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  lt; 0.05); significant differences were found among groups B, C, and D (P  lt; 0.05), but no significant difference between groups A and B, and among groups E, F, and G (P  gt; 0.05). After induction in vitro, the cell growth showed a progressive increase, significant difference was found among groups (P  lt; 0.05); the percentage of NF-200 positive cells in group B1 was significantly higher than that in the other 3 groups (P  lt; 0.05); the percentage of GFAP positive cells in group B1 was significantly lower than that in the other 3 groups (P  lt; 0.05), but no significant difference among groups A1, C1, and D1 (P  gt; 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.

Citation： YANG Yunkang,JIANG Dianming,YANG Mingming.. RESEARCH OF HUMAN KERATINOCYTE GROWTH FACTOR PROMOTING PROLIFERATION AND DIFFERENTIATION OF HUMAN NEURAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(10): 1246-1251. doi: 10.7507/1002-1892.20130272 Copy