tools are crucial for evaluating the
electrical functions along the development of organoids.55,60,61 At single-cell and
intracellular levels, conventional whole-cell patch clamp can record ionic currents in in-
dividual cells and properties of cell membrane inside brain organoids at millisecond
temporal resolution. This information reflects the functional characteristics of neurons
and proves the existence of excitatory neurons.34 However, high-resolution single-cell
characterization may lose attention on the network-wide information. Thus, microelec-
trode array (MEA) techniques have been developed and commercialized to detect ex-
tra- and inter-cellular electrical features of multiple neurons simultaneously at the
mesoscopic level. The spatial resolution reaches 1 mm.62 Using MEA recording, it
was found that periodic and regular nested events consistently increased over the
span of several months in brain organoids.27 Furthermore, calcium imaging has also
shown great potential in probing neural activity of brain organoids at the whole
network scale, based on the distinctive properties of transient calcium ion fluxes in neu-
rons at static and under stimulation. It demonstrated the human-mimic neural network
activities existed in stem-cell-derived brain organoids.63

Apart from acquiring informative outputs from organoids under regular conditions,
electrophysiological recording probes can also capture electrical signal variance un-
der stimulation, which, after computational processing, can be reckoned to orga-
noid responses toward external stimuli. Currently, there are multiple modalities to
manipulate brain organoids, based on electrical, thermal, mechanical, optical, and
chemical stimulation, as well as genetic engineering.

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Figure 3. Converging machine learning and brain organoid modeling with closed-loop BCI
ML augments brain organoids in predicting determinants of input, improving acquisition and mining of output data, and decoding the mapping
functions between the input and output of brain organoids. Conversely, brain organoids can also facilitate the next generation of ML and AI. In sum, with
the feedback of BCI, dynamic closed-loop control can be accomplished that converges ML and brain organoid modeling. Figure created with
BioRender.com.

Recently, Park et al.64 introduced a soft, shape-matched 3D MEA platform that pro-
vided multifunctional neural-silicon interfaces on brain organoids. The system
enabled electrical, optical, chemical, and thermal stimulation to cortical organoids,
and more importantly, it acquired evoked neural activities that were similar to in vivo
modulation systems. Likewise, it was demonstrated that neuronal plasticity related
to memory formation and consolidation existed in brain organoids by high-fre-
quency electrical stimulation and optogenetic stimulation (Figure 2).19,65,66

Chemical and genetic modulation are also broadly used in brain organoid studies. The
influences of chemical exposures on fetal neurodevelopmental process can be inter-
preted through analyzing features of developing organoids exposed to toxic molecules
or drugs like nicotine,67 cocaine,68 and cannabinoid.69 Disturbed electrical features were
acquired of neural circuits when electrodes were paired with drug and optogenetic stim-
ulation.19,70 Moreover, genetic perturbations are enabled by CRISPR-Cas9 to discover
therapeutic gene targets that determine brain development and dysfunction. In addi-
tion, mechanical and genetic manipulations are both utilized in organoids to investigate
the physics of folding of human brains, a significant feature of brain development.71,72

BRAIN ORGANOIDS AS DISEASE MODELS

Changes in the human genome, long-term developmental characteristics, and orga-
nizational structure have led to limitations in the use of traditional model organisms
to study human neurological and psychiatric diseases. The foregoing research shows
that brain organoids facilitate the study of specific phenotypes in the human environ-
ment by combining two important determinants of human identity—the human
genome and associated human developmental timelines.13 Therefore, the brain
(or cerebral) organoid modeling technique offers an effective choice owing to
their high visibility, tailored ability, numbering-up modeling capacity, and rapid re-
sponses toward stimuli and perturbation. Successes have been reported in

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Table 2. Main neurological diseases modeled by using human-stem-cell-derived brain organoids

Disease Cell line and genetic background Organoid types Phenotypes in organoids Reference
Neurodegenerative disease
Alzheimer hPSCs carrying PSEN2N141I point cerebral organoid high ratio of Ab42/Ab40; smaller Yin and
disease (AD) mutation size; disrupted calcium VanDongen73
homeostasis
hPSCs with specific APOE cerebral organoid presence of Ab and Tau Hernández et al.74
genotypes
hPSCs induced from AD patients cerebral organoid apoptosis and synaptic loss; Zhao et al.75
with increased Ab amounts;
APOE ε3/ε3 or ε4/ε4 genotype exacerbated tauopathy
DE9/wild-type (WT)-hiPSC line cerebral organoid aberrantly increased electrical Ghatak et al.76
bearing activity
the PSEN1 DE9 mutation; M146V/
WT
and amyloid precursor protein
(APP)swe/WT
hiPSC lines bearing the PSEN1
M146V and
APP Swedish mutation
PITRM1-knockout hPSCs cortical organoid increased APP and phospho-tau Pérez et al.77
levels
hPSCs carrying duplications in the cerebral organoid high level of Ab and Tau Raja et al.78
gene for
APP (APPDp1-1 and APPDp2-3)
normal hPSCs cerebral organoid induces Ab-like pathology and Chen et al.79
exposed to serum elevated p-Tau level synaptic loss
of AD patients and impairs neural network
Parkinson’s LRRK2 mutation in human iPSCs midbrain organoid a decrease in the number and Smits et al.80
disease (PD) complexity of midbrain-type and Kim et al.81
dopamine (mDA) neurons
in disease organoids
DNAJC6 ablation in hESCs midbrain organoid mDA neuron degeneration, Wulansari et al.82
pathologic a-synuclein
aggregation, increase of intrinsic
neuronal firing frequency, and
mitochondrial and lysosomal
dysfunctions
Neurodevelopmental disease (NDD)
Autosomal recessive hPSCs with WDR62 mutations cerebral Organoid smaller organoid sizes with Zhang et al.83
primary microcephaly reduced surface areas compared
(MCPH) with controls
normal hPSCs forebrain organoid a significant reduction in both VZ Qian et al.28
exposed to ZIKVM and neuronal layer thickness
Rett syndrome (RTT) hPSCs induced from RTT patients cerebral organoid impaired neurogenesis in RTT Mellios et al.84
with patient-derived 3D cerebral
MeCP2 mutated organoids
Miller-Dieker syndrome hPSCs generated from MDS cerebral organoid a mitotic defect in outer radial glia Bershteyn et al.85
(MDS) patients
hPSCs generated from MDS forebrain organoid premature neurogenesis Iefremova et al.86
patients
Down syndrome (DS) hPSCs generated from DS patients forebrain organoid specific subclasses of GABAergic Xu et al.87
interneurons; increased OLIG2
expression
hPSCs generated from DS patients cerebral organoid extracellular diffuse and fibrillar Ab Alic et al.88
deposits, hyperphosphorylated
and pathologically conformed Tau,
and premature neuronal loss
hPSCs generated from DS patients cerebral organoid diminished proliferation and Tang et al.89
decreased expression of layer II
and IV markers in cortical neurons
in the subcortical regions; reduced
size of the organoids
(Continued on next page)

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Table 2. Continued
Disease Cell line and genetic background Organoid types Phenotypes in organoids Reference
Hypoxia normal hPSCs human cortical specific defects in intermediate Pașca et al.90
spheroids (hCSs) progenitors
exposed to low
oxygen concentrations
Neuropsychiatric disease
Major depressive disorder hPSCs from 16p11.2 patient cortical organoid organoid size recapitulating Urresti et al.91
(MDD) macrocephaly and microcephaly
phenotypes
Schizophrenia hPSCs from schizophrenia cerebral organoid dispersion of proliferating cells in Stachowiak
patients the cortex and blockade of cortical et al.92
neuronal development; nuclear (n)
FGFR1 abundantly expressed by
developing subcortical cells but
depleted from the neuronal
committed cells (NCCs) of the
cortical zone
Brain tumor
Glioblastoma organoids / patient-derived maintenance of parental tumor Jacob et al.93
glioblastoma cellular heterogeneity, gene
organoids expression, and mutations
hESC line H9; normal hPSCs cerebral organoids invasive tumor-like structures Ogawa et al.94
co-culture with GSC and Goranci-
spheroids and GSCs Buzhala et al.95

mimicking various brain diseases, where therapeutic trials have been explored.
Here, we list main neurological disease models by using brain organoids (Table 2).

Neurodegenerative disease
Neurodegenerative diseases, including AD, Parkinson’s disease (PD), and Hunting-
ton’s disease (HTD), are receiving incremental attention with the growth of the aging
population worldwide. AD pathology has been created in human iPSCs-derived
brain organoids, characterized by extracellular Ab plaques and intracellular neurofi-
brillary tangles (NFTs).96 However, these AD organoids were induced by iPSCs
deliberately overexpressing a part of the profile of known variants in presenilin-1
(PSEN1), presenilin-2 (PSEN2), and amyloid precursor protein (APP) genes.97
Although these models simulated the pathology of familial AD (fAD), they failed
to model sporadic AD (sAD), which constitutes over 95% of the later onset AD
cohort. Recently, Chen et al.79 exposed healthy-brain organoids to patient serum
and created the pathological features of sAD. However, this modeling method is
not fully investigated in either efficacy or feasibility.

PD is the second most prevalent neurodegenerative disease after AD. At the cellular
level, PD is pathologically characterized by a-syn misfolding and aggregation,
disruption of the autophagy-lysosomal system, mitochondrial dysfunction, ER stress,
or dysregulated calcium homeostasis.98,99 These mechanisms act synergistically in
the death of substantia nigra (SN) dopamine (DA) neurons, resulting in the patient’s
bradykinesia, stiffness, postural abnormality, and resting tremor.100 Most reported
PD organoids were created from midbrain organoids80 derived from iPSCs with spe-
cific mutations, such as LRRK2 G2019S variant81 and DNAJC6 mutations.82 Alterna-
tively, PD-patient-derived iPSCs without profiled mutations were also proven to
develop brain organoids with typical PD characteristics.80,101

Neurodevelopmental disease (NDD)

Most congenital neurodevelopmental disorders are caused by specific genetic vari-
ants and develop at an early stage. Brain organoids are well suited as developmental

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models for mechanism study of NDD. Autosomal-recessive primary microcephaly

(MCPH) is a congenital brain disorder characterized by reduced brain size. Mutations
in WDR62 are the second most common genetic cause of human MCPH. Generation
of WDR62 gene-deficient hPSCs using the CRISPR-Cas9 technology successfully
induced microcephaly-mimicking brain organoids, manifested by reduced numbers
of neural progenitor cells (NPCs) and reduced brain organoid sizes.83 Exposure of
brain organoids to Zika virus also caused microcephaly.102 Rett syndrome (RTT) is a
neurodevelopmental disorder of intellectual disability caused by mutations in the
X-linked gene methyl-CpG-binding protein 2 (MECP2). RTT patient iPSC-derived
brain organoids explained how deficits in MECP2 function affected neurogenesis by
modulating microRNAs (miRNAs).84 Moreover, brain organoid mimics for other neu-
rodevelopmental disorders, such as Miller-Dieker syndrome,85,86 Sandhoff disease,103
Down syndrome,88,104 and hypoxia90 have also been extensively studied. In addition,
mutations in the small guanosine triphosphatase (GTPase) gene RAB39b have been
found associated with X-linked macrocephaly, autism spectrum disorder (ASD), and
intellectual disability, which might also be modeled using brain organoids.105

Infectious disease
Human brain organoids provide an unprecedented opportunity to study the suscep-
tibility and cell tropism of viral infections and their consequences in the nervous sys-
tem. Human forebrain organoids were first used to study ZIKV infection that results in
increased incidence of neonatal microcephaly.106 Brain organoids exposed to ZIKV
display features of microcephaly.102 Severe acute respiratory syndrome coronavirus
2 (SARS-COV-2) is the culprit of the global COVID-19 pandemic in 2020. Many pa-
tients infected with SARS-COV-2 developed neurological symptoms, such as dizzi-
ness, sensory disturbances, Guillain-Barre, skeletal and neuromuscular syndromes,
encephalopathy, cerebrovascular disease, altered mental status, and seizures.107
Previous studies have demonstrated that SARS-COV-2 has neurotropism, and the
MAP2-positive neurons of brain organoids are present in ACE2, the receptor for
the SARS-CoV-2 viral entry.108 In addition, the virus also has strong tropism to the
choroid plexus epithelial cells. The virus enters the CNS as a result of blood-brain-
barrier (BBB) damage, as demonstrated in brain organoids.109

Neuropsychiatric disease
Many neuropsychiatric disorders, such as autism, schizophrenia, bipolar disorder,
and major depressive disorder (MDD), have important social and medical implica-
tions. Several studies have used neurons differentiated from patient-derived iPSCs
to study psychiatric disorders.110 Cortical organoids as models of autism spectrum
disorders were generated from patient iPSCs with 16p11.2 copy number variation
(CNV).91 The CNV dosage affected brain organoid development by disrupting
neuronal maturation and the ratio of neurons to neural progenitors. Schizo-
phrenia-patient-derived organoids mimicked the first trimester of brain develop-
ment in utero and revealed that nuclear (n)FGFR1 was abundantly expressed by
developing subcortical cells, but not derived from neurons in the cortical zone.
Depletion in neuronal committed cells (NCCs) resulted in early cortical dysplasia
in organoids.92 Single-cell sequencing of twins-derived cerebral organoids with bi-
polar disorder reveals an excitation-inhibitory imbalance in the developing brain.111
Further, Wang et al.112 demonstrated that the constitutive activity of serotonin re-
ceptor six caused depression-like electrophysiological features in mice and structure
changes in brain organoids. However, brain organoids have intrinsic flaws to model
MDD because of their lack of mature neural networks and circuits. Therefore, they
have not yet been widely used in studies of neuropsychiatric diseases. Nevertheless,

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many neuropsychiatric disorders exhibit deficits in the postnatal process, which

might be mimicked and manipulated in brain organoids.

Brain tumor
Patient-derived xenografts and tumor spheroids have greatly advanced the under-
standing of brain tumorigenesis.113 Recently, glioblastoma organoids (GBOs)
were generated to understand the inter- and intra-tumor heterogeneity.93 However,
these GBOs did not recapitulate the critical features of glioma stem cells (GSCs) that
invade brain tissues to cause tumor spread. Co-culture of GSCs with cerebral orga-
noids or GBOs mimicked the process of GSCs invasion and evaluated molecules to
impede or inhibit this process.93–95 Collectively, brain organoids as basis of glioma
models can be used to explore the underlying mechanism and manipulation of GSCs
interacting with neurons. However, the current brain organoids lack sufficiently
mature neurons, astrocytes, microglia, endothelial cells, and vasculature; they
cannot fully mimic the tumor microenvironment of gliomas in humans. Therefore,
advanced organoids comprising all major types of neurons and non-neuronal types
of cells and vasculature simultaneously in brain organoids are in high demand to bet-
ter mimic the glioma environment in vitro.

Although it is still controversial whether brain organoids can be used as a drug-

screening platform, there are many works that have demonstrated the efficacy of
brain organoids in drug evaluation. Park et al.114 developed a logic network-based,
drug-screening platform for AD that combined mathematical simulation, AD patho-
logical feature extraction, and brain organoid modeling. They used 1,300 organoids
from 11 patients to construct a high-content screening system and to test 11 US
Food and Drug Administration (FDA)-approved drugs that permeated BBB. Wata-
nabe et al.115 used ZIKV-infected brain organoids to test whether antiviral drugs
could attenuate the teratogenic effects of ZIKV infection on cortical development.
And brain organoid invasion assays have allowed to differentiate the behavior of pri-
mary and recurrent GSCs to provide different diagnosis and treatment options for
primary and recurrent patients.95 The batch consistency of brain organoids has
been a long-standing controversy in drugs screening. Velasco et al.38 have improved
the method of producing large numbers of brain organoids. scRNA-seq results
showed that 95% of the organoids had indistinguishable proportions of different
cell types.

CONVERGING MACHINE LEARNING AND BRAIN ORGANOID

MODELING
Machine learning (ML) enables computers to learn from experiences and process
and analyze data more rapidly and robustly than manual operations. Moreover,
mapping function between the input and output can be learned via supervised ML
and then used to predict the outcomes. Combining ML algorithms with brain orga-
noids offers an emerging opportunity in biotechnology that benefits brain organoids
and artificial intelligence mutually (Figure 3).116,117 Though the practices of ML on
brain organoids remain limited, the opportunities can be prospected from successes
proven in organoids other than brain.

ML can assist in predicting determinants for manipulation of brain organoids. Brain

organoids can be regarded as intelligent cores with simplified biophysical neural
network of human brains, given modulation can induce corresponding responses.
Genetic modulation of organoids might trigger the presence of disease-related fea-
tures or changes of specific development trajectory. It is laborious and inefficient to

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find the potential risk genes manually; however, this can be achieved quickly and
efficiently through computational approaches, like artificial intelligence (AI)-based
technologies, which decreases the off-target probability and better controls the
gene editing toward the designated.118 More importantly, to activate or deactivate
neurons more precisely, it is useful to localize and classify neurons before inputting
electrical or optical stimuli. Improved localization and classification of neurons was
accomplished by converging deep learning and biophysical modeling.119 Other
than in aid of better input of brain organoids, ML can also facilitate analyzing output
data for assessment of brain organoids.

ML can augment data acquisition and mining from mono- to multi-scale and modal-
ity. Deep learning can improve time-lapse signal-to-noise ratio, which enables
acquisition of calcium imaging data at higher spatiotemporal resolution.120 A similar
strategy has been leveraged to image organoids.121 As for data mining, a combina-
tion of high content imaging and ML has been implemented into quantitate analysis
of organoids at cellular, multicellular, and whole-organoid scales.122,123 Moreover,
multi-scale and multimodality comparative analysis of the molecular, cellular,
spatial, cytoarchitectural, and organoid-wide properties of brain organoids was em-
powered by neural-network-based image analysis.124 Alternatively, trained neural
networks, capable of predicting the age of preterm infant, were applied to predict
developmental time of brain organoids.27 Also, machine-learning-aided analysis
can assure the regionalization and molecular signature of region-specific brain orga-
noid.36 Besides, hidden information is embedded among different modalities and
scales. Data of organoids can be fused together to reduce redundance and derivate
unanticipated outcomes, where transfer learning might come in handy by learning
the experiences cross-scale and cross-modality and predict new tasks.

In addition, ML can predict responses of brain organoids by learning the mapping

function between the output and input data of organoids. It was reported that ML
approaches could differentiate a given genetic mutation from the control by study-
ing the electrical signals from iPSC-derived organoids.125 Likewise, ML classifier was
applied on mapping the relationship between the input data (e.g., drug stimulation)
and the high-content output data (e.g., evaluation of response to drugs) to predict
neurotoxin-induced perturbations.126 Undoubtedly, ML algorithms, on the basis of
sufficient data for training, can establish target functions between digitalized manip-
ulation (input signals to organoids) and multi-omics analysis (output signals from
organoids) of brain organoids. It will be extremely beneficial to foresee the dynamics
of brain organoid networks in response to varying patterns of manipulation,
providing hints for mechanism of manipulating the brain organoids toward putative
results.

In turn, the convergence of brain organoids modeling and ML might offer insights for
the next generation of ML and AI. Brain organoid is a product on the interface of
biology and engineering and aids to understand the biophysical constrains of human
brain. Researchers argue that using structure-function theory and brain organoids
can deepen the understanding of human intelligence.127 In such case, brain organo-
ids may contribute impressively to inspire neuroscientists in setting up the frame-
work of artificial neural networks with more appropriate physical constraints, thus
providing cues for innovating brain-inspired computing algorithms. It is crucial to
note that the convergence of brain organoids modeling and ML is also promising
in enlightening the design of neuromorphic hardware. Recently, a photonic hard-
ware that mimicked neurons and synapses was reported capable of self-learning.128
Under this circumstance, it is possible that hardware inspired by brain organoids can

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fulfill the demand of fast, efficient, and low-energy computing for the next genera-
tion of ML and AI.

CONCLUSION AND PROSPECT

Great efforts have been made in fine-tuning the current protocols and disease
modeling of brain organoids. It is promising that the technical concerns, like repro-
ducibility, long-term culture, and neuronal activity of brain organoids can be over-
come someday. Essentially, the ethical and moral concerns also draw the attention
of scientists and have been heatedly debated.129–132 The discussions mainly focus
on whether the brain organoids could or have consciousness, ethical issues of trans-
plantations in rodents and other species, or so-called human-animal chimeras and
the consent and ownership of brain organoids.133 Fortunately, suitable ethical
frameworks and guidelines are being widely discussed so as to provide guidance
for research about human brain organoids.134,135

After overcoming the concerns above, the ultimate goal, translating human brain in a
dish, might be achieved in this reductionist way. It is possible to interface brain or-
ganoids with integrated functional neural probes in brain-computer (or machine)
interface (BCI) to output signals of organoids (Figure 3), then give commands to ma-
chines to perform tasks fulfilling consciousness of human brains. In turn, BCI struc-
ture can also implement the signaling-commanding loop from machine to brain,
which is analogous to the scenario of using organoids as processing cores or training
targets. Brain organoids are non-invasive, trial-and-error testing models that cannot
only offer high-throughput choices but enable real-time recording and in-depth
feature extraction with ML algorithms. Many invasive experimental trials that cannot
be performed in human brains may be progressed in organoids with intelligent feed-
back of data acquisition-feature extraction-decision making-organoid optimization.
For example, the biofeedback of brain organoids with varying neuromodulation pat-
terns may improve deep-brain stimulations (DBSs), a technique applied to regulate
disordered neural activity in clinical treatment of PD. In this manner, the closed-loop
BCI made up of neuronal culture, machinery system, and ML for predictive analysis
shows tremendous potential in carrying out pilot clinical trials, validating novel treat-
ments, and exploring plausible explanations for diverse neurological disorders.

ACKNOWLEDGMENTS
The work was supported by the National Natural Science Foundation of China (grant
numbers: 61971255 and 82111530212), the Natural Science Foundation of Guang-
dong Province (grant number: 2021B1515020092), the Shenzhen Science and
Technology Innovation Commission (grant numbers: WDZC20200821141349001,
RCYX20200714114736146, KCXFZ20201221173207022, and KCXFZ202002011
01050887), and the Shenzhen Bay Laboratory Fund (grant number: SZBL20200
90501014).

AUTHOR CONTRIBUTIONS
H.Z. and Y.F. reviewed the literature and provided a substantial contribution to dis-
cussions of the content. S.M., H.Z., Y.F., and J.T. contributed to paper writing and
revising. S.M. conceived and advised on this work. All authors contributed to
discussion.

DECLARATION OF INTERESTS
The authors declare no competing interests.

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REFERENCES
1. Teratani, T., Mikami, Y., Nakamoto, N.,
Suzuki, T., Harada, Y., Okabayashi, K.,
Hagihara, Y., Taniki, N., Kohno, K., Shibata, S.,
et al. (2020). The liver-brain-gut neural arc
maintains the T(reg) cell niche in the gut.
Nature 585, 591–596. https://doi.org/10.
1038/s41586-020-2425-3.
2. Morais, L.H., Schreiber, H.L.t., and
Mazmanian, S.K. (2021). The gut microbiota-
brain axis in behaviour and brain disorders.
Nat. Rev. Microbiol. 19, 241–255. https://doi.
org/10.1038/s41579-020-00460-0.
3. Zhang, X., Lei, B., Yuan, Y., Zhang, L., Hu, L.,
Jin, S., Kang, B., Liao, X., Sun, W., Xu, F., et al.
(2020). Brain control of humoral immune
responses amenable to behavioural
modulation. Nature 581, 204–208. https://doi.
org/10.1038/s41586-020-2235-7.
4. Arlotta, P. (2018). Organoids required! A new
path to understanding human brain
development and disease. Nat. Methods 15,
27–29. https://doi.org/10.1038/nmeth.4557.
5. Kelava, I., and Lancaster, M.A. (2016). Stem
cell models of human brain development.
Cell Stem Cell 18, 736–748. https://doi.org/
10.1016/j.stem.2016.05.022.
6. Lui, J.H., Hansen, D.V., and Kriegstein, A.R.
(2011). Development and evolution of the
human neocortex. Cell 146, 18–36. https://
doi.org/10.1016/j.cell.2011.06.030.
7. Watanabe, K., Kamiya, D., Nishiyama, A.,
Katayama, T., Nozaki, S., Kawasaki, H.,
Watanabe, Y., Mizuseki, K., and Sasai, Y.
(2005). Directed differentiation of
telencephalic precursors from embryonic
stem cells. Nat. Neurosci. 8, 288–296. https://
doi.org/10.1038/nn1402.
8. Eiraku, M., Watanabe, K., Matsuo-Takasaki,
M., Kawada, M., Yonemura, S., Matsumura,
M., Wataya, T., Nishiyama, A., Muguruma, K.,
and Sasai, Y. (2008). Self-organized formation
of polarized cortical tissues from ESCs and its
active manipulation by extrinsic signals. Cell
Stem Cell 3, 519–532. https://doi.org/10.
1016/j.stem.2008.09.002.
9. Kadoshima, T., Sakaguchi, H., Nakano, T.,
Soen, M., Ando, S., Eiraku, M., and Sasai, Y.
(2013). Self-organization of axial polarity,
inside-out layer pattern, and species-specific
progenitor dynamics in human ES cell-
derived neocortex. Proc. Natl. Acad. Sci. USA
110, 20284–20289. https://doi.org/10.1073/
pnas.1315710110.
10. Zhang, S.C., Wernig, M., Duncan, I.D., Brüstle,
O., and Thomson, J.A. (2001). In vitro
differentiation of transplantable neural
precursors from human embryonic stem cells.
Nat. Biotechnol. 19, 1129–1133. https://doi.
org/10.1038/nbt1201-1129.
11. Ying, Q.L., Stavridis, M., Griffiths, D., Li, M.,
and Smith, A. (2003). Conversion of embryonic
stem cells into neuroectodermal precursors in
adherent monoculture. Nat. Biotechnol. 21,
183–186. https://doi.org/10.1038/nbt780.
12. Di Lullo, E., and Kriegstein, A.R. (2017). The
use of brain organoids to investigate neural
development and disease. Nat. Rev.
14 Cell Reports Physical Science 3, 100974, July 20, 2022
Neurosci. 18, 573–584. https://doi.org/10.
1038/nrn.2017.107.
13. Sidhaye, J., and Knoblich, J.A. (2021). Brain
organoids: an ensemble of bioassays to
investigate human neurodevelopment and
disease. Cell Death Differ. 28, 52–67. https://
doi.org/10.1038/s41418-020-0566-4.
14. Del Dosso, A., Urenda, J.P., Nguyen, T., and
Quadrato, G. (2020). Upgrading the
physiological relevance of human brain
organoids. Neuron 107, 1014–1028. https://
doi.org/10.1016/j.neuron.2020.08.029.
15. Chiaradia, I., and Lancaster, M.A. (2020). Brain
organoids for the study of human
neurobiology at the interface of in vitro and
in vivo. Nat. Neurosci. 23, 1496–1508. https://
doi.org/10.1038/s41593-020-00730-3.
16. Pașca, S.P. (2018). The rise of three-
dimensional human brain cultures. Nature
553, 437–445. https://doi.org/10.1038/
nature25032.
17. Lancaster, M.A., Renner, M., Martin, C.A.,
Wenzel, D., Bicknell, L.S., Hurles, M.E.,
Homfray, T., Penninger, J.M., Jackson, A.P.,
and Knoblich, J.A. (2013). Cerebral organoids
model human brain development and
microcephaly. Nature 501, 373–379. https://
doi.org/10.1038/nature12517.
18. Lancaster, M.A., Corsini, N.S., Wolfinger, S.,
Gustafson, E.H., Phillips, A.W., Burkard, T.R.,
Otani, T., Livesey, F.J., and Knoblich, J.A.
(2017). Guided self-organization and cortical
plate formation in human brain organoids.
Nat. Biotechnol. 35, 659–666. https://doi.org/
10.1038/nbt.3906.
19. Quadrato, G., Nguyen, T., Macosko, E.Z.,
Sherwood, J.L., Min Yang, S., Berger, D.R.,
Maria, N., Scholvin, J., Goldman, M., Kinney,
J.P., et al. (2017). Cell diversity and network
dynamics in photosensitive human brain
organoids. Nature 545, 48–53. https://doi.
org/10.1038/nature22047.
20. Giandomenico, S.L., Mierau, S.B., Gibbons,
G.M., Wenger, L.M.D., Masullo, L., Sit, T.,
Sutcliffe, M., Boulanger, J., Tripodi, M.,
Derivery, E., et al. (2019). Cerebral organoids
at the air-liquid interface generate diverse
nerve tracts with functional output. Nat.
Neurosci. 22, 669–679. https://doi.org/10.
1038/s41593-019-0350-2.
21. Ormel, P.R., Vieira de Sá, R., van Bodegraven,
E.J., Karst, H., Harschnitz, O., Sneeboer,
M.A.M., Johansen, L.E., van Dijk, R.E.,
Scheefhals, N., Berdenis van Berlekom, A.,
et al. (2018). Microglia innately develop within
cerebral organoids. Nat. Commun. 9, 4167.
https://doi.org/10.1038/s41467-018-06684-2.
22. Pham, M.T., Pollock, K.M., Rose, M.D., Cary,
W.A., Stewart, H.R., Zhou, P., Nolta, J.A., and
Waldau, B. (2018). Generation of human
vascularized brain organoids. Neuroreport
29, 588–593. https://doi.org/10.1097/wnr.
0000000000001014.
xca, A.M., Sloan, S.A., Clarke, L.E., Tian, Y.,
23. Pas
Makinson, C.D., Huber, N., Kim, C.H., Park,
J.Y., O’Rourke, N.A., Nguyen, K.D., et al.
(2015). Functional cortical neurons and
astrocytes from human pluripotent stem cells
Perspective
in 3D culture. Nat. Methods 12, 671–678.
https://doi.org/10.1038/nmeth.3415.
24. Sloan, S.A., Darmanis, S., Huber, N., Khan,
T.A., Birey, F., Caneda, C., Reimer, R., Quake,
S.R., Barres, B.A., and Pasxca, S.P. (2017).
Human astrocyte maturation captured in 3D
cerebral cortical spheroids derived from
pluripotent stem cells. Neuron 95, 779–
790.e6. https://doi.org/10.1016/j.neuron.
2017.07.035.
25. Marton, R.M., Miura, Y., Sloan, S.A., Li, Q.,
Revah, O., Levy, R.J., Huguenard, J.R., and
Pașca, S.P. (2019). Differentiation and
maturation of oligodendrocytes in human
three-dimensional neural cultures. Nat.
Neurosci. 22, 484–491. https://doi.org/10.
1038/s41593-018-0316-9.
26. Madhavan, M., Nevin, Z.S., Shick, H.E.,
Garrison, E., Clarkson-Paredes, C., Karl, M.,
Clayton, B.L.L., Factor, D.C., Allan, K.C.,
Barbar, L., et al. (2018). Induction of
myelinating oligodendrocytes in human
cortical spheroids. Nat. Methods 15, 700–706.
https://doi.org/10.1038/s41592-018-0081-4.
27. Trujillo, C.A., Gao, R., Negraes, P.D., Gu, J.,
Buchanan, J., Preissl, S., Wang, A., Wu, W.,
Haddad, G.G., Chaim, I.A., et al. (2019).
Complex oscillatory waves emerging from
cortical organoids model early human brain
network development. Cell Stem Cell 25, 558–
569.e7. https://doi.org/10.1016/j.stem.2019.
08.002.
28. Qian, X., Nguyen, H.N., Song, M.M., Hadiono,
C., Ogden, S.C., Hammack, C., Yao, B.,
Hamersky, G.R., Jacob, F., Zhong, C., et al.
(2016). Brain-region-specific organoids using
mini-bioreactors for modeling ZIKV exposure.
Cell 165, 1238–1254. https://doi.org/10.1016/
j.cell.2016.04.032.
29. Qian, X., Su, Y., Adam, C.D., Deutschmann,
A.U., Pather, S.R., Goldberg, E.M., Su, K., Li,
S., Lu, L., Jacob, F., et al. (2020). Sliced human
cortical organoids for modeling distinct
cortical layer formation. Cell Stem Cell 26,
766–781.e9. https://doi.org/10.1016/j.stem.
2020.02.002.
30. Popova, G., Soliman, S.S., Kim, C.N., Keefe,
M.G., Hennick, K.M., Jain, S., Li, T., Tejera, D.,
Shin, D., Chhun, B.B., et al. (2021). Human
microglia states are conserved across
experimental models and regulate neural
stem cell responses in chimeric organoids.
Cell Stem Cell 28, 2153–2166.e6. https://doi.
org/10.1016/j.stem.2021.08.015.
31. Mansour, A.A., Gonçalves, J.T., Bloyd, C.W.,
Li, H., Fernandes, S., Quang, D., Johnston, S.,
Parylak, S.L., Jin, X., and Gage, F.H. (2018). An
in vivo model of functional and vascularized
human brain organoids. Nat. Biotechnol. 36,
432–441. https://doi.org/10.1038/nbt.4127.
32. Shi, Y., Sun, L., Wang, M., Liu, J., Zhong, S., Li,
R., Li, P., Guo, L., Fang, A., Chen, R., et al.
(2020). Vascularized human cortical organoids
(vOrganoids) model cortical development
in vivo. PLoS Biol. 18, e3000705. https://doi.
org/10.1371/journal.pbio.3000705.
33. Cakir, B., Xiang, Y., Tanaka, Y., Kural, M.H.,
Parent, M., Kang, Y.J., Chapeton, K.,
Patterson, B., Yuan, Y., He, C.S., et al. (2019).

Perspective
Engineering of human brain organoids with a
functional vascular-like system. Nat. Methods
16, 1169–1175. https://doi.org/10.1038/
s41592-019-0586-5.
34. Birey, F., Andersen, J., Makinson, C.D., Islam,
S., Wei, W., Huber, N., Fan, H.C., Metzler,
K.R.C., Panagiotakos, G., Thom, N., et al.
(2017). Assembly of functionally integrated
human forebrain spheroids. Nature 545,
54–59. https://doi.org/10.1038/nature22330.
35. Jo, J., Xiao, Y., Sun, A.X., Cukuroglu, E., Tran,
H.D., Göke, J., Göke, J., Tan, Z.Y., Saw, T.Y.,
Tan, C.P., et al. (2016). Midbrain-like
organoids from human pluripotent stem cells
contain functional dopaminergic and
neuromelanin-producing neurons. Cell Stem
Cell 19, 248–257. https://doi.org/10.1016/j.
stem.2016.07.005.
36. Huang, W.K., Wong, S.Z.H., Pather, S.R.,
Nguyen, P.T.T., Zhang, F., Zhang, D.Y., Zhang,
Z., Lu, L., Fang, W., Chen, L., et al. (2021).
Generation of hypothalamic arcuate
organoids from human induced pluripotent
stem cells. Cell Stem Cell 28, 1657–1670.e10.
https://doi.org/10.1016/j.stem.2021.04.006.
37. Xiang, Y., Tanaka, Y., Cakir, B., Patterson, B.,
Kim, K.Y., Sun, P., Kang, Y.J., Zhong, M., Liu,
X., Patra, P., et al. (2019). hESC-derived
thalamic organoids form reciprocal
projections when fused with cortical
organoids. Cell Stem Cell 24, 487–497.e7.
https://doi.org/10.1016/j.stem.2018.12.015.
38. Velasco, S., Kedaigle, A.J., Simmons, S.K.,
Nash, A., Rocha, M., Quadrato, G., Paulsen,
B., Nguyen, L., Adiconis, X., Regev, A., et al.
(2019). Individual brain organoids
reproducibly form cell diversity of the human
cerebral cortex. Nature 570, 523–527. https://
doi.org/10.1038/s41586-019-1289-x.
39. Marton, R.M., and Pașca, S.P. (2020).
Organoid and assembloid technologies for
investigating cellular crosstalk in human brain
development and disease. Trends Cell Biol.
30, 133–143. https://doi.org/10.1016/j.tcb.
2019.11.004.
40. Kelley, K.W., and Pașca, S.P. (2022). Human
brain organogenesis: toward a cellular
understanding of development and disease.
Cell 185, 42–61. https://doi.org/10.1016/j.cell.
2021.10.003.
xca, S.P. (2019). Assembling human brain
41. Pas
organoids. Science 363, 126–127. https://doi.
org/10.1126/science.aau5729.
42. Bagley, J.A., Reumann, D., Bian, S., Lévi-
Strauss, J., and Knoblich, J.A. (2017). Fused
cerebral organoids model interactions
between brain regions. Nat. Methods 14,
743–751. https://doi.org/10.1038/nmeth.
4304.
43. Xiang, Y., Tanaka, Y., Patterson, B., Kang, Y.J.,
Govindaiah, G., Roselaar, N., Cakir, B., Kim,
K.Y., Lombroso, A.P., Hwang, S.M., et al.
(2017). Fusion of regionally specified hPSC-
derived organoids models human brain
development and interneuron migration. Cell
Stem Cell 21, 383–398.e7. https://doi.org/10.
1016/j.stem.2017.07.007.
44. Miura, Y., Li, M.Y., Birey, F., Ikeda, K., Revah,
O., Thete, M.V., Park, J.Y., Puno, A., Lee, S.H.,
Porteus, M.H., and Pașca, S.P. (2020).
Generation of human striatal organoids and
cortico-striatal assembloids from human
pluripotent stem cells. Nat. Biotechnol. 38,
1421–1430. https://doi.org/10.1038/s41587-
020-00763-w.
45. Kasai, T., Suga, H., Sakakibara, M., Ozone, C.,
Matsumoto, R., Kano, M., Mitsumoto, K.,
Ogawa, K., Kodani, Y., Nagasaki, H., et al.
(2020). Hypothalamic contribution to pituitary
functions is recapitulated in vitro using 3D-
cultured human iPS cells. Cell Rep. 30, 18–
24.e5. https://doi.org/10.1016/j.celrep.2019.
12.009.
46. Andersen, J., Revah, O., Miura, Y., Thom, N.,
Amin, N.D., Kelley, K.W., Singh, M., Chen, X.,
Thete, M.V., and Walczak, E.M. (2020).
Generation of functional human 3D cortico-
motor assembloids. Cell 183, 1913–1929.e26.
https://doi.org/10.1016/j.cell.2020.11.017.
47. Cederquist, G.Y., Asciolla, J.J., Tchieu, J.,
Walsh, R.M., Cornacchia, D., Resh, M.D., and
Studer, L. (2019). Specification of positional
identity in forebrain organoids. Nat.
Biotechnol. 37, 436–444. https://doi.org/10.
1038/s41587-019-0085-3.
48. Renner, M., Lancaster, M.A., Bian, S., Choi, H.,
Ku, T., Peer, A., Chung, K., and Knoblich, J.A.
(2017). Self-organized developmental
patterning and differentiation in cerebral
organoids. EMBO J. 36, 1316–1329. https://
doi.org/10.15252/embj.201694700.
49. Abud, E.M., Ramirez, R.N., Martinez, E.S.,
Healy, L.M., Nguyen, C.H.H., Newman, S.A.,
Yeromin, A.V., Scarfone, V.M., Marsh, S.E.,
Fimbres, C., et al. (2017). iPSC-derived human
microglia-like cells to study neurological
diseases. Neuron 94, 278–293.e9. https://doi.
org/10.1016/j.neuron.2017.03.042.
50. Song, L., Yuan, X., Jones, Z., Vied, C., Miao, Y.,
Marzano, M., Hua, T., Sang, Q.X.A., Guan, J.,
Ma, T., et al. (2019). Functionalization of brain
region-specific spheroids with isogenic
microglia-like cells. Sci. Rep. 9, 11055. https://
doi.org/10.1038/s41598-019-47444-6.
51. Lin, Y.T., Seo, J., Gao, F., Feldman, H.M., Wen,
H.L., Penney, J., Cam, H.P., Gjoneska, E., Raja,
W.K., Cheng, J., et al. (2018). APOE4 causes
widespread molecular and cellular alterations
associated with alzheimer’s disease
phenotypes in human iPSC-derived brain cell
types. Neuron 98, 1141–1154.e7. https://doi.
org/10.1016/j.neuron.2018.05.008.
52. Bennett, M.L., Song, H., and Ming, G.L. (2021).
Microglia modulate neurodevelopment in
human neuroimmune organoids. Cell Stem
Cell 28, 2035–2036. https://doi.org/10.1016/j.
stem.2021.11.005.
53. Wörsdörfer, P., Dalda, N., Kern, A., Krüger, S.,
Wagner, N., Kwok, C.K., Henke, E., and Ergün,
S. (2019). Generation of complex human
organoid models including vascular networks
by incorporation of mesodermal progenitor
cells. Sci. Rep. 9, 15663. https://doi.org/10.
1038/s41598-019-52204-7.
54. Matsui, T.K., Tsuru, Y., Hasegawa, K., and
Kuwako, K.I. (2021). Vascularization of human
brain organoids. Stem Cells 39, 1017–1024.
https://doi.org/10.1002/stem.3368.
55. Poli, D., Magliaro, C., and Ahluwalia, A. (2019).
Experimental and computational methods for
Cell Reports Physical Science 3, 100974, July 20, 2022
ll
OPEN ACCESS
the study of cerebral organoids: a review.
Front. Neurosci. 13, 162. https://doi.org/10.
3389/fnins.2019.00162.
56. Dekkers, J.F., Alieva, M., Wellens, L.M.,
Ariese, H.C.R., Jamieson, P.R., Vonk, A.M.,
Amatngalim, G.D., Hu, H., Oost, K.C.,
Snippert, H.J.G., et al. (2019). High-resolution
3D imaging of fixed and cleared organoids.
Nat. Protoc. 14, 1756–1771. https://doi.org/
10.1038/s41596-019-0160-8.
57. Atamian, A., Cordón-Barris, L., and Quadrato,
G. (2021). Taming human brain organoids one
cell at a time. Semin. Cell Dev. Biol. 111,
23–31. https://doi.org/10.1016/j.semcdb.
2020.05.022.
58. Tanaka, Y., Cakir, B., Xiang, Y., Sullivan, G.J.,
and Park, I.H. (2020). Synthetic analyses of
single-cell transcriptomes from multiple brain
organoids and fetal brain. Cell Rep. 30, 1682–
1689.e3. https://doi.org/10.1016/j.celrep.
2020.01.038.
59. Kanton, S., Boyle, M.J., He, Z., Santel, M.,
Weigert, A., Sanchı́s-Calleja, F., Guijarro, P.,
Sidow, L., Fleck, J.S., Han, D., et al. (2019).
Organoid single-cell genomic atlas uncovers
human-specific features of brain
development. Nature 574, 418–422. https://
doi.org/10.1038/s41586-019-1654-9.
60. Pelkonen, A., Pistono, C., Klecki, P., Gómez-
Budia, M., Dougalis, A., Konttinen, H.,
Stanová, I., Fagerlund, I., Leinonen, V.,
Korhonen, P., and Malm, T. (2021). Functional
characterization of human pluripotent stem
cell-derived models of the brain with
microelectrode arrays. Cells 11, 106. https://
doi.org/10.3390/cells11010106.
61. Tasnim, K., and Liu, J. (2022). Emerging
bioelectronics for brain organoid
electrophysiology. J. Mol. Biol. 434, 167165.
https://doi.org/10.1016/j.jmb.2021.167165.
62. Hong, G., and Lieber, C.M. (2019). Novel
electrode technologies for neural recordings.
Nat. Rev. Neurosci. 20, 330–345. https://doi.
org/10.1038/s41583-019-0140-6.
63. Sakaguchi, H., Ozaki, Y., Ashida, T.,
Matsubara, T., Oishi, N., Kihara, S., and
Takahashi, J. (2019). Self-organized
synchronous calcium transients in a cultured
human neural network derived from cerebral
organoids. Stem Cell Rep. 13, 458–473.
https://doi.org/10.1016/j.stemcr.2019.05.029.
64. Park, Y., Franz, C.K., Ryu, H., Luan, H., Cotton,
K.Y., Kim, J.U., Chung, T.S., Zhao, S., Vazquez-
Guardado, A., Yang, D.S., et al. (2021). Three-
dimensional, multifunctional neural interfaces
for cortical spheroids and engineered
assembloids. Sci. Adv. 7, eabf9153. https://
doi.org/10.1126/sciadv.abf9153.
65. Zafeiriou, M.P., Bao, G., Hudson, J., Halder,
R., Blenkle, A., Schreiber, M.K., Fischer, A.,
Schild, D., and Zimmermann, W.H. (2020).
Developmental GABA polarity switch and
neuronal plasticity in Bioengineered
Neuronal Organoids. Nat. Commun. 11, 3791.
https://doi.org/10.1038/s41467-020-17521-w.
66. Osaki, T., and Ikeuchi, Y. (2021). Complex
activity and short-term memories in
reciprocally connected cerebral organoids.
Preprint at bioRxiv. https://doi.org/10.1101/
2021.02.16.431387.
15
 ll
OPEN ACCESS
67. Wang, Y., Wang, L., Zhu, Y., and Qin, J. (2018).
Human brain organoid-on-a-chip to model
prenatal nicotine exposure. Lab Chip 18,
851–860. https://doi.org/10.1039/c7lc01084b.
68. Lee, C.T., Chen, J., Kindberg, A.A., Bendriem,
R.M., Spivak, C.E., Williams, M.P., Richie, C.T.,
Handreck, A., Mallon, B.S., Lupica, C.R., et al.
(2017). CYP3A5 mediates effects of cocaine
on human neocorticogenesis: studies using
an in vitro 3D self-organized hPSC model with
a single cortex-like unit.
Neuropsychopharmacology 42, 774–784.
https://doi.org/10.1038/npp.2016.156.
69. Ao, Z., Cai, H., Havert, D.J., Wu, Z., Gong, Z.,
Beggs, J.M., Mackie, K., and Guo, F. (2020).
One-stop microfluidic assembly of human
brain organoids to model prenatal cannabis
exposure. Anal. Chem. 92, 4630–4638. https://
doi.org/10.1021/acs.analchem.0c00205.
70. Shiri, Z., Simorgh, S., Naderi, S., and
Baharvand, H. (2019). Optogenetics in the era
of cerebral organoids. Trends Biotechnol. 37,
1282–1294. https://doi.org/10.1016/j.tibtech.
2019.05.009.
71. Li, Y., Muffat, J., Omer, A., Bosch, I., Lancaster,
M.A., Sur, M., Gehrke, L., Knoblich, J.A., and
Jaenisch, R. (2017). Induction of expansion
and folding in human cerebral organoids. Cell
Stem Cell 20, 385–396.e3. https://doi.org/10.
1016/j.stem.2016.11.017.
72. Karzbrun, E., Kshirsagar, A., Cohen, S.R.,
Hanna, J.H., and Reiner, O. (2018). Human
brain organoids on a chip reveal the physics of
folding. Nat. Phys. 14, 515–522. https://doi.
org/10.1038/s41567-018-0046-7.
73. Yin, J., and VanDongen, A.M. (2021).
Enhanced neuronal activity and asynchronous
calcium transients revealed in a 3D organoid
model of alzheimer’s disease. ACS Biomater.
Sci. Eng. 7, 254–264. https://doi.org/10.1021/
acsbiomaterials.0c01583.
74. Hernández, D., Rooney, L.A., Daniszewski, M.,
Gulluyan, L., Liang, H.H., Cook, A.L., Hewitt,
A.W., and Pébay, A. (2022). Culture
variabilities of human iPSC-derived cerebral
organoids are a major issue for the modelling
of phenotypes observed in alzheimer’s
disease. Stem Cell Rev. Rep. 18, 718–731.
https://doi.org/10.1007/s12015-021-10147-5.
75. Zhao, J., Fu, Y., Yamazaki, Y., Ren, Y., Davis,
M.D., Liu, C.C., Lu, W., Wang, X., Chen, K.,
Cherukuri, Y., et al. (2020). APOE4
exacerbates synapse loss and
neurodegeneration in Alzheimer’s disease
patient iPSC-derived cerebral organoids. Nat.
Commun. 11, 5540. https://doi.org/10.1038/
s41467-020-19264-0.
76. Ghatak, S., Dolatabadi, N., Trudler, D., Zhang,
X., Wu, Y., Mohata, M., Ambasudhan, R.,
Talantova, M., and Lipton, S.A. (2019).
Mechanisms of hyperexcitability in
Alzheimer’s disease hiPSC-derived neurons
and cerebral organoids vs isogenic controls.
Elife 8, e50333. https://doi.org/10.7554/eLife.
50333.
77. Pérez, M.J., Ivanyuk, D., Panagiotakopoulou,
V., Di Napoli, G., Kalb, S., Brunetti, D., Al-
Shaana, R., Kaeser, S.A., Fraschka, S.A.K.,
Jucker, M., et al. (2021). Loss of function of the
mitochondrial peptidase PITRM1 induces
proteotoxic stress and Alzheimer’s disease-
16 Cell Reports Physical Science 3, 100974, July 20, 2022
like pathology in human cerebral organoids.
Mol. Psychiatry 26, 5733–5750. https://doi.
org/10.1038/s41380-020-0807-4.
78. Raja, W.K., Mungenast, A.E., Lin, Y.T., Ko, T.,
Abdurrob, F., Seo, J., and Tsai, L.H. (2016).
Self-organizing 3D human neural tissue
derived from induced pluripotent stem cells
recapitulate alzheimer’s disease phenotypes.
PLoS One 11, e0161969. https://doi.org/10.
1371/journal.pone.0161969.
79. Chen, X., Sun, G., Tian, E., Zhang, M.,
Davtyan, H., Beach, T.G., Reiman, E.M.,
Blurton-Jones, M., Holtzman, D.M., and Shi, Y.
(2021). Modeling sporadic alzheimer’s
disease in human brain organoids under
serum exposure. Adv. Sci. (Weinh) 8,
e2101462. https://doi.org/10.1002/advs.
202101462.
80. Smits, L.M., Reinhardt, L., Reinhardt, P.,
Glatza, M., Monzel, A.S., Stanslowsky, N.,
Rosato-Siri, M.D., Zanon, A., Antony, P.M.,
Bellmann, J., et al. (2019). Modeling
Parkinson’s disease in midbrain-like
organoids. NPJ Parkinsons Dis. 5, 5. https://
doi.org/10.1038/s41531-019-0078-4.
81. Kim, H., Park, H.J., Choi, H., Chang, Y., Park,
H., Shin, J., Kim, J., Lengner, C.J., Lee, Y.K.,
and Kim, J. (2019). Modeling G2019S-LRRK2
sporadic Parkinson’s disease in 3D midbrain
organoids. Stem Cell Rep. 12, 518–531.
https://doi.org/10.1016/j.stemcr.2019.01.020.
82. Wulansari, N., Darsono, W.H.W., Woo, H.J.,
Chang, M.Y., Kim, J., Bae, E.J., Sun, W., Lee,
J.H., Cho, I.J., Shin, H., et al. (2021).
Neurodevelopmental defects and
neurodegenerative phenotypes in human
brain organoids carrying Parkinson’s disease-
linked DNAJC6 mutations. Sci. Adv. 7,
eabb1540. https://doi.org/10.1126/sciadv.
abb1540.
83. Zhang, W., Yang, S.L., Yang, M., Herrlinger, S.,
Shao, Q., Collar, J.L., Fierro, E., Shi, Y., Liu, A.,
Lu, H., et al. (2019). Modeling microcephaly
with cerebral organoids reveals a WDR62-
CEP170-KIF2A pathway promoting cilium
disassembly in neural progenitors. Nat.
Commun. 10, 2612. https://doi.org/10.1038/
s41467-019-10497-2.
84. Mellios, N., Feldman, D.A., Sheridan, S.D., Ip,
J.P.K., Kwok, S., Amoah, S.K., Rosen, B.,
Rodriguez, B.A., Crawford, B., Swaminathan,
R., et al. (2018). MeCP2-regulated miRNAs
control early human neurogenesis through
differential effects on ERK and AKT signaling.
Mol. Psychiatry 23, 1051–1065. https://doi.
org/10.1038/mp.2017.86.
85. Bershteyn, M., Nowakowski, T.J., Pollen, A.A.,
Di Lullo, E., Nene, A., Wynshaw-Boris, A., and
Kriegstein, A.R. (2017). Human iPSC-derived
cerebral organoids model cellular features of
lissencephaly and reveal prolonged mitosis of
outer radial glia. Cell Stem Cell 20, 435–
449.e4. https://doi.org/10.1016/j.stem.2016.
12.007.
86. Iefremova, V., Manikakis, G., Krefft, O., Jabali,
A., Weynans, K., Wilkens, R., Marsoner, F.,
Brändl, B., Müller, F.J., Koch, P., and Ladewig,
J. (2017). An organoid-based model of
cortical development identifies non-cell-
autonomous defects in wnt signaling
contributing to Miller-Dieker syndrome. Cell
Perspective
Rep. 19, 50–59. https://doi.org/10.1016/j.
celrep.2017.03.047.
87. Xu, R., Brawner, A.T., Li, S., Liu, J.J., Kim, H.,
Xue, H., Pang, Z.P., Kim, W.Y., Hart, R.P., Liu,
Y., and Jiang, P. (2019). OLIG2 drives
abnormal neurodevelopmental phenotypes
in human iPSC-based organoid and chimeric
mouse models of Down syndrome. Cell Stem
Cell 24, 908–926.e8. https://doi.org/10.1016/
j.stem.2019.04.014.
88. Alic, I., Goh, P.A., Murray, A., Portelius, E.,
Gkanatsiou, E., Gough, G., Mok, K.Y.,
Koschut, D., Brunmeir, R., Yeap, Y.J., et al.
(2021). Patient-specific Alzheimer-like
pathology in trisomy 21 cerebral organoids
reveals BACE2 as a gene dose-sensitive AD
suppressor in human brain. Mol. Psychiatry 26,
5766–5788. https://doi.org/10.1038/s41380-
020-0806-5.
89. Tang, X.Y., Xu, L., Wang, J., Hong, Y., Wang,
Y., Zhu, Q., Wang, D., Zhang, X.Y., Liu, C.Y.,
Fang, K.H., et al. (2021). DSCAM/PAK1
pathway suppression reverses neurogenesis
deficits in iPSC-derived cerebral organoids
from patients with Down syndrome. J. Clin.
Invest. 131, 135763. https://doi.org/10.1172/
jci135763.
90. Pașca, A.M., Park, J.Y., Shin, H.W., Qi, Q.,
Revah, O., Krasnoff, R., O’Hara, R., Willsey,
A.J., Palmer, T.D., and Pașca, S.P. (2019).
Human 3D cellular model of hypoxic brain
injury of prematurity. Nat. Med. 25, 784–791.
https://doi.org/10.1038/s41591-019-0436-0.
91. Urresti, J., Zhang, P., Moran-Losada, P., Yu,
N.K., Negraes, P.D., Trujillo, C.A., Antaki, D.,
Amar, M., Chau, K., Pramod, A.B., et al. (2021).
Cortical organoids model early brain
development disrupted by 16p11.2 copy
number variants in autism. Mol. Psychiatry 26,
7560–7580. https://doi.org/10.1038/s41380-
021-01243-6.
92. Stachowiak, E.K., Benson, C.A., Narla, S.T.,
Dimitri, A., Chuye, L.E.B., Dhiman, S.,
Harikrishnan, K., Elahi, S., Freedman, D.,
Brennand, K.J., et al. (2017). Cerebral
organoids reveal early cortical
maldevelopment in schizophrenia-
computational anatomy and genomics, role
of FGFR1. Transl. Psychiatry 7, 6. https://doi.
org/10.1038/s41398-017-0054-x.
93. Jacob, F., Salinas, R.D., Zhang, D.Y., Nguyen,
P.T.T., Schnoll, J.G., Wong, S.Z.H., Thokala,
R., Sheikh, S., Saxena, D., Prokop, S., et al.
(2020). A patient-derived glioblastoma
organoid model and biobank recapitulates
inter- and intra-tumoral heterogeneity. Cell
180, 188–204.e22. https://doi.org/10.1016/j.
cell.2019.11.036.
94. Ogawa, J., Pao, G.M., Shokhirev, M.N., and
Verma, I.M. (2018). Glioblastoma model using
human cerebral organoids. Cell Rep. 23,
1220–1229. https://doi.org/10.1016/j.celrep.
2018.03.105.
95. Goranci-Buzhala, G., Mariappan, A., Gabriel,
E., Ramani, A., Ricci-Vitiani, L., Buccarelli, M.,
D’Alessandris, Q.G., Pallini, R., and
Gopalakrishnan, J. (2020). Rapid and efficient
invasion assay of glioblastoma in human brain
organoids. Cell Rep. 31, 107738. https://doi.
org/10.1016/j.celrep.2020.107738.

Perspective
96. Venkataraman, L., Fair, S.R., McElroy, C.A.,
Hester, M.E., and Fu, H. (2022). Modeling
neurodegenerative diseases with cerebral
organoids and other three-dimensional
culture systems: focus on Alzheimer’s disease.
Stem Cell Rev. Rep. 18, 696–717. https://doi.
org/10.1007/s12015-020-10068-9.
97. Lanoiselée, H.M., Nicolas, G., Wallon, D.,
Rovelet-Lecrux, A., Lacour, M., Rousseau, S.,
Richard, A.C., Pasquier, F., Rollin-Sillaire, A.,
Martinaud, O., et al. (2017). APP, PSEN1, and
PSEN2 mutations in early-onset Alzheimer
disease: a genetic screening study of familial
and sporadic cases. PLoS Med. 14, e1002270.
https://doi.org/10.1371/journal.pmed.
1002270.
98. Stefanis, L. (2012). a-Synuclein in Parkinson’s
disease. Cold Spring Harb. Perspect. Med. 2,
a009399. https://doi.org/10.1101/
cshperspect.a009399.
99. Surmeier, D.J., Obeso, J.A., and Halliday,
G.M. (2017). Selective neuronal vulnerability in
Parkinson disease. Nat. Rev. Neurosci. 18,
101–113. https://doi.org/10.1038/nrn.2016.
178.
100. Michel, P.P., Hirsch, E.C., and Hunot, S. (2016).
Understanding dopaminergic cell death
pathways in Parkinson disease. Neuron 90,
675–691. https://doi.org/10.1016/j.neuron.
2016.03.038.
101. Galet, B., Cheval, H., and Ravassard, P. (2020).
Patient-derived midbrain organoids to
explore the molecular basis of Parkinson’s
disease. Front. Neurol. 11, 1005. https://doi.
org/10.3389/fneur.2020.01005.
102. Qian, X., Nguyen, H.N., Jacob, F., Song, H.,
and Ming, G.L. (2017). Using brain organoids
to understand Zika virus-induced
microcephaly. Development 144, 952–957.
https://doi.org/10.1242/dev.140707.
103. Allende, M.L., Cook, E.K., Larman, B.C.,
Nugent, A., Brady, J.M., Golebiowski, D.,
Sena-Esteves, M., Tifft, C.J., and Proia, R.L.
(2018). Cerebral organoids derived from
Sandhoff disease-induced pluripotent stem
cells exhibit impaired neurodifferentiation.
J. Lipid Res. 59, 550–563. https://doi.org/10.
1194/jlr.M081323.
104. Gough, G., O’Brien, N.L., Alic, I., Goh, P.A.,
Yeap, Y.J., Groet, J., Nizetic, D., and Murray,
A. (2020). Modeling Down syndrome in cells:
from stem cells to organoids. Prog. Brain Res.
251, 55–90. https://doi.org/10.1016/bs.pbr.
2019.10.003.
105. Zhang, W., Ma, L., Yang, M., Shao, Q., Xu, J.,
Lu, Z., Zhao, Z., Chen, R., Chai, Y., and Chen,
J.F. (2020). Cerebral organoid and mouse
models reveal a RAB39b-PI3K-mTOR
pathway-dependent dysregulation of cortical
development leading to macrocephaly/
autism phenotypes. Genes Dev. 34, 580–597.
https://doi.org/10.1101/gad.332494.119.
106. Coelho, A.V.C., and Crovella, S. (2017).
Microcephaly prevalence in infants born to
Zika virus-infected women: a systematic
review and meta-analysis. Int. J. Mol. Sci. 18,
1714. https://doi.org/10.3390/ijms18081714.
107. Khatoon, F., Prasad, K., and Kumar, V. (2020).
Neurological manifestations of COVID-19:
available evidences and a new paradigm.
J. Neurovirol. 26, 619–630. https://doi.org/10.
1007/s13365-020-00895-4.
108. Song, E., Zhang, C., Israelow, B., Lu-Culligan,
A., Prado, A.V., Skriabine, S., Lu, P., Weizman,
O.E., Liu, F., Dai, Y., et al. (2021).
Neuroinvasion of SARS-CoV-2 in human and
mouse brain. J. Exp. Med. 218, e20202135.
https://doi.org/10.1084/jem.20202135.
109. Pellegrini, L., Albecka, A., Mallery, D.L.,
Kellner, M.J., Paul, D., Carter, A.P., James,
L.C., and Lancaster, M.A. (2020). SARS-CoV-2
infects the brain choroid plexus and disrupts
the blood-CSF barrier in human brain
organoids. Cell Stem Cell 27, 951–961.e5.
https://doi.org/10.1016/j.stem.2020.10.001.
110. Wang, M., Zhang, L., and Gage, F.H. (2020).
Modeling neuropsychiatric disorders using
human induced pluripotent stem cells.
Protein Cell 11, 45–59. https://doi.org/10.
1007/s13238-019-0638-8.
111. Sawada, T., Chater, T.E., Sasagawa, Y.,
Yoshimura, M., Fujimori-Tonou, N., Tanaka,
K., Benjamin, K.J.M., Paquola, A.C.M., Erwin,
J.A., Goda, Y., et al. (2020). Developmental
excitation-inhibition imbalance underlying
psychoses revealed by single-cell analyses of
discordant twins-derived cerebral organoids.
Mol. Psychiatry 25, 2695–2711. https://doi.
org/10.1038/s41380-020-0844-z.
112. Wang, Q., Dong, X., Hu, T., Qu, C., Lu, J.,
Zhou, Y., Li, J., and Pei, G. (2021). Constitutive
activity of serotonin receptor 6 regulates
human cerebral organoids formation and
depression-like behaviors. Stem Cell Rep. 16,
75–88. https://doi.org/10.1016/j.stemcr.2020.
11.015.
113. da Hora, C.C., Schweiger, M.W., Wurdinger,
T., and Tannous, B.A. (2019). Patient-derived
glioma models: from patients to dish to
animals. Cells 8, 177. https://doi.org/10.3390/
cells8101177.
114. Park, J.C., Jang, S.Y., Lee, D., Lee, J., Kang, U.,
Chang, H., Kim, H.J., Han, S.H., Seo, J., Choi,
M., et al. (2021). A logical network-based
drug-screening platform for Alzheimer’s
disease representing pathological features of
human brain organoids. Nat. Commun. 12,
280. https://doi.org/10.1038/s41467-020-
20440-5.
115. Watanabe, M., Buth, J.E., Vishlaghi, N., de la
Torre-Ubieta, L., Taxidis, J., Khakh, B.S.,
Coppola, G., Pearson, C.A., Yamauchi, K.,
Gong, D., et al. (2017). Self-organized cerebral
organoids with human-specific features
predict effective drugs to combat Zika virus
infection. Cell Rep. 21, 517–532. https://doi.
org/10.1016/j.celrep.2017.09.047.
116. Singh, A.V., Maharjan, R.S., Kanase, A.,
Siewert, K., Rosenkranz, D., Singh, R., Laux, P.,
and Luch, A. (2021). Machine-learning-based
approach to decode the influence of
nanomaterial properties on their interaction
with cells. ACS Appl. Mater. Interfaces 13,
1943–1955. https://doi.org/10.1021/acsami.
0c18470.
117. Singh, A.V., Maharjan, R.S., Jungnickel, H.,
Romanowski, H., Hachenberger, Y.U.,
Reichardt, P., Bierkandt, F., Siewert, K.,
Gadicherla, A., Laux, P., and Luch, A. (2021).
Evaluating particle emissions and toxicity of
3D pen printed filaments with metal
Cell Reports Physical Science 3, 100974, July 20, 2022
ll
OPEN ACCESS
nanoparticles as additives: in vitro and in silico
discriminant function analysis. ACS Sust.
Chem. Eng. 9, 11724–11737. https://doi.org/
10.1021/acssuschemeng.1c02589.
118. Badai, J., Bu, Q., and Zhang, L. (2020). Review
of artificial intelligence applications and
algorithms for brain organoid research.
Interdiscip. Sci. 12, 383–394. https://doi.org/
10.1007/s12539-020-00386-4.
119. Buccino, A.P., Kordovan, M., Ness, T.V.,
Merkt, B., Häfliger, P.D., Fyhn, M.,
Cauwenberghs, G., Rotter, S., and Einevoll,
G.T. (2018). Combining biophysical modeling
and deep learning for multielectrode array
neuron localization and classification.
J. Neurophysiol. 120, 1212–1232. https://doi.
org/10.1152/jn.00210.2018.
120. Li, X., Zhang, G., Wu, J., Zhang, Y., Zhao, Z.,
Lin, X., Qiao, H., Xie, H., Wang, H., Fang, L.,
and Dai, Q. (2021). Reinforcing neuron
extraction and spike inference in calcium
imaging using deep self-supervised
denoising. Nat. Methods 18, 1395–1400.
https://doi.org/10.1038/s41592-021-01225-0.
121. McAleer, S., Fast, A., Xue, Y., Seiler, M., Tang,
W., Balu, M., Baldi, P., and Browne, A.W.
(2020). Deep machine learning-assisted
multiphoton microscopy to reduce light
exposure and expedite imaging. Preprint at
arXiv. https://doi.org/10.48550/arXiv.2011.
06408.
122. Gritti, N., Lim, J.L., Anlasx, K., Pandya, M.,
Aalderink, G., Martı́nez-Ara, G., and Trivedi, V.
(2021). Morgana: accessible quantitative
analysis of organoids with machine learning.
Development 148, dev.199611. https://doi.
org/10.1242/dev.199611.
123. Beghin, A., Grenci, G., Rajendiran, H., Delaire,
T., Raffi, S.B.M., Blanc, D., De Mets, R., Ong,
H.T., Acharya, V., and Sahini, G. (2021). High
content 3D imaging method for quantitative
characterization of organoid development
and phenotype. Preprint at bioRxiv. https://
doi.org/10.1101/2021.03.26.437121.
124. Albanese, A., Swaney, J.M., Yun, D.H., Evans,
N.B., Antonucci, J.M., Velasco, S., Sohn, C.H.,
Arlotta, P., Gehrke, L., and Chung, K. (2020).
Multiscale 3D phenotyping of human cerebral
organoids. Sci. Rep. 10, 21487. https://doi.
org/10.1038/s41598-020-78130-7.
125. Hasib, M., Lybrand, Z., Estevez, V.N., Hsieh, J.,
and Huang, Y. (2019). In Charactering hESCs
Organoids from Electrical Signals with
Machine Learning (IEEE), pp. 1–4.
126. Monzel, A.S., Hemmer, K., Kaoma, T., Smits,
L.M., Bolognin, S., Lucarelli, P., Rosety, I.,
Zagare, A., Antony, P., Nickels, S.L., et al.
(2020). Machine learning-assisted
neurotoxicity prediction in human midbrain
organoids. Parkinsonism Relat. Disord. 75,
105–109. https://doi.org/10.1016/j.parkreldis.
2020.05.011.
127. Silva, G.A., Muotri, A.R., and White, C. (2020).
Understanding the human brain using brain
organoids and a structure-function theory.
Preprint at bioRxiv. https://doi.org/10.1101/
2020.07.28.225631.
128. Feldmann, J., Youngblood, N., Wright, C.D.,
Bhaskaran, H., and Pernice, W.H.P. (2019). All-
optical spiking neurosynaptic networks with
17
 ll
OPEN ACCESS
self-learning capabilities. Nature 569,
208–214. https://doi.org/10.1038/s41586-019-
1157-8.
129. Farahany, N.A., Greely, H.T., Hyman, S.,
Koch, C., Grady, C., Pașca, S.P., Sestan,
N., Arlotta, P., Bernat, J.L., Ting, J., et al.
(2018). The ethics of experimenting with
human brain tissue. Nature 556, 429–432.
https://doi.org/10.1038/d41586-018-
04813-x.
130. Sawai, T., Hayashi, Y., Niikawa, T., Shepherd,
J., Thomas, E., Lee, T.L., Erler, A., Watanabe,
M., and Sakaguchi, H. (2022). Mapping the
ethical issues of brain organoid research and
application. AJOB Neurosci. 13, 81–94.
18 Cell Reports Physical Science 3, 100974, July 20, 2022
https://doi.org/10.1080/21507740.2021.
1896603.
131. Lavazza, A. (2021). Potential ethical problems
with human cerebral organoids:
consciousness and moral status of future
brains in a dish. Brain Res. 1750, 147146.
https://doi.org/10.1016/j.brainres.2020.
147146.
132. Koplin, J.J., and Savulescu, J. (2019). Moral
limits of brain organoid research. J. Law Med.
Ethics 47, 760–767. https://doi.org/10.1177/
1073110519897789.
133. Chen, H.I., Song, H., and Ming, G.L. (2019).
Applications of human brain organoids to
Perspective
clinical problems. Dev. Dyn. 248, 53–64.
https://doi.org/10.1002/dvdy.24662.
134. Samarasinghe, R.A., Miranda, O.A., Buth, J.E.,
Mitchell, S., Ferando, I., Watanabe, M.,
Allison, T.F., Kurdian, A., Fotion, N.N.,
Gandal, M.J., et al. (2021). Identification of
neural oscillations and epileptiform changes
in human brain organoids. Nat. Neurosci. 24,
1488–1500. https://doi.org/10.1038/s41593-
021-00906-5.
135. Barnhart, A.J., and Dierickx, K. (2022). The
many moral matters of organoid models: a
systematic review of reasons. Med. Health
Care Philos. https://doi.org/10.1007/s11019-
022-10082-3.