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While traditional 2D monolayer cultures have been widely vascular scaffolds, or co-culture with microglia is under
used for NDD research, they lack the spatial complexity development to address these limitations.
necessary to replicate critical developmental processes Collectively, these innovations position brain
such as cell migration, polarization, and intricate cell-cell organoids as a transformative model for investigating
interactions. 40 NDDs, offering unprecedented opportunities to elucidate
Brain organoids represent a groundbreaking advancement disease mechanisms, personalize medicine, and accelerate
in modeling human neurodevelopment, overcoming many therapeutic discovery.
limitations of animal models and 2D culture systems. These
3D structures recapitulate key aspects of human brain 3. Brain organoids: 3D self-organized
development, including cytoarchitecture, gene expression models for neurodevelopmental research
profiles, and epigenomic signatures. Derived from and disease modeling
8,11
hPSCs, organoids are formed by allowing cells to aggregate Brain organoids are 3D, self-organized cellular structures
into embryoid bodies (EBs), which, under specific culture generated in vitro, serving as powerful models for studying
conditions, differentiate into neuroectodermal tissue. 41,42 49
Neural progenitor cells (NPCs) within these structures neurodevelopment and neural function. Derived from
proliferate, expand the epithelial architecture, and sequentially iPSCs, these models can differentiate into specific brain
transition through neurogenic and gliogenic phases. regions, such as the dorsal or ventral forebrain, in response
43
to developmental signaling factors. Alternatively, through
The complexity and cellular diversity of brain organoids unguided differentiation, they can give rise to diverse
depend on the morphogens used during differentiation. neural and non-neural cell lineages. 15,50
Unguided protocols generate cerebral organoids containing
various brain regions, while guided protocols employ 3D organoid culture technology has significantly
specific morphogen gradients to establish distinct regional advanced developmental neurobiology by providing
identities. 44,45 Advanced approaches, including fusion physiologically relevant models that more closely mimic
organoids (assembloids), enable the study of interregional human brain development than traditional animal models,
interactions, while in vivo transplantation into rodent brains which often exhibit structural, functional, and developmental
facilitates vascularization and neuronal circuit maturation. 14 differences. 51,52 Unlike 2D monolayer cultures, brain
organoids replicate many characteristics of human brain
Compared to 2D cultures, brain organoids provide a
more physiologically relevant environment by mimicking tissue, including early cellular organization, cell-type
diversity, and cell-cell interactions.
This structural
42,53
the 3D structure, temporal dynamics, and spatial resemblance to in vivo tissue enables the study of neuronal
organization of the developing brain, which are essential and glial interactions during early development. Under
46
for studying processes such as neurogenesis, lamination, optimal differentiation conditions, brain organoids contain
and long-range connectivity. In contrast, 2D systems mature, functional neurons, and astrocytes, as confirmed
are limited by their inability to support complex tissue by immunohistological and electrophysiological analyses.
architecture and often fail to capture disease phenotypes Their ability to model connectivity between different brain
that depend on multicellular or layered organization. 47
regions allows researchers to study network formation
Compared to in vivo animal models, organoids offer and synaptic activity. Given their genetic and structural
species-specific insights into human brain development and compatibility with the human brain, brain organoids provide
pathology. Rodent brains differ significantly from human an essential platform for investigating NDDs, particularly
brains in terms of size, structure, cortical organization, and those involving large-scale structural rearrangements and
gene regulatory networks. Moreover, transgenic models complex genetic interactions. Patient-derived iPSCs enable
54
often do not fully replicate the polygenic or epigenetic the generation of organoids that replicate disease phenotypes,
contributions to NDDs observed in humans. 13,48 facilitating the study of congenital brain malformations and
8,55
Organoids also allow for high-throughput screening neural circuit dysfunctions. Furthermore, organoids
and genetic manipulation in a patient-specific context, offer a scalable and accessible alternative to human brain
which is often impractical or ethically constrained in tissue, allowing researchers to explore uniquely human
animal models. Furthermore, they enable the study of early aspects of brain formation and evolution, thus broadening
developmental processes that are inaccessible in vivo, such our understanding of neurodevelopmental processes and
50,56
as the emergence of human-specific progenitor populations disorders.
like outer radial glia. 13,48 Nevertheless, organoids also face The generation of human brain organoids from human
challenges, including variability in differentiation protocols, iPSCs and human ESCs under 3D culture conditions relies
lack of vascularization and immune components, and on either unguided or guided differentiation methods
limited maturation. Integration with microfluidic systems, (Table 1). Unguided differentiation exploits the intrinsic

Volume 1 Issue 3 (2025) 4 doi: 10.36922/OR025100010