Objective      To improve a method for isolating and culturing glial progenitors for transplantation

and study in vivo function of the engrafted glial progenitors in cortex in single cell level by two-photon imaging.       Methods      Neural stem cells (NSCs) were isolated from the embryonic cortex

and cultured in vitro. Then

the cells were induced into glial progenitors and astrocytes. In order to detect the cellular morphology

Nestin

A

2

B

5

and GFAP as markers of NSCs

glial progenitors and astrocytes

respectively

were detected. In order to detect the cellular function

calcium responses of the astrocytes to ATP and thapsigargin were recorded by confocal imaging. The glial progenitors were engrafted into the cerebral cortex of adult mice

and the morphology of the engrafted cells was observed by immunostaining after 12 weeks. In vivo two-photon imaging was adopted to observe calcium signals of the engrafted cells stimulated with ATP after 4 weeks.       Results      The percentage of glial progenitors (A

2

B

5

-positive cells) derived from the NSCs accounted for (87.4±3.4)%

while that of astrocytes (GFAP-positive cells) derived from the glial progenitors aclounted for (83.4±3.3)%. The astrocytes derived from the NSCs could respond to ATP and thapsigargin. The engrafted glial progenitors could survive and differentiate into the astrocytes after 12 weeks. In vivo two-photon imaging showed that ATP could induce the engrafted cells in the cortex to generate calcium signals after 4 weeks.       Conclusion      The engrafted glial progenitors derived from the NSCs can survive for at least 12 weeks and differentiate into the astrocytes in the cortex. In addition

the engrafted glial progenitors function in the cortex after 4 weeks.