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Table 1. Comparative analysis of unguided and guided differentiation: Key characteristics and insights into cell lineage and function
Characteristic Unguided differentiation Guided differentiation References
Differentiation control Low–relies on intrinsic signaling High–controlled by specific growth factors and small 42
pathways molecules
Cellular heterogeneity High–multiple neural and non-neural Lower–more uniform cell composition 63
cell lineages present
Reproducibility Variable–significant batch-to-batch More consistent due to directed differentiation 64
variation
Brain region specificity Mixed populations–various brain Targeted differentiation into specific brain regions 65
regions may arise randomly
Use in disease modeling Limited–higher variability makes precise More effective–greater control over disease-relevant 15,42
modeling challenging features
Developmental staging Broad–includes a range of progenitor Defined–controlled development of specific 63,66
and mature cell types neuronal subtypes
Tissue organization Less structured–random spatial More structured–mimics laminar organization of 42,63
organization of cells the brain
Duration of maturation Long–maturation can be slow and Shorter–optimized conditions accelerate 15,42
heterogeneous development
Microenvironment Highly variable–dependent on Precisely controlled by external signaling factors 67
influence spontaneous differentiation signals
Applications in drug Limited–high variability affects assay More suitable–consistent cellular composition aids 68
screening reproducibility in screening
Potential for neural Unpredictable–randomized cell Higher–directed differentiation improves synaptic 42,69
circuit studies arrangements limit functional organization
connectivity
Scalability and Challenging–high variability hampers More feasible–standardized protocols improve 42,70
standardization large-scale production scalability

signaling potential of pluripotent stem cell aggregates, while subsequent exposure to factors such as WNT3A,
allowing spontaneous generation of brain organoids SHH, BMP7, and FGF8 enables the formation of specific
containing multiple cell lineages. When grown in a stromal brain regions. 72-74 Early studies demonstrated that
gel suspension such as Matrigel, these organoids can ESCs could self-organize into polarized cortical tissue
develop into various brain regions, including the dorsal resembling human brain structures. 75,76 This approach was
and ventral forebrain, midbrain, hindbrain, hippocampus, refined to develop dorsal forebrain organoids in suspension
retina, and choroid plexus. 57,58 However, the stochastic culture without requiring an extracellular matrix (ECM),
nature of differentiation results in variability in composition yielding organoids that contain both deep and superficial
and organization, posing challenges for systematic studies cortical neurons. Over several months, these organoids
and reproducibility. Early developmental conditions mature to resemble postnatal brain structures, with
59
influence organoid formation, potentially leading to non- rotating bioreactors further enhancing their ability to
physiological cellular interactions if certain populations replicate key features of cortical development, including
are selectively favored or eliminated. 60,61 To mitigate progenitor organization, neurogenesis, and glial cell layer
variability and enhance consistency in neural induction, formation. 62,77
researchers have introduced small molecules and fibrous Guided differentiation has facilitated the generation of
microfilaments, yet the heterogeneity of unguided brain organoids representing various regions, such as the
organoids remains a limitation in disease modeling and dorsal forebrain cortex, ventral forebrain, hippocampus,
drug screening. 62,63 thalamus, hypothalamus, midbrain, and cerebellum. 78,79
Guided differentiation directs aggregated pluripotent These region-specific organoids are instrumental in brain
stem cells toward an ectodermal fate, promoting the assembly studies, allowing the investigation of interneuron
formation of region-specific organoids through the migration, neuronal projections, and oligodendrocyte
use of small molecules and growth factors that induce development. 15,80 While astrocytes and oligodendrocyte
neural differentiation. 57,71 The inhibition of the bone progenitors emerge in cortical organoids after long-term
morphogenetic protein (BMP)/transforming growth culture, mature oligodendrocytes remain challenging to
factor-beta signaling pathway facilitates neural induction, establish. 81-83 Researchers have developed region-specific

Volume 1 Issue 3 (2025) 5 doi: 10.36922/OR025100010