Ex Vivo Manipulation Strategies for Directed Stem Cell Differentiation
DOI:
https://doi.org/10.65649/hmrj2g17Keywords:
Directed Differentiation, Stem Cells, Centrosome, Centriole, Cell Fate Determination, Regenerative Medicine, Ex Vivo ManipulationAbstract
Directed differentiation of stem cells into functional somatic cell types ex vivo remains a cornerstone of regenerative medicine, yet conventional approaches relying solely on soluble factor modulation have reached a plateau in efficiency, homogeneity, and functional maturation. This review synthesizes classical and emerging manipulation strategies, with emphasis on the centrosome as an underappreciated but critical regulator of cell fate decisions. Classical methods—including growth factor-mediated chemical induction, matrix-engineered scaffolds, and coculture systems—are systematically evaluated for their strengths and limitations. Genetic engineering approaches utilizing transcription factor overexpression and CRISPR/Cas9-mediated reporter generation are discussed as tools for enhancing purity and enabling real-time fate monitoring. Physical stimulation through bioreactors delivering pulsatile flow, mechanical stretch, and electrical fields is examined for its capacity to engage mechanotransductive pathways that converge on the centrosome. The core of this review introduces pioneering strategies targeting centrioles and associated cell fate determination systems (CAFDs), including pharmacological modulation of centriolar kinases (PLK4, NEK2, Aurora A), optogenetic control of CAFD localization, nanoparticle-mediated delivery to centrosomes, magnetic manipulation of centrosome position, and pharmacological stimulation of ciliogenesis. An integrative protocol for generating functional dopaminergic neurons is presented as a proof-of-concept, sequentially incorporating centriole priming, dual SMAD inhibition, ciliogenesis-enhanced ventral specification, mechanical maturation, and reporter-based purification. Critical quality assessment methodologies—super-resolution imaging, ciliogenesis assays, spindle orientation analysis, single-cell omics, and transplantation validation—are outlined. Finally, key challenges including safety validation, persistent heterogeneity, scalability limitations, and ethical considerations are addressed. The emerging paradigm positions the centriole not as a static structural element but as a dynamic, manipulable target for biotechnological engineering of cell fate. Integration of centrosome-directed approaches with predictive digital twin modeling promises to accelerate the transition from generating incompletely differentiated populations toward creating truly functional, mature, and safe transplantable products for regenerative medicine.
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