Developmental stage–dependent effects of cerebrospinal fluid on hair follicle stem cell proliferation, morphology, and neural-associated gene expression

Hair follicle stem cells (HFSCs), owing to their accessibility and neural-like differentiation potential, represent a promising model for studying stem cell responses to central nervous system (CNS) microenvironments. Cerebrospinal fluid (CSF), a dynamic component of the CNS, contains stage-dependent bioactive factors that reflect developmental and functional states; however, their effects on peripheral stem cells remain unclear. This study investigated how CSF from distinct developmental stages modulates the viability, cellular metabolic activity, morphology, and neural-associated gene expression of rat HFSCs. HFSCs isolated from rat whisker pad skin were cultured in medium supplemented with 20% CSF collected from postnatal day 5 (P5) or adult rats and compared with cells maintained in 20% fetal bovine serum (FBS). Cell viability, cellular metabolic activity, neurite-like morphology, and gene expression were evaluated using FDA staining, MTT assay, crystal violet staining, and qRT-PCR, respectively. P5 CSF maintained HFSC viability and cellular metabolic activity at levels comparable to the FBS control and supported neurite-like extension. In contrast, adult CSF significantly reduced cellular metabolic activity and neurite-like extension length. Gene expression analysis demonstrated increased β-III Tubulin and PDGFR-α expression in P5 CSF-treated cells, whereas adult CSF upregulated MAP2 and Nestin expression. In addition, doublecortin, synapsin, and PSD-95 exhibited an increasing trend in adult CSF-treated cells, with synapsin showing significant elevation. These findings demonstrate that CSF exerts developmental stage–specific effects on HFSC behavior, with P5 CSF supporting cellular viability, metabolic activity, and neural-associated features, while adult CSF favors markers associated with neuronal maturation. Collectively, these findings highlight the potential utility of HFSCs as an accessible in vitro model for investigating how physiological and pathological changes in CSF composition influence stem cell behavior and neural-associated properties.