Changes in HtrA1 Expression during Neural Differentiation of P19 Embryonal Carcinoma Cells and Rat Bone Marrow-Derived Mesenchymal Stem Cells.

OBJECTIVE: High temperature requirement protease A1 (HtrA1) serves as a regulator of fibroblast growth factor (FGF) signaling by degrading proteoglycans attached to FGF ligands on the cell surface. This degradation releases FGF ligands into the extracellular space, facilitating their binding to cognate receptors and subsequent neural induction, as demonstrated in Xenopus embryos. Whether HtrA1 plays a similar function in mammalian neural development remains unknown. As a first step toward addressing this, the present study aims to quantify HtrA1 expression before and after in vitro neural differentiation in mammalian cells, establishing whether neural differentiation is accompanied by changes in HtrA1 at the mRNA level. MATERIALS AND METHODS: In this experimental study, P19 cells were aggregated into embryoid bodies (EBs) using the hanging-drop method (500 cells/drop) for 2 days, cultured in α-minimum essential medium eagle (αMEM) supplemented with 3% fetal bovine serum (FBS) for an additional 2 days, and subsequently neurally induced with 1 μM retinoic acid (RA) for 4 days. In parallel, rat bone marrow-derived mesenchymal stem cells (BM-MSCs) were subjected to a preinduction step in Dulbecco's modified Eagle medium-low glucose (DMEM-LG) supplemented with 20% FBS and 1 mM β-mercaptoethanol (β-ME), followed by neural induced in DMEM-LG containing 200 μM butylated hydroxyanisole (BHA) and 2% dimethyl sulfoxide (DMSO). Finally, the expression levels of neural marker genes and HtrA1 were evaluated by quantitative real-time polymerase chain reaction (RT-qPCR) in P19 cells and rat BM-MSCs, with or without neural induction. RESULTS: Differentiated cells exhibited a neural morphology with long, extensive processes. RT-qPCR confirmed significantly elevated Map2 expression in neural-induced groups (P19: 149.66 ± 10.24; BM-MSCs: 4.42 ± 0.53) and increased HtrA1 expression (P19: 4.14 ± 0.19; BM-MSCs: 8.20 ± 0.15) versus uninduced controls. CONCLUSION: HtrA1 expression correlates with neural differentiation in both embryonic and adult stem cells, suggesting that HtrA1 may contribute to neural lineage commitment.