Sc 16143

Cells were treated with the preinduction medium containing 10⁻⁷ mol/L all-trans-retinoic acid (ATRA, Sigma) and 10 ng/ml FGF2 (Gibco) for 18 h, and then with modified neuronal medium (MNM) for 36 h. The expression of neurofilament medium polypeptide (1 : 100, sc-16143, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and neuron-specific anolase (1 : 100, sc-292097, Santa Cruz Biotechnology) in induced MSCs was detected by immunofluorescence staining.

En-PSCs (passage 6) were assessed for their multipotency using adipogenic, osteogenic, and neural-like differentiation assays. The cells were seeded at a density of 2 × 10⁴/cm² in 24-well plates. Growth media was replaced with the appropriate differentiation medium when cells reached 90% confluency. Adipogenic and osteogenic induction media (Gibco) were used for differentiation. After 30 days, cells were stained with oil red O (Sigma) or alizarin red S (Gibco) to identify lipid droplets or calcium deposition, respectively. For neural-like differentiation, pre-induction media containing 10⁻⁷ mol/L all-trans-retinoic acid (ATRA; Sigma) and 10 ng/ml bFGF was added to cells for 24 h followed by the addition of modified neuronal medium for 6 h. Neural-like differentiation was detected via immunofluorescent staining for neurofilament medium polypeptide (NF-M; 1:100, sc-16143, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and neuron-specific enolase (NSE; 1:100, sc-292097, Santa Cruz Biotechnology).

The multi-lineage differentiation potential of human UC-MSCs was checked by adipogenic, osteogenic and neural-like differentiation assays at the fourth passage. Adipogenesis was induced by adipogenic induction medium (Gibco) for 14 days and confirmed by Oil red O staining to show intracellular lipid accumulation. Osteogenesis was induced by osteogenic induction medium (Gibco) for 28 days and calcium deposition was shown by Alizarin red staining. For neural-like differentiation, UC-MSCs seeded on poly-L-lysine-coated coverslips in a 24-well culture plate were treated with pre-induction medium containing 10^-7 M all-trans-retinoic acid (ATRA; Sigma-Aldrich, St. Louis, MO, USA) and 10 ng/ml bFGF (Gibco) for 24 h and then with modified MNM medium for 36 h. The cells were co-incubated with the anti-NSE antibody (1:100, sc-292097, Santa Cruz Biotechnology) and the anti-NF-M antibody (1:100, sc-16143, Santa Cruz Biotechnology) to confirm their differentiation into neural-like cells.