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distribution in larger cultures, the risk of aberrant vessel cells. A key example is the overexpression of ETS variant
formation, and the need for precise temporal and dosage 2 (ETV2), which promotes the formation of complex
regulation due to their transient bioactivity. vascular-like networks within COs (Figure 5D). ETV2-
expressing cells exhibit essential BBB characteristics,
3.1.3. Organoid fusion for vascularization such as nutrient transport and TJ formation, facilitating
Organoid fusion is a promising strategy to enhance organoid maturation. Cakir et al. demonstrated this
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vascularization in BOs by combining distinct cell populations approach by genetically modifying hESCs to express ETV2
to better mimic the NVU (Figure 5C). Song et al. under a doxycycline-inducible promoter. By overexpressing
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demonstrated this approach by fusing neural progenitor the ETV2 transcription factor in hESCs, ECs were induced,
spheroids, EC spheroids, and induced pluripotent stem cell and a 20% ETV2 expression cell ratio was optimized to
(iPSC)-derived mesenchymal stem cells to form hybrid NVU successfully generate V-Organoids. In V-Organoids, ECs
assembloids. These assembloids expressed key BBB markers, formed stable vascular networks, which persisted for
such as glucose transporter 1, TJ protein zona occluden 1 30, 70, and 120 days of culture, with significantly greater
(ZO-1), and matrix metalloproteinases, while promoting vessel area and network complexity compared to control
VEGF-A secretion and cortical tissue development. Ahn organoids. In addition, V-Organoids exhibited BBB-like
et al. utilized hiPSCs to generate blood vessel organoids characteristics, confirmed by TJ markers such as α-ZO-1,
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and investigated their interaction with COs. The results occludin, and kinase insert domain receptor. After 120 days
demonstrate that blood vessel organoids could infiltrate COs of culture, V-Organoids showed reduced apoptosis and
and form functional vessel-like structures. These structures significantly lower HIF-1α levels, indicating that the
were composed not only of ECs (cluster of differentiation vascular network effectively supported oxygen and nutrient
31-positive [CD31 ]) but were also surrounded by transport. Electrophysiological patch-clamp experiments
+
smooth muscle cells (smooth muscle actin-positive) and revealed that neurons in V-Organoids could generate
pericytes (PDGFR-positive), exhibiting molecular marker multiple action potentials at 80 – 90 days, unlike those
characteristics of the BBB. Similarly, Sun et al. fused brain in control organoids. Single-cell RNA sequencing further
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and vascular organoids to form neurovascular assembloids, demonstrated that V-Organoids exhibited gene expression
enabling the study of neurovascular interactions (Figure 5B). patterns resembling those of human fetal brain development
The fused organoids developed functional vascular networks at 16 – 19 weeks, while control organoids were closer to
that coexisted with neurons and promoted the generation the 10 – 12-week stage. This method provides a powerful
of neural progenitor cells. In addition, the fused organoids tool for refining BOs’ models by generating accurate BBB
exhibited structures resembling the BBB, and microglial cells, components and promoting functional vascularization, all
on immune stimulation, displayed synapse phagocytosis, while allowing precise control over cell differentiation.
indicating their role in immune regulation. These studies Gene editing techniques offer high precision and the
highlight the feasibility of vascularizing BOs through potential for consistent induction of vascular features
vascular organoids, offering a new model for investigating across organoid batches. Scalability is feasible, as gene-
neurovascular diseases. However, challenges such as the edited stem cells can be expanded and differentiated into
absence of a perfusion system – leading to necrotic cores in organoids with vascular potential. However, challenges
the organoids – still persist. include ensuring the efficiency and specificity of gene edits,
Organoid fusion has shown the potential to generate more as well as addressing ethical considerations. Standardizing
physiologically relevant tissue architectures. However, its gene editing protocols and conducting thorough validation
scalability remains constrained by the efficiency of the fusion are essential for reproducibility and scalability.
process and the challenges associated with sustaining viability
and functionality in larger fused organoids. Reproducibility 3.2. Biomaterial for vascularized BO
is further impacted by variations in organoid size, cellular Biomaterials can facilitate cell growth within organoids and
composition, and the precision of spatial alignment during regulate the microenvironment, thereby influencing the
fusion. Advances in bioengineering technologies, including development of neural networks and vascular structures
microfabrication and bioprinting, offer promising strategies within the organoids. As a result, biomaterials offer a
to improve the scalability, standardization, and structural promising approach for generating vascularized BOs.
consistency of this approach. Recent advancements in biomaterials, including the use
of natural and synthetic hydrogels, have contributed to
3.1.4. Gene editing for vascularized BOs improved maturation and vascularization of BOs. This
Gene editing offers a promising strategy for inducing section focuses on key developments in the biomaterials
vascularization in BOs, particularly through transcription field that may enhance the formation of BOs, particularly
factor-mediated differentiation of hESCs into vascular their capacity to support both neural and vascular

Volume 1 Issue 2 (2025) 11 doi: 10.36922/or.8162