Biological

Psychiatry Review

Transplantation Strategies to Enhance Maturity

and Cellular Complexity in Brain Organoids
Meiyan Wang, Fred H. Gage, and Simon T. Schafer

ABSTRACT
Human brain organoids are 3-dimensional cell aggregates that are generated from pluripotent stem cells and reca-
pitulate features of the early developing human brain. Brain organoids mainly consist of cells from the neural lineage,
such as neural progenitor cells, neurons, and astrocytes. However, current brain organoid systems lack functional
vasculature as well as other non-neuronal cells that are indispensable for oxygen and nutrient supply to the orga-
noids, causing cell stress and formation of a necrotic center. Attempts to utilize intracerebral transplantation ap-
proaches have demonstrated successful vascularization of brain organoids and robust neurodifferentiation. In this
review, we summarize recent progress and discuss ethical considerations in the field of brain organoid
transplantation.
https://doi.org/10.1016/j.biopsych.2023.01.004

Brain organoids are self-organizing 3-dimensional cultures of
mainly neuroectodermal origin that resemble features of the
developing human brain. These culture systems are usually
derived from stem cells, such as embryonic stem cells (ESCs)
or induced pluripotent stem cells (iPSCs), that have the po-
tential to differentiate into any type of cell of the human body.
The fact that brain organoids can be generated from iPSCs
derived from human subjects presents the unprecedented
opportunity for using these models to study process dynamics
pertaining to the unique characteristics of human brain
development and human-specific brain disorders. Brain orga-
noid models have been successfully used in various applica-
tions (1–4). However, despite a high correlation of cellular
diversification between brain organoid models and the devel-
oping human brain (5), current protocols still face a number of
limitations, including incomplete maturation and cellular stress.
Of note, a recent study (6) demonstrated that cell stress is
limited to a defined subpopulation of cells in the organoids and
does not affect neuronal specification or maturation. Never-
theless, the lack of a microvasculature system results in low
oxygen levels and reduced nutrient supply to the center of the
organoid, rendering the organoid’s core prone to variable de-
grees of necrosis (7,8).
To overcome these limitations, various approaches to allow
vascularization of brain organoids are the subjects of active
research, including in vitro co-culture strategies using vascular
cells (9,10) or fusion to blood vessel organoids (11,12), the
modification of culture conditions to generate vascular cells
(13), genome-editing technologies to ectopically induce endo-
thelial cells (14), and “organoid-on-a-chip” microfluidic prepa-
rations (15). Interestingly, some of these studies reported that
the formation and/or presence of vascular-like structures in
brain organoids resulted in attenuated apoptosis and acceler-
ated neuronal maturation, even in the absence of blood flow.
Another study explored a specific xenotransplantation
strategy, by which human brain organoids were transplanted
into highly vascularized brain regions of an animal host to
vascularize the developing organoid tissue. In a recent study,
Mansour et al. (7) transplanted cerebral organoids derived from
human ESCs into the retrosplenial cortex of immunodeficient
mice and reported highly efficient infiltration with blood vessels
and the presence of functional circulation within the organoid
grafts.
There are several benefits for organoid models to feature an
active and physiologically perfused vasculature. One advan-
tage is prevention of necrosis and cell death that severely
compromise the integrity and long-term culture of organoid
models. Another benefit is that it supplies the organoids with
nutrients that are otherwise missing from current in vitro par-
adigms. The presence of a vascular system within brain
organoids provides great value, not only because it supplies
nutrients but also because it may enable faithful modeling of
the invasion/extravasation and interaction with different non-
neuronal cell types.
In this review, we focus on the benefits and purpose of
transplantation strategies to incorporate functional vascular
systems and to allow the generation of more complex organoid
models for the study of cellular phenotypes underlying in vivo–
like physiology.
NEURAL TRANSPLANTS OR NEURAL CHIMERAS?
Neural and non-neuronal cells can be transplanted directly into
the brain of a nonhuman animal during development or
adulthood to allow the investigation of these cells under quasi-
physiological conditions (Figure 1A). As an example, trans-
planted neural precursor cells can mature into functional
neurons and integrate into the host nervous system to form
functional connections. The grafted single neuronal cells are

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Brain Organoid Transplantation
capable of receiving synaptic input from and providing syn-
aptic input to host neurons. Different types of glia have also
been shown to be able to integrate into and interact with host
cells following transplantation. A special and new form of
neural transplantation is the concept of brain organoid trans-
plantation, in which whole-brain organoids grown up to a
certain stage in vitro are transplanted into an animal host to
allow functional integration and adequate blood supply for
extended periods of time (Figure 1A).
Human neural chimeras, in contrast, are a special form of
transplant in which donor cells are introduced into a nonhuman
host prior to gastrulation (16). Here, human donor cells inter-
mingle with host cells and populate the nervous system from
the onset of neurogenesis to develop side-by-side within the
host, enabling some level of integration (Figure 1B). Particular
cell types of the host may be genetically ablated, thus
permitting donor cells to contribute to forming a tissue or or-
gan. An example of such an approach is blastocyst comple-
mentation (Figure 1B) (17). Extensive contribution of donor
cells to an animal host’s forebrain has been reported following
neural blastocyst complementation in mice (17).
The distinction between neural cell or organoid transplants
and neural chimeras is vaguely defined and largely based on
whether the cells introduced into the host animal remain
limited to the nervous system or contribute to other organs as
well. In a recent consensus study report, the National Academy
of Sciences suggested that a useful dividing line could be the
gastrula stage of embryogenesis, because many scientists in
the field view the introduction of exogenous cells to an or-
ganism prior to gastrulation as generating chimeras and after
gastrulation as generating transplants (16). Of note, most chi-
meras are generated using donors from the same species. The
formation of human neural chimeras in mice has not been re-
ported to date.
BRAIN ORGANOID TRANSPLANTATIONS
The first studies that attempted to induce vascularization of
brain organoids in vivo took advantage of an intracerebral
transplantation technique originally developed for trans-
planting fetal or adult tissue into the rodent brain (18–20). This
technique allowed sufficient infiltration of the transplanted
brain organoids by the vasculature system of the host, which
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Figure 1. Schematic illustrating the distinction
between neural chimeras and neural transplants. (A)
Neural and glial cells or brain organoids can be
directly transplanted into the brain of a fetal or adult
animal host. (B) Neural chimeras, in contrast, are a
special form of transplant in which donor cells are
introduced into the host prior to gastrulation.
could be observed after 7 to 10 days in vivo; extensive
vascularization was observed after 14 days after engraftment
(Figure 2) (7). Importantly, using two-photon microscopy, the
authors were able to demonstrate that the organoid-infiltrating
blood vessels showed robust blood flow. The survival of the
organoid transplants was highly dependent on successful
vascularization (7); very limited cell death and decreased
cellular stress were observed in brain organoids in vivo (7,21).
In line with this finding, a recent study showed that cellular
stress pathways ectopically activated in cortical organoids
in vitro could impair the proper specification of cellular sub-
types and maturation of neural progenitors (22). When trans-
planted into the cortices of immunocompromised mouse pups,
dissociated organoids exhibited increased levels of cell sub-
type specification in both outer radial glia and newborn neu-
rons. Although the group only analyzed dissociated organoid
cells in vivo (22), the results suggest that the transplantation
was—at least in part—sufficient to alleviate the cell stress and
subtype defects observed in vitro. Nevertheless, a more
detailed analysis is needed to assess the differences between
dissociated and whole organoid grafts in vivo. It is important to
highlight another study that reported enhanced survival and a
higher degree of vascularization in cerebral organoid grafts as
compared with transplants using dissociated neural progenitor
cells (23). Engrafted organoids featured an increased number
of neurons and showed progressive transition from the
Figure 2. Schematic illustrating vascularization of brain organoids in vivo,
following engraftment.
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progenitor-dominated, rosette-rich organoid tissue to a more
mature tissue that predominantly consisted of neurons and
astrocytes in vivo (7). Further research is needed to assess the
intrinsic and environmental drivers that permit sufficient
vascularization of brain organoids in vivo. Alternatively, intro-
duction of human vasculature prior to transplantation appears
to be a promising strategy to potentially replace the host
vasculature with human cells. Although not sufficiently
explored, in vitro vascularized brain organoids displayed some
level of functional perfusion by the animal host on trans-
plantation (9,14).
Neuronal network activity in the engrafted organoids has
been assessed using different approaches. Longitudinal
measurements of AAV8-CaMKII-jRGECO1a–labeled neurons
in the organoid grafts by two-photon calcium imaging revealed
recurrent and spontaneous rhythmic calcium transients (7).
Moreover, extracellular recordings using multielectrode arrays
identified neuronal action potentials and synchronized
neuronal network activity (7). Cross-correlation analysis of
neuronal activity in the same recording sites revealed a more
active and coordinated network in late-stage grafts, suggest-
ing progressive neuronal maturation (7). An independent study
used whole-cell patch-clamp recordings and showed that
human glutamatergic neurons indeed acquired electrophysio-
logical properties indicative of progressive cellular maturation
that occurred within the graft over time, following trans-
plantation (24). Together, these data suggest robust neuro-
differentiation potential and extended maturation of human
brain organoids in vivo.
Extensive axonal growth of organoid grafts and long-
distance axonal projections from the grafts to distant targets
of the host brain have been reported. Human brain organoids
extended axons through the cortical layers and the corpus
callosum (7), along the corticospinal tract (25), and to the basal
brain regions (24). Viral tracing and optogenetics have identi-
fied synaptic connections between human cerebral organoids
and mouse host neurons (7,24). Interestingly, in response to
environmental stimuli, neurons in organoid grafts demon-
strated state-dependent firing changes (7). Mice with cerebral
organoid grafts have also been shown to feature increased
startle fear responses to auditory conditioned stimuli (24).
These data suggest that human brain organoids can integrate
and form functional circuits with the host brain.
Other studies have also reported improved lesion repair
through organoid grafting in mouse and rat models of brain
injuries (26–28). In a rat model of traumatic brain injury,
engrafted cortical organoids supported reconstruction of
damaged motor cortex and led to improved neurologic motor
function and reduced brain damage (28). Similarly, in a cerebral
artery occlusion model of stroke, engrafted cortical organoids
supported motor cortex region–specific reconstruction in rats
(27). In both models, cells from engrafted cortical organoids
displayed extensive migration into different brain regions along
the corpus callosum (27,28). Using a controlled cortical impact
model for experimental brain injury, increased vascularization
and reduced glial scarring in the lesion area were observed
following brain organoid transplantation, suggesting that the
presence of the organoid graft was able to promote neural
repair (26). Despite these promising findings, additional
research on the effects that brain organoid grafts exert on the
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damaged brain environment and the potential for controlling is
warranted.
ETHICAL CONSIDERATIONS FOR HUMAN NEURAL
TRANSPLANTS AND CHIMERAS
While advancements in brain organoid research have made
them an ever more valuable tool for clinicians and scientists,
the very features that make them more similar to the complex
human brain have raised some ethical concerns. This is, in
part, based on the use of human materials and the concern
that transplanting humanlike tissue into an animal host may
result in enhancement of brain functions due to partial hu-
manization of the host.
The National Academy of Sciences has recently released a
consensus study report from the Committee on Ethical, Legal,
and Regulatory Issues Associated with Neural Chimeras and
Organoids to address ethical concerns and to summarize as-
pects related to governance and oversight of research with
human-animal chimeras (16). According to this report, the is-
sues specific to this form of transplantation include concerns
about general animal welfare, about the distinctions between
human beings and animals, and about enhancement of brain
functions and consciousness.
There are a range of positions on these important issues
that require discussion among people with different perspec-
tives. Thus, the research on human transplants and chimeras is
subject to a wide range of national and international oversight
mechanisms that regulate various aspects of this field. These
include, but are not limited to, a collection of federal and state
laws and regulations, professional society guidelines (e.g., In-
ternational Society for Stem Cell Research and Guidelines for
Stem Cell Research and Clinical Translation), nonbinding
consensus studies by scientific academies (e.g., National
Academy of Sciences), and various conference reports. The
legal requirements pertaining to the use of human tissues and
stem cells and the use and welfare of nonhuman animals are
often implemented by research oversight committees at an
institutional level. In general, if research in any biotechnological
area raises substantial ethical and regulatory concerns, a
3-tiered oversight structure has been proposed and recom-
mended by many National Academies committees for the
guidance of institutional committees. Most of the research
involving human neural transplants falls into the first-tier
category because it presents no new ethical or regulatory
concerns beyond those that the current oversight committee is
charged with addressing. Research that falls into the second
tier can only proceed after additional review and approval
because it may raise additional ethical concerns beyond what
the current oversight system can address (protection of human
subjects and animal welfare). As an example, research that
renders a brain more humanlike, particularly when trans-
planting human cells into the brains of nonhuman primates,
may fall into this category. The third-tier category encom-
passes research that should not be permitted at present; this
includes experiments that are currently forbidden under federal
law, such as the introduction of human ESCs or iPSCs into the
blastocyst of a nonhuman animal.
As indicated above, the institutional oversight committees
for human neural transplant and chimera research have

Brain Organoid Transplantation
specific regulatory charges to ensure the protection of human
subjects and animal welfare. If an enhancement of brain
functions or the acquisition of new capacities is suspected in
animals used as a host, additional training to determine which
conditions and environments are preferred by the animals may
be required for the evaluators. Veterinary evaluations may be
required to include ongoing monitoring for changes in specific
behaviors (e.g., activity, feeding, and socializing) and assess-
ment of physiological parameters (e.g., blood pressure, cortisol
level, and heart rate) to ensure animal welfare.
As the research on neural transplants and chimeras de-
velops, further discussion of oversight is needed to address
the ethical concerns that do not fall within the scope or
expertise of the existing committees, offering opportunities for
much broader discussions on a national and international level.
OUTLOOK
Studying prenatal human brain development remains a chal-
lenge due to limited access to live human brain tissue and
ethical constraints on research using human embryos. Brain
organoids offer a promising avenue to overcome these limi-
tations by allowing researchers to study human brain devel-
opment directly in vitro. Great progress has been made in the
last decade to model human disorders in a dish, but challenges
remain that need to be overcome when attempting to translate
clinical observations made in vivo to molecular and cellular
phenotypes observed in vitro.
Recent studies that performed similar organoid xeno-
transplantation experiments in mice (23) and rats (29)
demonstrated that the cellular and molecular state of maturity
was enhanced as compared with in vitro organoids. Trans-
planted neurons displayed increased morphological
complexity and synaptic properties as compared with their
in vitro counterparts. However, further work is needed to
disentangle the exact mechanisms that support organoid
maturation and complexity in vivo. Moreover, it remains to be
explored whether the increased maturity would reveal molec-
ular and cellular changes in complex neuropsychiatric and
neurological disorders that are otherwise difficult to investigate
in other cell models.
While current brain organoids allow us to recapitulate, in
particular, those developmental processes that emerge from
cells of the neuroectoderm, they still lack other cell types that
are of non-ectodermal origin. Incorporation of cell types
forming the neurovascular unit and blood vessels (14,30) are
forthcoming approaches to increase the cellular composition
of these models. Additionally, most brain organoid systems
lack microglial cells and, in most cases, they develop with
limited or underrepresented amounts of oligodendrocytes and
astrocytes. Moreover, brain organoid models that allow
specification of anatomically defined subclasses of human
astrocytes have yet to be developed. It is also worth noting
that cerebral organoids generated through unguided ap-
proaches can spontaneously develop microglia-like cells that
possibly emerge from mesodermal contributions within these
structures (31). Additionally, short-term co-culture experiments
have demonstrated that the incorporation of microglia-like
cells into organoids appears feasible (32–35). However, it is
not known whether these cells are similar to the ones that
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originate from the yolk sac in vivo. The variability of microglia-
like cells within cerebral organoids and the lack of a complete
recapitulation of the resting ramified state observed in vivo
warrant further investigation.
As highlighted in this review, transplantation strategies can
provide an alternative tool for investigating human cells under
in vivo–like conditions. However, concerns about the contri-
bution of host cells to the human graft should be considered
when generating these models because they could potentially
alter the system or cell type under investigation. Thus, forward
engineering of ever more sophisticated human organoid
models would provide a path toward investigating the inter-
action between different human non-neuronal and neural cells
under in vivo–like conditions. Previous studies have shown
that human glial progenitor cells can be transplanted and that
they generate functional astrocytes when transplanted
neonatally into murine hosts [e.g., (36)]. Neonatal engraftment
of human glial progenitor cells allows differentiation into both
astrocytes and myelinogenic oligodendrocytes in a hypo-
myelinated environment (37–39). Engrafted human glial pro-
genitor cells may even outcompete the resident glial
progenitors and dominate the host brain (36,37,39). Using such
human glial transplants revealed species-specific glial prop-
erties (36) as well as disease-associated changes in astrocyte
and oligodendrocyte differentiation (37,39). Similarly, trans-
plantation of ESC/iPSC-derived microglial or hematopoietic
progenitors into neonatal or postnatal mouse brains resulted in
a context-dependent differentiation of microglia (40–43).
However, generating these glial transplant models required
expression of human-specific factors such as CSF1 (colony-
stimulating factor 1), which was achieved by utilizing mice
that harbored a knockin of human CSF1 into the murine locus
(40,41,43) or constitutive CSF1 overexpression (42). The ESC/
iPSC-derived microglia acquired complex morphologies spe-
cific to microglia and gained gene expression signatures
shown to be induced by the in vivo brain environment (40–43).
Collectively, these data suggest that engrafted human glial
cells are functional in the mouse brain. In the future, grafting
human brain organoids that contain a diverse set of resident
human glial cells may provide an in vivo environment ideal for
studying human neuron-glia interactions.
A more complete in vivo brain organoid model offers many
advantages over current in vitro protocols regarding the
feasibility of investigating long-term phenotypes. We advocate
that, rather than replacing existing models, future research
should focus on developing complementary strategies. It will
be the combination of both purposeful reduction and more
authentic, complex model systems that will provide the means
to start disentangling the genetic, molecular, and cellular
complexity underlying human disorders.
ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported by the American Heart Association and the Paul G.
Allen Frontiers Group (Grant No. 19PABHI34610000 [to FHG]), National
Institute of Mental Health (Grant No. U19-MH106434 [to FHG]), JPB Foun-
dation, Annette C. Merle-Smith, Robert andMary Jane Engman Foundation,
Lynn and Edward Streim, and the Ray and Dagmar Dolby Family Fund (to
FHG). MW is supported by a NARSAD Young Investigator Grant from the
Brain and Behavior Research Foundation. STS is supported by a NARSAD
Young Investigator Grant from the Brain and Behavior Research Foundation
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and a priority program grant from the German Research Foundation (DFG
SPP 2395 [to STS]) under award No. 500300695.
We thank members of the Gage Lab for discussions, M.L. Gage for
editorial suggestions and B. Coyne for administrative assistance.
Figures were created on BioRender.com.
The authors report no biomedical financial interests or potential conflicts
of interest.
ARTICLE INFORMATION
From the Laboratory of Genetics, The Salk Institute for Biological Studies, La
Jolla, California (MW, FHG, STS); Department of Psychiatry and Psycho-
therapy, Medical School, Technical University of Munich, Munich, Germany
(STS); and Center for Organoid Systems and Tissue Engineering, Technical
University of Munich, Garching, Germany (STS).
Address correspondence to Simon T. Schafer, Ph.D., at simon.schafer@
tum.de, or Meiyan Wang, Ph.D., at mwang@salk.edu.
Received Jun 15, 2022; revised Nov 23, 2022; accepted Jan 9, 2023.
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