Development of quantitative PCR methods to analyse neural progenitor cell culture state.
Stem cells have significant potential for tissue engineering and regeneration, and neural stem and progenitor cells have proven promising for neuroregeneration in numerous animal disease and injury models. However, improved approaches must be developed to culture, expand and control the cells. Therefore the development of enhanced methods to quantify cell differentiation would significantly aid both in the basic investigation of cell-fate control mechanisms and in the optimization and validation of cell culture and expansion conditions. Quantitative reverse transcription-PCR methods were developed to quantify cell differentiation state by monitoring the expression of several cell-lineage-specific markers. These methods provide more rapid and readily quantitative results when compared with immunostaining. These methods were also applied in a preliminary investigation of cell-culture conditions, and it was found that regular feeding of cells with fresh medium is necessary to maintain them in an undifferentiated and highly proliferative state. The present study may aid both basic efforts to study the control of neural stem and progenitor differentiation as well as endeavours to optimize cell culture and expansion conditions for biomedical applications.
Abranches
Elsa
E
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
O'Neill
Analeah
A
Robertson
Matthew J
MJ
Schaffer
David V
DV
Cabral
Joaquim M S
JM
D001483:Base Sequence; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002455:Cell Division; D003470:Culture Media; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D009420:Nervous System / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering
D003470:Culture Media; D017931:DNA Primers