BACKGROUND:
Y-27632 is a Rho-associated coil-formed
protein kinase inhibitor that can regulate the
self-renewal of
stem cells, promote clonal formation and
cell survival, and regulate and protect neuronal
cell growth and development. How to improve the differentiation
efficiency of
embryonic stem cells into
neuron-like
cells is highly important for nerve
injury repair and
nerve regeneration.
OBJECTIVE:
To investigate the effect of Y-27632 on the differentiation
efficiency and function of
human embryonic stem cells into
neuron-like
cells.
METHODS:
After
resuscitation,
human embryonic stem cells at passage 16 were subjected to morphological
observation and
staining identification. The
embryoid bodies were prepared by
suspension culture, and after 8 days of incubation, the
cells were cultured in Sato medium containing different concentrations of Y-27632 (0, 5, 10, 20, 40 μmol/L) for 10 days and identification by
staining. After induction for 18 days, the differentiation
efficiency and
neurite outgrowth were identified by
immunofluorescence staining. RESULTS AND
CONCLUSION:
The
human embryonic stem cells co-expressed Oct4 and SSEA-3
stem cell specific markers. After
suspension culture for 8 days and further adherent
culture for 10 days, the
cells could be differentiated into
neuron-like
cells with neurogenic morphology and expressing Tuj-1. Y-27632, especially at a concentration of 10 μmol/L, not only promoted
cell proliferation (a significant increase in adherent
cells an Tuj-1 positive
cells), but also facilitated
cell differentiation into
neurons.
Immunofluorescence staining findings showed that 10 μmol/L Y-27632 significantly increased the number of Tuj-1 positive
cells and
neurites and the length of
neurites after 18 days of differentiation. These results indicate that Y-27632 not only promotes the differentiation of
human embryonic stem cells into
neuron-like
cells, but also accelerates
neurite outgrowth.