Trends in

Molecular Medicine OPEN ACCESS

Review

Pushing the boundaries of brain organoids to

study Alzheimer’s disease
Jonas Cerneckis, 1,2 Guojun Bu, 3 and Yanhong Shi 1,2,*

Progression of Alzheimer’s disease (AD) entails deterioration or aberrant function Highlights

of multiple brain cell types, eventually leading to neurodegeneration and cogni- Induced pluripotent stem cell (iPSC)
tive decline. Defining how complex cell–cell interactions become dysregulated technology has enabled modeling of
in AD requires novel human cell-based in vitro platforms that could recapitulate human diseases using patient-derived
cells with relevant genetic background.
the intricate cytoarchitecture and cell diversity of the human brain. Brain
organoids (BOs) are 3D self-organizing tissues that partially resemble the iPSC-derived 3D brain organoids self-
human brain architecture and can recapitulate AD-relevant pathology. In this re- organize and resemble the
cytoarchitecture of in vivo brain tissue,
view, we highlight the versatile applications of different types of BOs to model AD making them attractive tools for model-
pathogenesis, including amyloid-β and tau aggregation, neuroinflammation, my- ing Alzheimer’s disease (AD).
elin breakdown, vascular dysfunction, and other phenotypes, as well as to accel-
erate therapeutic development for AD. Brain organoids (BOs) contain multiple
human brain cell types, enabling the
study of cell–cell interactions that drive
AD progression.
Brain organoids recapitulate cellular diversity of the human brain
Human iPSC-derived BOs may reveal
A groundbreaking development of the induced pluripotent stem cell (iPSC) technology has en-
human-specific disease phenotypes
abled disease modeling in vitro using patient-derived cellular models [1–3]. Human iPSC-based and molecular mechanisms that are not
disease models overcome the difficulty of obtaining primary human tissues, such as brain tissue, recapitulated in animal models.
as well as the issue of species divergence when using animal models [4,5]. Although 2D cell cul-
ture models have dominated in vitro research, more complex cellular assemblies are required to
faithfully recapitulate multifaceted pathogenesis of AD. The emergence of human organoid tech-
nology bridges the gap between 2D models and the complex 3D environment in vivo. BOs are 3D
stem-cell-derived tissues that mimic the cellular composition and structure of the human brain [6–
9]. BOs can be engineered to contain the diversity of human brain cell types, including neurons,
astrocytes, oligodendrocytes, microglia, pericytes, and epithelial cells (Box 1). BOs also exhibit
complex cytoarchitecture and electrophysiological properties, facilitating the study of neurode-
generation and cognitive decline (Box 2).

In this review, we begin by considering the immense complexity of cell–cell interactions that de-
teriorate during AD progression; we also discuss the importance and advantages of using
human cell-based models for studying AD. Subsequently, we draw on the recent advances in de-
1
veloping various BO models, including neural organoids, myelinoids, neuroimmune organoids Department of Neurodegenerative
Diseases, Beckman Research Institute
(NIOs), blood–brain barrier (BBB) organoids, and choroid plexus (see Glossary) organoids
of City of Hope, Duarte, CA 91010, USA
(ChPOs), to highlight their potential for modeling AD pathogenesis and developing novel thera- 2
Irell and Manella Graduate School of Bi-
peutics for AD. ological Sciences, Beckman Research
Institute of City of Hope, Duarte, CA
91010, USA
Cell–cell interactions in Alzheimer’s disease 3
SciNeuro Pharmaceuticals, Rockville,
AD is a progressive neurodegenerative disease that presents with cognitive decline and is the MD 20850, USA
most common cause of dementia [10]. Most AD cases are sporadic (sAD), whereas autosomal
dominant familial AD (fAD) comprises about 1% of all AD cases and is caused by mutations in
PSEN1, PSEN2, and APP [11]. The neuron-centric amyloid hypothesis has been the leading the-
ory for how AD develops and progresses, postulating that amyloid-β (Aβ) accumulation and *Correspondence:yshi@coh.org (Y. Shi).

Trends in Molecular Medicine, August 2023, Vol. 29, No. 8 https://doi.org/10.1016/j.molmed.2023.05.007 659
© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Box 1. Techniques for brain organoid differentiation

Glossary
Numerous BO differentiation protocols to derive neural structures of the desired brain region and incorporate different cel- Astrogliosis: astrocyte activation and
lular components have been developed [9,104–106]. To obtain BOs, PSCs are aggregated into embryoid bodies and in- proliferation in response to a damaging
duced towards the neuroectoderm lineage [39]. Signaling molecules can be added to promote guided differentiation, insult or brain injury.
patterning the BO towards a specified regional identity; alternatively, unguided differentiation yields neural organoids that Cerebral amyloid angiopathy: a
contain patches of tissue of various brain regional identities [39,106]. medical condition characterized by
amyloid accumulation on the walls of the
Enrichment of BOs with glial cells necessitates cell type-specific strategies, given the variable developmental timing and brain vasculature.
ontogeny of glial cells [105]. Astrocytes arise only after the neurogenic-to-gliogenic transition of radial glia in the developing Choroid plexus: a brain tissue com-
human brain [105]. Similarly, astrocytes spontaneously arise following long-term culture of BOs [62,107]. Given the prised of a network of blood vessels and
protracted nature of human brain myelination [23], additional signaling cues are required to induce oligodendrocyte pro- secretory epithelium that produces
genitor cell (OPC) and oligodendrocyte development in BOs. Supplementation of signaling molecules required for cerebrospinal fluid.
oligodendrogenesis, including platelet-derived growth factor AA, insulin-like growth factor 1, and thyroid hormone (T3), CRISPR interference (CRISPRi): a
promotes OPC and oligodendrocyte differentiation as well as neuron myelination in specialized BOs termed myelinoids molecular biology technique to suppress
[66–68]. Alternatively, ectopic expression of SOX10 and OLIG2 promotes directed differentiation of OPCs and oligoden- gene expression using the CRISPR/
drocytes in the myelinoid [108]. Myelination can be improved by patterning the myelinoid identity towards the ventral spinal Cas9 platform (generally performed in a
cord, where oligodendrogenesis begins during brain development [68]. Microglia and brain vasculature arise from the me- high-throughput manner).
soderm, whereas mesoderm specification is generally suppressed during guided BO differentiation. Therefore, iPSC-de- Genome-wide association studies
rived microglia can be seeded onto differentiating BOs to allow infiltration [70]. Microglia-containing neuroimmune (GWAS): a genetics methodology to
organoids can also be derived by ectopic expression of the microglial fate-determining transcription factor PU.1 during identify genetic alterations that are linked
BO differentiation [71]. Alternatively, iPSC-derived microglia progenitors can be mixed with neural progenitors before to a specific disease.
BO differentiation [73]. Microglia may also develop innately during unguided BO differentiation; however, the reproducibility Glutamatergic neurons: excitatory
of this approach remains to be clarified [109]. Vascularized BOs can be obtained by ectopic expression of ETV2 during BO neurons that use glutamate as a neuro-
differentiation [79]. Alternatively, integration of pericytes into differentiating BOs yields pericyte-containing BOs [82]. Self- transmitter.
organized iBBB can be obtained by mixing astrocytes, pericytes, and endothelial cells in a 3D hydrogel or assembling High-content screening: a method-
these cells into a spheroid [30,80]. Finally, a combination of WNT and BMP signaling pathway activation has been applied ology to identify cellular and subcellular
to generate ChPOs [84,85]. phenotypes in a high-throughput man-
ner.
Hyperexcitability: increased probabil-
toxicity initiate a cascade of events culminating in neurodegeneration and cognitive decline [11]. ity of neuronal activation upon stimula-
Extensive efforts to target Aβ pathology have resulted in recent accelerated approvals for mono- tion.
clonal antibodies aducanumab and lecanemab by the FDA [12–14]. Hyperphosphorylation and Microgliosis: inflammatory activation
of microglia in response to a damaging
insult or brain injury.
Box 2. Modeling electrophysiology of Alzheimer’s disease using brain organoids Neuroectoderm: a type of tissue in a
developing embryo that gives rises to
Human brain electrophysiological activity reflects neuronal network connectivity and cognitive functions. Although electro-
the central nervous system.
physiological recordings are cost-effective and noninvasive, identification of electrophysiological markers as a diagnostic
Neuroinflammation: chronic activa-
tool for AD remains a challenge [110]. Nevertheless, novel insights into electrophysiological activity of AD-afflicted neuronal
tion of the innate immune system of the
networks may be obtained by recording neuronal activity of 3D human BOs that form relatively complex neuronal networks
brain.
as compared to 2D neuronal cultures. Neuronal activity of cortical BOs increases over the course of differentiation and ex-
Nodes of Ranvier: intermittent areas of
hibits alternating periods of synchronized events and quiescence, reminiscent of early brain development in vivo [111].
a myelinated axon that lack myelin and
Other modifications, such as BO vascularization or microglia infiltration, also facilitate BO electrophysiological maturation
promote saltatory conduction of action
[71,72,79,112,113]. For example, neuroimmune organoids exhibit increased frequency of oscillatory bursts and
potentials.
synchronization as compared to microglia-lacking BOs [71,72,112].
Organ-on-a-chip: a microfluidics
device mimicking in vivo tissue architec-
Emerging evidence indicates that BOs derived from AD patients also recapitulate neuronal network dysfunction character-
ture.
istic of AD patients. For example, neuronal hyperexcitability and increased prevalence of epileptiform activity are evident
Oscillatory bursts: rhythmic electrical
in AD patients [114]. Neuronal hyperexcitability has also been document in fAD, sAD, and ApoE4 BOs [50,115,116]. Fur-
activity of neuronal networks.
thermore, cortical and ganglionic eminence assembloids integrate excitatory and inhibitory neurons, enabling the emer-
Positron emission tomography: a
gence of more complex electrophysiology [117]. Such assembloids exhibit epileptiform-like activity in a disease setting,
noninvasive imaging methodology to
whereas aberrant neuronal activity can be modulated pharmacologically [117]. Deterioration of BO neuronal activity may
detect tissue metabolic activity.
also be used as a readout for cognitive decline; therefore, it is important to characterize how various AD-relevant insults
Radial glia: progenitor cells of the
affect BO neuronal activity over the course of disease progression. Notably, serum exposure and HSV-1 infection both re-
developing nervous system that give rise
sult in impaired neuronal activity of BOs [62,90].
to neurons and macroglia.

2D microelectrode arrays (MEAs) are often used to record neuronal activity in whole or sliced BOs [62,111,118]. However,
engineered solutions to record BO neuronal activity across the BO surface area and without the need for slicing would fa-
cilitate spatiotemporal recording during BO differentiation and beyond. Novel self-folding MEA caps overcome the con-
straints of 2D MEAs and can wrap around organoids of different sizes in 3D space [119]. Cyborg organoids integrate
stretchable mesh nanoelectronics arrays across the organoid to further increase the interface area between neurons
and electrodes [120]. Such 3D electrode interfaces will enable investigators to assess how various AD-relevant genetic
and environmental insults drive the deterioration of neuronal networks over time.

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aggregation of the microtubule-associated protein tau is another hallmark of AD [10]. In recent

years, tau has become a major focus in AD research, given the emerging evidence that tau pa-
thology can develop and progress independently of Aβ [15]. Moreover, spatial and temporal cor-
relation between tau pathology and neurodegeneration is much stronger than that between Aβ
pathology and neurodegeneration, highlighting the importance of tau in disease progression
[15]. In addition to neuronal Aβ and tau pathology, genome-wide association studies
(GWAS), high-throughput profiling of gene expression, and functional experiments have revealed
remarkable complexity of genetic risk factors, aging, cellular states, and cell–cell interactions that
together drive AD pathogenesis [16–22]. Novel hypotheses for AD progression, including the cel-
lular phase, apolipoprotein (Apo)E cascade, myelin breakdown and neuroinflammation hypoth-
eses have emerged [17,18,23,24]. As the strongest genetic risk factor for AD, the E4 variant of
APOE has been linked to dysfunctional lipid metabolism, impaired myelination, heightened neu-
roinflammation, and vascular breakdown, among other phenotypes, exemplifying the immense
complexity of cellular interactions that go awry in AD [18,21,25]. As the primary source of the se-
creted ApoE protein, astrocytes orchestrate brain lipid metabolism that is essential for neuronal
physiology, injury repair, and myelin remodeling [17]. Astrocytes also maintain systemic homeo-
stasis of the neuronal circuitry, remodel synapses, and form the glia limitans membrane at the
BBB. In AD, astrocytes undergo reactive astrogliosis, leading to deterioration of their supportive
functions, increased proinflammatory cytokine secretion, and neurotoxicity [17,26]. Oligodendro-
cytes provide metabolic support to neurons and produce the myelin sheath for neuron electrical
insulation [17]. The myelin breakdown hypothesis postulates the central importance of myelin in-
tegrity to brain homeostasis, whereas myelin breakdown drives neurodegeneration and cognitive
decline in AD [23,27]. Excessive myelin debris may also activate microglia, initiating neuroinflam-
mation [27]. As the brain resident macrophages, microglia survey the brain environment, prune
synapses, phagocytose debris, and mount an immune response to a pathogenic stimulus [17].
Microglial ability to remove toxic Aβ debris indicates a protective role in AD; however, at later
stages of the disease, microglia drive neuroinflammation and may even propagate Aβ into unaf-
fected brain regions [28]. Finally, brain vasculature coordinates energy metabolism and forms the
BBB that protects the privileged brain environment from peripheral neurotoxic factors [17]. More-
over, the ChP forms the blood–cerebrospinal fluid (CSF) barrier and secretes CSF that facilitates
metabolite exchange across the brain and serves a protective function [29]. Yet, deterioration of
the brain vasculature in AD manifests as vascular loss, cortical hypoperfusion, BBB breakdown,
and cerebral amyloid angiopathy, among other phenotypes [22,30,31]. Oligomeric Aβ drives
capillary constriction and pericyte dysfunction, whereas profiling of the CSF proteome reveals
dozens of dysregulated proteins in AD patients [32,33]. All these cellular phenotypes converge
to drive neuronal network degeneration, neuron cell death, and ultimately, cognitive decline.
Therefore, defining how diverse cell–cell interactions deteriorate in AD and contribute to synaptic
loss and neuronal cell death is a pivotal goal for future therapeutic development.

Species divergence necessitates human iPSC-based models of Alzheimer’s

disease
Species differences between human and mouse brains have been a significant hurdle in translat-
ing rodent-based findings into viable AD therapies in the clinic [16]. High-throughput profiling
studies have revealed considerable divergence in transcriptional programs of the brain cell
types, differences in cortical cell organization, and notably increased cell–cell contacts in the
human cortex [34,35]. The divergence in gene expression profiles is particularly apparent in
non-neuronal cells; for example, human microglia are more enriched for expression of AD risk
genes than mouse microglia [36]. Transcriptional divergence also implies phenotypic differences.
Indeed, human and mouse astrocytes exhibit contrasting programs of energy metabolism, re-
sponse to inflammatory stimulation, and susceptibility to oxidative stress [37]. Divergent

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oligodendrocyte gene expression profiles manifest in notable differences in myelin proteomes

[38]. Species differences are also evident in the brain vasculature. Brain endothelial cells (BECs)
and pericytes exhibit among the most divergent transcriptional profiles between human and mouse
brain cell types [22]. These differences are amplified in a disease setting; for example, there is little
overlap of differentially expressed genes between BECs isolated from AD patients and an AD
mouse model when compared with healthy controls [22]. Consequently, considerable species
divergence has hindered the elucidation of human-specific disease mechanisms and, by extension,
therapeutic development for AD. BO technology enables modeling of AD in all-human cellular
systems, so that human-relevant molecular mechanisms of AD pathogenesis can be uncovered.

Brain organoid models of Alzheimer’s disease

Unravelling the complexity of AD pathogenesis requires diverse BO models that resemble distinct
structures of the human brain and incorporate specialized cell types. In this section, we highlight
the applications of different BO models to study various aspects of AD pathogenesis.

Neural organoids
Neural or cerebral organoids are the most widely used 3D pluripotent-stem-cell-derived models
of the human brain tissue to study brain diseases, including AD [6,39]. Neural organoids derived
from iPSCs of fAD patients robustly develop AD-relevant pathology, including Aβ aggregation
and tau hyperphosphorylation [40]. These neural organoids also exhibit synaptic loss, impaired
neuronal activity (Box 2), and neuronal cell death, in addition to other phenotypes, such as
endosomal dysfunction [40]. Tau pathology is also evident in neural organoids that express mu-
tant tau variants associated with frontotemporal dementia but relevant in a broader context of
tauopathies, including AD [41]. For example, mutant tau-V337M neural organoids accumulate
phosphorylated tau (p-tau) and exhibit impaired proteostasis, neuroinflammation, dysregulated
lipid metabolism, and selective loss of glutamatergic neurons [42,43]. Moreover, Aβ and tau
pathology can be induced in a controllable manner, such as by treating neural organoids with
an amyloidogenesis activator Aftin-5 or by injecting adeno-associated virus (AAV) carrying a mu-
tant tau expression cassette [44,45]. In this way, both independent and synergistic effects of Aβ
and tau pathology on AD progression may be investigated using neural organoids [46,47]. An im-
portant consideration is whether the conformation of tau aggregates in neural organoids resem-
bles that of in vivo inclusions. Indeed, iPSC-derived neurons lack tau isoform diversity of the
human brain, which may affect tau oligomerization and fibrillization properties in neural organoids
[48]. AAV-mediated expression of a mutant tau-P301L that is more prone to aggregation than
wild-type tau may overcome this limitation, leading to formation of tau oligomers and fibrils rem-
iniscent of late tau pathology in AD [45]. Finally, self-propagation of aggregated tau can be
modeled using astrocyte-neuron spheroids, where p-tau pathology is readily propagated from
neurons pre-treated with tau to untreated neurons [49].

Although AD-relevant pathology can be induced by overt dysfunction or manipulation of the am-
yloid and tau cascades, neural organoids can also reveal subtle contributions of genetic and en-
vironmental risk factors for sAD. Neural organoids derived from iPSCs of sAD patients secrete
more Aβ40, Aβ42, and p-tau than do neural organoids derived from healthy controls, indicating
neuronal dysfunction [50]. The secreted amounts of Aβ40, Aβ42, and p-tau correlate with the
Aβ burden detected by positron emission tomography in corresponding sAD patients, indi-
cating the sensitivity of the BO technology for modeling sAD [50]. Neural organoids carrying ge-
netic risk variants, such as APOE4, and their isogenic corrected controls can also be engineered
by CRISPR/Cas9 editing (Figure 1). Isogenic disease models eliminate the confounding effects of
patient-to-patient variation and reveal genetic risk factor-specific phenotypes; this technique is
often applied to generate isogenic iPSC lines carrying different APOE variants [51]. To study

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Figure 1. Generation and applications of isogenic APOE3 and APOE4 brain organoids (BOs). Patient-derived fibroblasts
are first obtained from a skin biopsy of a carrier of APOE4. Fibroblasts are then reprogrammed into induced pluripotent stem cells
(iPSCs), and a single nucleotide point mutation (cytosine-to-thymine) is introduced using CRISPR/Cas9-based genetic editing,
resulting in a missense mutation and exchange of arginine (Arg) for cysteine (Cys), generating the APOE3 variant. Subsequently,
unedited APOE4 iPSCs and isogenic edited APOE3 iPSCs are differentiated into BOs following well-established protocols. The
resulting BOs share the same genetic background except for the APOE variant, enabling mechanistic studies of the roles of ApoE
isoforms in lipid metabolism, neuroinflammation, cellular stress, and other processes, without the confounding effects of patient-to-
patient variation. Abbreviation: sgRNA, single guide RNA.

the impact of ApoE isoforms on AD progression using neural organoids, robust astrocyte differ-
entiation is required (Figure 2A, Key figure); indeed, immature neural organoids express little
APOE because they lack astrocytes [51]. As the organoids mature, radial glia and astrocytes se-
crete ApoE that is diffusively distributed throughout the 3D space of the organoid [51,52]. APOE4

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Key figure
Strategies for obtaining glial cell-enriched brain organoids (BOs)

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Figure 2. (A) Long-term culture of neural organoids promotes astrocyte differentiation as radial glia progenitors undergo the
neurogenic-to-gliogenic transition. Long-term maintenance of neural organoids can be improved by slicing spherical
organoids and maintaining sliced organoids to allow efficient nutrient and oxygen exchange. Astrocyte-enriched neural
organoids can be used to study astrocyte lipid metabolism and astrogliosis in Alzheimer’s disease (AD), among other
roles. (B) Supplying key signaling molecules required for oligodendrocyte progenitor cell (OPC) and oligodendrocyte
differentiation, including platelet-derived growth factor AA (PDGF-AA), insulin-like growth factor 1 (IGF-1), and thyroid
hormone (T3), promotes OPC and oligodendrocyte differentiation in myelinoids. Patterning myelinoids towards ventral
spinal cord identity can further improve myelination. Oligodendrocyte-enriched myelinoids can be used to study the effects
of myelination and myelin breakdown in AD. (C) Given that microglia arise from a different germ layer (mesoderm) than do
neural tissues (ectoderm), generation of microglia-containing neuroimmune organoids (NIOs) can be achieved by seeding
induced pluripotent stem cell (iPSC)-derived microglia onto differentiating BOs. To obtain microglia, iPSCs are
differentiated into an erythromyeloid progenitor-like intermediate followed by microglia differentiation and maturation in the
presence of transforming growth factor β (TGF-β), interleukin 34 (IL-34), and macrophage colony-stimulating factor (M-
CSF). Microglia-enriched NIOs can be used to study phagocytosis of toxic debris and microgliosis in AD.

neural organoids accumulate more Aβ and p-tau than do APOE3 neural organoids although this
phenotype may depend on neural organoid maturation [51,53]. Neural organoids may thus be
used to investigate how ApoE4 interacts with tau to drive AD-relevant pathology, given the recent
observation that removal of neuronal ApoE4 suppresses tau pathology in vivo [25]. Neural

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organoids are also amenable to pharmacological manipulation, indicating their utility for testing
novel therapeutic candidates [42,49,54]. Therefore, tau-targeting therapies, such as the recently
developed tau-degrading intrabodies, as well as ApoE-targeting antibodies or antisense nucleo-
tides could be tested in neural organoids [55–58]. However, it is important to note that different
neural organoid differentiation protocols and variable organoid-to-organoid reproducibility
(Box 3) may affect the experimental findings obtained from neural organoid studies. For example,
Hernández et al. found that ApoE, Aβ42, and p-tau levels were highly variable within the groups of
APOE3 and APOE4 neural organoids; as a result, there was no statistically significant difference
between ApoE, Aβ42, and p-tau levels of isogenic APOE3 and APOE4 neural organoids [59]. This
discrepancy may also be attributed to the relatively mild ApoE isoform-dependent phenotypes as
compared to those stemming from causal disease mutations.

Neural organoids may also be used to define the effects of nongenetic risk factors on AD pathogen-
esis. For example, BBB leakage is a well-recognized AD risk factor that may be driven by aging
[60,61]. Exposure of neural organoids to human serum induces accumulation of insoluble Aβ aggre-
gates and p-tau, among other phenotypes, indicating that BBB leakage may precipitate AD-relevant
pathology [62]. Because AD is an age-related neurodegenerative disorder, the study of BBB leakage
that occurs in aged brains offers a way to incorporate age-relevant events when modeling AD in vitro
[60–62]. Moreover, given that serum exposure results in widespread AD-relevant pathology, it is im-
portant to determine what specific peripheral factors induce neuronal toxicity upon BBB breakdown
[62]. For example, liver-derived ApoE4 has recently been shown to trigger BBB breakdown, induce
astrogliosis, and impair memory in vivo [63]. Emerging reports also indicate a role for peripheral im-
mune cells in AD progression [31,64]. We anticipate that complex organoid models that integrate pe-
ripheral effectors will reveal novel mediators of neurotoxicity originating from outside the brain.

Myelinoids
Although neural organoids robustly develop neurons and astrocytes, additional strategies to in-
troduce other AD-relevant cell types are required (Box 1). Cortical myelinoids contain oligoden-
drocyte progenitor cells (OPCs) and oligodendrocytes as well as exhibit a degree of myelination

Box 3. Rigor and reproducibility of brain organoid technology

To reach the full potential of modeling AD using BO technology, it is critical to ensure rigorous differentiation of organoids
that faithfully and reproducibly recapitulate key features of the human brain. However, self-organization in vitro, especially
when relying on unguided BO differentiation, exhibits substantial organoid-to-organoid variation [59,121,122]. BOs de-
rived by unguided differentiation not only contain an assortment of cells from various brain regions, but also develop
non-ectodermal cell types, including microglia [39,109]. Thus, while unguided differentiation may increase the cell type di-
versity, it also affects reproducibility across individual BOs and may confound experimental findings. On the contrary,
guided BO differentiation yields highly reproducible BOs. Single-cell sequencing of forebrain organoids reveals remarkable
reproducibility of cell types with variation comparable with that of human brain development [121–123]. Therefore, un-
guided BO differentiation may be more suitable for exploratory experiments, whereas guided BO differentiation should
be used for single-cell high-throughput profiling studies and drug screening applications, among others.

Rigor of the experiments using BO models also depends on how well BOs recapitulate the in vivo environment and
whether BOs exhibit any phenotypes that could confound the experimental readout in the context of AD. BOs exhibit el-
evated expression of genes that regulate the pathways of endoplasmic reticulum stress and glycolysis [93]. Moreover, the
lack of organoid vascularization is associated with internal hypoxia and increased hypoxia-inducible factor (HIF)-1α levels
as compared to vascularized organoids [79]. These phenotypes have also been implicated in AD progression and thus
may confound experimental findings. For example, HIF-1α signaling orchestrates microglia metabolic reprogramming from
oxidative phosphorylation to glycolysis upon encounter of Aβ, whereas AD patient-derived neurons undergo Warburg ef-
fect-like glycolytic reprogramming that may be dependent on HIF-1α [124,125]. Internal BO necrosis may also activate in-
flammatory responses of microglia and astrocytes upon debris encounter. Therefore, investigators should be aware of
these intrinsic BO limitations as well as the ways to overcome them. For example, improved nutrient and oxygen exchange
using a microfluidic device leads to increased glucose consumption, lactate secretion, cell proliferation, and neuronal ac-
tivity as well as reduced necrosis and HIF-1α+ area in BOs [94]. Similarly, BO slicing reduces necrosis and enables long-
term organoid maintenance [95,96].

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[65–67]. Ventral spinal cord myelinoids (Figure 2B) further develop lamellae of compact myelin
wrapped around neurons with comparable myelin thickness to that in vivo as well as nodes of
Ranvier and paranodal junctions [68]. Whether myelinoids with improved myelination but pat-
terned towards the spinal cord identity are applicable to AD research remains to be shown.
Given the accumulating evidence for the importance of myelin breakdown in AD, we anticipate
that myelinoids will become an effective platform for examining disease-associated phenotypes,
underlying molecular mechanisms, and therapeutic strategies targeting myelination programs
[23,27]. For example, APOE4 expression is associated with impaired myelination, whereas 2-
hydroxypropyl-β-cyclodextrin, a compound that promotes cholesterol efflux, has recently been
shown to improve myelination in APOE4-targeted replacement mice in vivo [69]. Whether such
a beneficial effect can be demonstrated in human cell-derived 3D myelinoids that exhibit complex
myelination patterns remains to be determined.

Neuroimmune organoids
NIOs (Figure 2C and Box 1) contain microglia that survey their environment, prune synapses,
phagocytose debris, and respond to injury [70–74]. NIOs can be used to model clearance of
AD-relevant debris as well as inflammatory responses of microglia that contribute to neuroinflam-
mation in the brain. Upon oligomeric Aβ42 treatment, microglia become activated and phagocy-
tose Aβ42 in NIOs, whereas gene expression profiling reveals enrichment for lysosome and
phagocytosis-related pathways [71]. Given that several genetic risk factors for sAD, such as var-
iants of TREM2, are microglia specific, manipulation of their expression in NIOs may reveal novel
disease phenotypes [75]. Cakir et al. developed a CRISPR interference (CRISPRi)-based
strategy to suppress expression of various sAD risk genes in microglia in a high-throughput man-
ner [71]. The authors found that suppression of TREM2 or SORL1 was associated with increased
organoid injury upon oligomeric Aβ42 treatment, indicating protective roles of triggering receptor
expressed on myeloid cells (TREM)2 and sortilin-related receptor (SORL)1 against amyloid-
related toxicity [71]. Engineered microfluidic devices could further improve characterization of mi-
croglia behavior in response to AD-relevant pathology. Tubular NIOs with a hollow core can be
constantly perfused to improve nutrient and oxygen exchange as well as deliver toxic substances
to the organoid [76]. Moreover, NIOs may be used to clarify how microglia propagate tau pathol-
ogy, whereas exposure of NIOs to primary or iPSC-derived peripheral immune cells may reveal
microglia-immune cell interactions that contribute to AD progression [77,78].

Blood-brain barrier organoids

3D models of the brain vasculature include vascularized BOs, pericyte-containing BOs, in vitro
BBB (iBBB), and organs-on-a-chip (Figure 3A,C and Box 1) [30,79–82]. Vascularized BOs de-
velop perfusable CD31+ endothelial tubes while maintaining normal cortical organization, contain
tight junction proteins and have higher transendothelial resistance than control BOs [79]. Similarly,
iBBB exhibits restricted paracellular permeability and active export of small-molecule compounds
[30,80]. Therefore, BBB organoid models can be used to evaluate drug permeability across the
BBB as well as determine the effects of a particular drug on BBB integrity [30,80]. BBB organoid
models also develop AD-relevant pathology: exposure of vascularized BOs and the iBBB to Aβ
disrupts tight junctions and results in amyloid accumulation reminiscent of cerebral amyloid
angiopathy, respectively [30,79]. Moreover, APOE4 iBBB accumulates more amyloid deposits
than APOE3 or ApoE-depleted controls [30]. A BBB organ-on-a-chip enables further functional
compartmentalization of cell types that form the neurovascular unit, whereas uncoupled perfu-
sion of vascular and brain compartments facilitates modeling of nutrient exchange characteristic
of the BBB [81,83]. Overall, in vitro BBB models may be used to study the molecular mechanisms
driving pericyte and endothelial cell dysfunction, breakdown of the selective barrier, impaired glu-
cose metabolism, and other phenotypes relevant to AD.

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Figure 3. Human organoid technology for modeling brain vascular systems. (A) Vascularized brain organoids (BOs)
can be engineered by transcription factor-driven directed endothelial cell differentiation during BO generation. Alternatively,
spheroids containing astrocytes, pericytes, and endothelial cells self-organize into distinct layers that form a blood–brain
barrier (BBB)-like structure. Finally, organ-on-a-chip devices combine microfluidics with in vitro cell culture, enabling the
desired layering of different BBB cell types to be achieved. (B) Differentiation of choroid plexus organoids (ChPOs) enables
modeling of the blood–cerebrospinal fluid (CSF) barrier and the production of CSF-like fluid. ChPOs may be used to study
the changes in CSF composition during Alzheimer’s disease (AD) progression as well as to uncover AD-relevant CSF
biomarkers. (C) Organoid models of brain vascular systems recapitulate key elements of the BBB and the blood–CSF
barrier. For example, BBB models contain endothelial cells, pericytes, and astrocytic end-feet as well as express genes
encoding tight junction and channel proteins, such as aquaporin (AQP)4 (AQP4). ChPOs contain secretory epithelium
producing CSF-like fluid as well as express genes encoding tight junction proteins, such as claudin 1 (CLDN1), and
proteins required for CSF secretion, such as AQP1 (AQP1). Therefore, the BBB and blood–CSF barrier models can be
used to study various aspects of vascular dysfunction associated with AD, and explore novel directions for AD research,
such as the roles of peripheral lipoproteins and the adaptive immune system in AD progression.

Choroid plexus organoids

Recently developed ChPOs form a blood–CSF barrier-like structure lined with secretory cuboidal
epithelium that produces CSF-like fluid into enclosed cysts [84] (Figure 3B,C and Box 1). ChPOs
express various genes encoding transporter, signaling, and junction proteins as well as proteins
required for CSF secretion, such as AQP1 [84,85]. Remarkably, permeability of the ChPO barrier
to various drugs correlates with that of the blood–CSF barrier in vivo, highlighting the potential of
using ChPOs for testing drug permeability and toxic drug accumulation before proceeding to clin-
ical trials [84]. Moreover, the CSF-like fluid produced by ChPOs contains AD-relevant factors,
such as ApoE [84]. Therefore, ChPOs may be used to study how various AD risk factors affect
the integrity of the blood–CSF barrier as well as the composition of the CSF-like fluid. Given
that peripheral ApoE4 has been shown to trigger BBB breakdown, it will be important to define
the impact of peripheral ApoE4 on the blood–CSF barrier as well [63]. Moreover, coculturing
ChPOs with T cells will facilitate modeling of T cell activity and dysfunction in the CSF of AD pa-
tients [64]. ChPOs may also aid identification of novel CSF biomarkers, which could both
inform research directions and facilitate more accurate stratification of patients who have different
types of dementia but share symptomatology and pathology similarities [10,16,33]. However,
biomarker discovery based on the CSF-like fluid produced by ChPOs should be rigorously
validated using CSF isolated from AD patients, given the limitations of in vitro CSF-like fluid
production.

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Organoid models of viral infection Clinician’s corner

Environmental risk factors may precipitate AD onset or accelerate its progression [16]. BOs have The etiology of sporadic AD is poorly
been used extensively to study viral infection, including severe acute respiratory syndrome coro- understood, whereas GWAS and
high-throughput profiling of the
navirus 2 (SARS-CoV-2) infection most recently [82,85–87]. Viruses may preferentially infect dif-
human brain have revealed immense
ferent cell types of the brain, illustrating the need for diverse BO models to evaluate viral and complexity of AD pathogenesis that in-
other environmental risk factors for AD [85,87]. For example, SARS-CoV-2 selectively infects volves multiple cell types and extensive
ChPOs and astrocyte-enriched BOs as well as infects ApoE4 neurons and astrocytes more read- cell–cell interactions.

ily than their ApoE3 counterparts [85,87]. However, it remains to be determined if there is a defin- Human organoid technology provides
itive link between SARS-CoV-2 and AD although reports supporting the idea are emerging [88]. sophisticated self-organizing human
Conversely, viruses of the Herpesviridae family have long been implicated in AD pathogenesis brain-like tissues for modeling these
cell–cell interactions in the context of
[16,89]. Infection of a neural progenitor cell-derived 3D brain tissue model with herpes simplex
AD. The organoid technology over-
virus (HSV)-1 induces formation of Aβ deposits, promotes astrocyte activation, and drives neuro- comes the limitations associated with
inflammation [90]. Similarly, infection with Zika virus accelerates Aβ aggregation and tau phos- using rodent models that exhibit sub-
phorylation in fAD BOs as well as leads to microglia and astrocyte activation in NIOs [73,91]. stantial species divergence in their cel-
lular programs compared with those of
These findings indicate that viral infection evokes AD-relevant pathology and warrant further in-
humans.
vestigation into the relationships between pathogenic insults, known risk factors for AD, and
other effectors that may trigger AD onset or worsen its progression. BO technology can be scaled to
develop high-content screening as-
says for drug discovery using
High-throughput drug screening using brain organoids engineered microarrays and auto-
In addition to drug development applications discussed throughout this review, BOs may also be mated imaging platforms to evaluate
applied for high-throughput drug screening, if BO scaling and reproducibility are optimized (Box disease pathology and response to
3). For example, engineered micropillar arrays may serve as scaffolds for efficient and highly re- therapeutic intervention.

producible generation of a large number of BOs suitable for drug screening [92]. Moreover, tissue Modeling viral infection using BOs may
clearing techniques enable high-content screening of organoid pathology by automated imag- rapidly inform research and medical
ing acquisition and analysis [50]. Park et al. developed a scalable platform to evaluate accumula- community of how novel viruses,
including SARS-CoV-2, infect human
tion of Aβ and p-tau in BOs using automated imaging in 96-well plates [50]. The authors
brain cell types and precipitate sec-
performed a proof-of-principle screening of a panel of drugs targeting AD-relevant cellular path- ondary pathology, such as neurode-
ways and robustly detected reduction in the Aβ and p-tau burden under most treatment condi- generation.
tions [50]. We anticipate that advancing automated imaging techniques will also be applied to
Organoids and organs-on-a-chip
assess AD-relevant pathology in various other organoids discussed throughout this review,
engineered to contain brain vascular
such as myelinoids to evaluate changes in the myelin volume [68]. systems can be used to quantitatively
evaluate drug permeability and toxic
Limitations of brain organoids for the study of Alzheimer’s disease drug accumulation in brain tissues be-
fore initiating clinical trials. Substantial
Although the exponential growth of the BO field has led to substantial improvements in BO differ- species divergence between mouse
entiation and characterization protocols in recent years, important limitations relevant to disease and human brain vasculature necessi-
modeling remain. BOs are primarily a neurodevelopmental model that recapitulates early human tates complementary evaluation of
brain development rather than the aging brain when AD manifests. Therefore, the extent to which drug pharmacokinetics in human
iPSC-based cellular models.
BOs can be used to model age-related neurodegeneration is not self-evident and requires rigor-
ous validation of the observed phenotypes using alternative approaches. These include immuno-
histochemical and transcriptomic analysis of primary human brain tissue as well as experiments
in vivo. Application of various techniques to model age-relevant events, such as BBB leakage
by serum exposure as discussed earlier, is likely to exacerbate neurodegenerative phenotypes
and strengthen the relevance of using BOs for the study of AD [62]. BOs are also a reductionist
model, resembling only certain aspects of the brain cytoarchitecture and function; for example,
neural organoids largely lack microglia, myelination, and the BBB. Although it remains challenging
to introduce these critical components into a single BO model, specialized BOs discussed
throughout this review – myelinoids, NIOs, BBB organoids, and ChPOs – offer a versatile platform
for modeling distinct AD-relevant phenotypes. The lack of vascularization in BOs contributes to
internal hypoxia and cellular stress that lead to necrosis and impaired cell subtype specification
[93]. Oxygen and nutrient exchange may be improved by using spinning bioreactors, engineering

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Trends in Molecular Medicine
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organoids-on-a-chip, introducing vascular cells, or simply slicing BOs (Box 3) [8,79,94–96]. Finally, Outstanding questions
organoid-to-organoid and batch-to-batch variation is a major issue that affects reproducibility of BO How does the interplay between
studies and is a barrier to high-throughput applications, such as drug screening. Variation can be neurons and glial cells drive
neuroinflammation in BO models of
mitigated by selecting guided BO differentiation approaches over those of unguided differentiation
AD?
(Box 3), generating BOs from a defined number of iPSCs using microscaffolds, and including a
quality control step to ensure correct neuroectoderm organization during differentiation [39,92]. What are the key events that promote
Similarly, careful assessment and reporting of the BO differentiation stage are also required to obtain microglial activation leading to
neuroinflammation in AD BOs?
reproducible results. For example, studying ApoE-related phenotypes requires sufficient BO matu-
ration to establish a prominent astrocyte compartment, given that astrocytes are the primary source How does a multitude of genetic and
of secreted ApoE [21,52]. Likewise, optimization of NIO differentiation protocols is required to en- environmental risk factors for AD inter-
act to drive disease progression in
sure consistent microglia density across different batches of NIOs [73].
BOs?

Concluding remarks and future directions How do common and rare APOE
Rapidly advancing human BO technology enables modeling of complex cell–cell interactions of variants affect myelin health and
integrity in myelinoids?
the human brain using easily obtainable and genetically modifiable in vitro models. BOs can be
engineered to contain the desired combinations of genetic risk variants and cell types, so that sci- How do various genetic and
entific hypotheses could be addressed in the most direct manner (see Outstanding questions). environmental risk factors for AD affect
BBB integrity in brain vasculature-
Unique advantages of BO models discussed throughout this review can be combined to derive
containing organoids?
organoids that recapitulate distinct cellular phenotypes associated with AD. For example, integra-
tion of microglia into myelinoids would enable modeling of microglia-mediated recycling of myelin How do APOE variants affect the
debris upon myelin breakdown as well as how remyelination could be invigorated by therapeutic integrity of the choroid plexus as well
as the composition of the CSF-like
intervention in AD patients. Novel approaches to promote BO maturation and specification will
fluid in choroid plexus organoids?
also facilitate the development of more complex BO models. For example, slicing BOs resolves
diffusion constraints and allows formation of an expanded cortical plate and nerve tracts, Can choroid plexus organoids
whereas integration of a Sonic hedgehog signaling center promotes topographical BO specifica- producing CSF-like fluid be used for
biomarker discovery?
tion [95–97]. The ability to assemble heterotypic regionally patterned BOs into assembloids will
enable modeling of the interactions between distal brain regions during AD progression; for ex- How can aging-associated pheno-
ample, cortical–hippocampal assembloids could be used to study tau propagation, modeling types be induced in BOs to study the
impact of aging on AD pathogenesis?
the in vivo pattern of tau spreading to the neocortex, as well as clarify the roles of ApoE4 and mi-
croglia in tau propagation [10,15,25,77]. Development of neural-vascular assembloids could fur- What are the roles of the adaptive
ther advance BO vascularization efforts and facilitate the study of the neurovascular unit [98]. immune system in AD pathogenesis,
Chimeric BOs, where only a specific cell type expresses a disease risk variant (reminiscent of con- and how can these roles be evaluated
experimentally using the organoid
ditional transgenic mouse models), may be used to define the cell type specificity in mediating the
technology?
effects of genetic risk variants for AD [30,99]. Advances in bioengineering of biomimetic scaffolds
and organs-on-chip may enable BO perfusion as well as assembly of multiorgan chips to uncover
effectors originating from distal organs, such as gut microbiota-derived metabolites, that contrib-
ute to AD progression [100,101]. Finally, BO transplantation in vivo will accelerate the efforts to
promote BO maturation and functionalization, including circuit integration, vascularization, and
microglia infiltration [102,103]. We envision that BO transplantation models will serve as drug de-
velopment platforms that combine the advantages of complex in vivo environment with the ad-
vantages of using human cell-derived BO models that have the potential to reveal human-
specific molecular mechanisms governing AD progression, thus accelerating therapeutic devel-
opment for AD.

Acknowledgments
The authors would like to thank Louise and Herbert Horvitz, the Christopher Family, the Judy and Bernard Briskin Fund, and
the Sidell Kagan Foundation for their generosity. This work was supported by the National Institute of Aging of the National
Institutes of Health R01 AG056305, RF1 AG061794, R01 AG072291, and RF1 AG079307 to Y.S. J.C. is a predoctoral
scholar in the Stem Cell Biology and Regenerative Medicine Research Training Program of the California Institute for Regen-
erative Medicine (CIRM). Figures 1, 2, and 3 were created with BioRender.com

Trends in Molecular Medicine, August 2023, Vol. 29, No. 8 669


OPEN ACCESS
Declaration of interests
G.B. is an employee of SciNeuro Pharmaceuticals. The remaining authors have no interests to declare.
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