Establishment of an immortalized GABAergic neuronal progenitor cell line from embryonic ventral mesencephalon in the rat.
Effective cell replacement therapies for neurological disease require neuron-restricted precursors as grafted cells. The problem of obtaining sufficient grafts for transplantation can be resolved by creating an appropriate immortalized cell line. In the present study, a thermally controlled immortalized GABAergic neuronal progenitor cell line (RMNE6) was established from E13 rat ventral mesencephalon cells immortalized using the temperature-sensitive mutant of SV40 large T antigen (ts-TAg). RMNE6 cells proliferated rapidly and expressed a neuron-like phenotype at the permissive temperature (33 degrees C), but eventually stopped growing at the non-permissive temperature (39 degrees C). Expression of the neuronal markers PSA-NCAM, beta-tubulin III and MAP2 by RMNE6 cells was confirmed by RT-PCR or immunocytochemistry. Furthermore, these cells exhibited functional GABAergic neuron properties, as evidenced by the expression of glutamate decarboxylase (GAD) as well as the synthesis and release of the neurotransmitter GABA in a calcium-dependent manner. Moreover, RMNE6 cells spontaneously expressed and secreted several neurotrophic factors, such as NGF, BDNF, NT-3, NT-4/5, and GDNF. The cells survived well and kept expression of SV40 Tag, GAD65/67 and GABA in the striatum, at least 28 days after being transplanted in the rat brain. Tumorigenesis assays confirmed the safety of the immortalized cell line in vivo. Taken together, the results support the use of RMNE6 cells as an ideal cell model for transplantation research aimed at the treatment and prevention of neurodegenerative disease.
Wang
Beibei
B
Beijing Institute for Neuroscience, Capital Medical University, Beijing Center of Neural Regeneration and Repair, Key Laboratory of Neurodegenerative diseases of Ministry of Education of China, Beijing 100069, China.
Zou
Xifeng
X
Zhang
Haiyan
H
Duan
Deyi
D
Ju
Lili
L
Jiang
Xiaohua
X
Sun
Xiaohong
X
Zhao
Chunli
C
Zhao
Huanying
H
Guo
Jin
J
Xu
Changlei
C
Gao
Erjing
E
Xu
Qunyuan
Q
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D015415:Biomarkers / Q000378:metabolism; D016380:Brain Tissue Transplantation / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D005968:Glutamate Decarboxylase / Q000378:metabolism; D008297:Male; D008636:Mesencephalon / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009414:Nerve Growth Factors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013696:Temperature; D014183:Transplantation, Heterologous; D014184:Transplantation, Homologous; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D015415:Biomarkers; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase