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Batch-to-batch inconsistencies can arise due to differences in integrity. It drives sprouting angiogenesis by activating
cell sourcing, culture conditions, and the timing of co-culture the VEGF–VEGF receptor (VEGFR) signaling pathway,
integration. Standardizing protocols and employing automated which initiates the Delta-like ligand 4–Notch cascade.
systems may enhance reproducibility and scalability. This cascade regulates ECs’ differentiation, ensuring the
proper roles of tip and stalk cells, which are essential for
3.1.2. Growth factor-induced vascularization vessel branching and stability in developing vascular
As described in Section 2.2, the vascularization process networks. 42,90 Beyond VEGF, BOs’ vascularization can
in the brain is regulated by multiple growth factors that be improved with key growth factors at different stages
facilitate blood vessel formation and stability. The addition of development. FGF supports early vascular and neural
of specific growth factors to BOs’ cultures has proven to be a growth, while the epidermal growth factor helps connect
crucial strategy for promoting vascularization (Figure 5B). vascular and neural cells. As vessels mature, PDGF and
Table 1 summarizes the primary growth factors strategies TGF-β stabilize them by recruiting smooth muscle cells and
that have been explored to enhance vascularization in BOs. forming supportive structures. ANG1 strengthens vessel
walls, insulin-like growth factor promotes cell survival, and
89
Ham et al. have made significant strides in introducing
vascular differentiation cues within BOs by cultivating bone morphogenetic protein organizes vascular structures.
embryoid bodies in a VEGF-enriched medium. Their These factors enhance vascular stability and integration, but
91,92
approach demonstrates that the addition of VEGF during further research is needed to confirm their functionality.
the neuroectodermal differentiation stage could induce While pro-angiogenic growth factors facilitate
the formation of blood vessel-like structures with essential controlled vascularization in BOs and can be scaled by
BBB characteristics. VEGF enhances vascular network modulating their concentrations and exposure durations,
complexity and stability in BOs while preserving neuronal their application presents challenges, including inconsistent

Table 1. Growth factors promoting vascularization in brain organoids
Growth Function Effect on vascularization Signaling pathway (s) References
factor
VEGF Stimulates endothelial cell Increases blood vessel VEGF/VEGFR–DLL4–Jagged–Notch (for 16,17,89,209
proliferation and migration and formation within organoids sprouting angiogenesis, key in endothelial
promotes sprouting angiogenesis tip cell guidance and vascular branching)
and vessel branching
FGF Promotes proliferation and Supports angiogenesis and FGF/FGFR, PI3K/AKT, and MAPK 22,42,210
differentiation of stem cells and stabilization of vascular (essential for angioblast formation and
endothelial cells networks vasculature differentiation)
EGF Enhances proliferation of neural Promotes both neural and EGFR/MAPK and Notch (involved in cell 158,211
progenitors and endothelial cells vascular growth, indirectly survival and neurovascular growth in early
supporting vascularized stages)
organoid development
PDGF Aids in the maturation of vascular Supports blood vessel PDGF/PDGFR, YAP–TAZ, and MAPK 84,211-213
smooth muscle cells maturation and stability (regulates pericyte recruitment and vessel
maturation)
TGF-β Involved in cell differentiation, Stabilizes vascular structures TGF-β/SMAD (integrin αVβ8–TGF-β 42,214,215
immune response, and ECM and promotes ECM deposition pathway important for vascular stability and
production ECM formation)
Angiopoietin Stabilizes blood vessels by Enhances blood vessel ANG–TIE2, MAPK, and PI3K (important 83,216
supporting pericyte–endothelial stabilization and maturity for vessel stabilization and survival of
cell interaction endothelial cells)
IGF Promotes cell survival, Supports vascular development IGF/IGF1R (PI3K-AKT and MAPK 217,218
proliferation, and differentiation and neural survival within pathways promoting cell growth and
organoids survival)
BMP Regulates stem cell differentiation Promotes vascular BMP/SMAD and WNT (interaction with 219
and vascular development differentiation and organization TGF-β for vascular differentiation)
in brain organoids
Abbreviations: AKT: Protein kinase B; ANG: Angiopoietin; BMP: Bone morphogenetic protein; DLL4: Delta-like 4 protein; ECM: Extracellular
matrix; EGF: Epidermal growth factor; EGFR: EGF receptor; FGF: Fibroblast growth factor; FGFR: FGF receptor; IGF: Insulin-like growth factor;
IGF1R: IGF 1 receptor; MAPK: Mitogen-activated protein kinase; PDGF: Platelet-derived growth factors; PDGFR: PDGF receptor;
PI3K: Phosphoinositide 3-kinase; TAZ: Transcriptional coactivator with PDZ-binding motif; TGF-β: Transforming growth factor beta; VEGF:
Vascular endothelial growth factor; VEGFR: VEGF receptor; YAP: Yes-associated protein.

Volume 1 Issue 2 (2025) 10 doi: 10.36922/or.8162