Objective      To investigate how to induce the retinal stem cells (RSCs) differentiation into retinal ganglion cells (RGCs) in vitro in order to provide the seed cells for the repair of optic nerve injury after cell transplantation.       Methods      RSCs were isolated from the retina of LE rats at embryonic days 14

and then identified by flow cytometry and immunofluorescence staining after sub-culturing for 2 culture generations. Then the obtained cells were cultured in 4 different w

ays

5%FBS (control)

BDNF (10 ng/mL)

DAPT (25 μmol/L)

and DAPT+BDNF for 14 d. Flow cytometry was used to identify specific marker CD90.1 (thy1.1) of mature RGCs and markers math5

and brn3b

and neural stem cells’ markers nestin

ki67 of DAPT+BDNF group at different time points.       Results      The obtained RSCs at passage 2 were strongly positive to nestin

chx10

and ki67

and some of them even to math5. The positive rate of thy1.1 was (11.38±1.58)%

(19.10±2.29)%

(23.90±1.09)% and (31.31±2.98)% respectively

in the RSCs cultured in the mediums containing 5%FBS

BDNF

DAPT

and DAPT+BDNF

with significant difference between any 2 groups (

P

0.05). DAPT+BDNF induction for 3 to 5 d resulted in positive expression of nestin

ki67

brn3b and math5 in some cells.       Conclusion      DAPT+BDNF enhances the differentiation of RSCs into RGCs in vitro

and the cells express RGCs relative markers and remain reproductive capacity after 3 to 5 days’ induction.