[Research of human keratinocyte growth factor promoting proliferation and differentiation of human neural stem cells].

OBJECTIVE

To study the effects of the human keratinocyte growth factor 2 (hKGF-2) on the survival and differentiation of human neural stem cells (hNSCs).

METHODS

The hNSCs at 17 passages preserved in liquid nitrogen were resuscitated and cultured for 7 days with normal methods to form neural spheres. The specific Nestin antigen and differentiated cells antigen were identified using immunohistochemistry technology. Some concentrated hNSCs were incubated in 12-well culture plate with 1 mL basic medium [(DMEM/F12 + N2 (1 : 100) + epidermal growth factor (EGF) (20 ng/mL)] and divided into 7 groups, 6 wells each group. hKGF-2 (0, 10, 30, 60, 90, and 120 ng/mL) and bFGF (10 ng/mL) were added in groups A (control), B, C, D, E, F, and G, respectively. The neurospheres and the cell number were recorded for analyzing growth and multiplication of neural spheres. Some concentrated hNSCs were incubated in 6-well culture plate (cover glass coated with polylysine) with 3 mL DMEM/F12 medium and divided into 4 groups, 6 wells each group. N2 (1 : 100), N2 (1 : 100) + hKGF-2 (90 ng/mL), FBS (1 : 20), and FBS (1 : 20) + hKGF-2 (90 ng/mL) were added in groups A1, B1, C1, and D1, respectively. Then, the growth and multiplication of neural spheres were observed during culture; the separated neural spheres was identified and analyzed with indirect immunofluorescence and flow cytometry.

RESULTS

Reanimated hNSCs could form neural spheres containing a lot of Nestin antigen; differentiated cells by induction expressed the specific antigens of neurofilament 200 (NF-200) and glial fibrillary acidic protein (GFAP). At 7 days after culture, enlarged neural spheres were observed in each group. The neurospheres and the cell number of hNSCs increased with increased concentration of hKGF-2, showing a gradually increasing tendency; they were significantly higher in groups E, F, and G than that in groups A, B, C, and D (P < 0.05); significant differences were found among groups B, C, and D (P < 0.05), but no significant difference between groups A and B, and among groups E, F, and G (P > 0.05). After induction in vitro, the cell growth showed a progressive increase, significant difference was found among groups (P < 0.05); the percentage of NF-200 positive cells in group B1 was significantly higher than that in the other 3 groups (P < 0.05); the percentage of GFAP positive cells in group B1 was significantly lower than that in the other 3 groups (P < 0.05), but no significant difference among groups A1, C1, and D1 (P > 0.05). At 14 days after culture, cell growth reached the peak, which were mainly astero-cells.

CONCLUSION

The hNSCs are pure after incubated to 17 passages in vitro. hKGF-2 can promote the clone and the growth of differentiated cells, and increase the proportion of neuron.