An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells.
BACKGROUND
The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro.


METHODS
Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation.


RESULTS
The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2 +/- 3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8 +/- 0.3 per plate in the control group (clonogenic assay, P < 0.01), and the ratio of nestin positive cells was (50.2 +/- 2.8)% in astrocyte-induced group and only (1.4 +/- 0.5)% in the control group (flow cytometry, P < 0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was (42.7 +/- 2.6)% in astrocyte-induced group and only (11.2 +/- 1.8)% in the control group (P < 0.01).


CONCLUSIONS
Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.
Zhou
Yu-feng
YF
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Fang
Feng
F
Fu
Jin-rong
JR
Dong
Yong-sui
YS
Ye
Du-yun
DY
Shu
Sai-nan
SN
Zhen
Hong
H
Li
Ge
G
D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology*; D051379:Mice; D009474:Neurons / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*