title: An agarose gel-based neurosphere culture system leads to enrichment of neuronal lineage cells in vitro.
abstract: Stem cell-based therapy holds great potential especially for neurological disorders. However, clinical applications await further understanding of many aspects of stem cell differentiation and development of technology enabling manipulation of stem cells into desired cell types in the central nervous system. Here, we developed a new method that leads to enrichment of neuronal lineage cells in neural stem cell cultures. The protocol involves cultivation of primary cells derived from the forebrains of rat E18 embryos above a layer of nonadhesive hard agarose gel in the form of neurospheres. In contrast to the neurospheres that were cultured above an anti-adhesive hydrogel layer, the primary cells that were cultured above a layer of agarose gel preferentially differentiated into β-III tubulin-positive neurons when allowed to undergo differentiation in vitro.In an effort to investigate the mechanism behind this observation, we found that the gene expression of a vertebrate neuronal determination gene (neurogenin1) was enhanced in the neurospheres that proliferated above a layer of agarose gel as compared with the control, and the gene expression level of neurogenin1 was quite well correlated with the rigidity of agarose gel. These results indicate that agarose gel can contribute, at least in part, to enrich neuronal progenitors and immature postmitotic neurons during neurosphere formation and may provide additional information to establish efficient protocols for the neural stem cell-based study.
authors.affiliation: Gyeonggi Bio-Center, Gyeonggi Institute of Science and Technology Promotion, Suwon, Gyeonggi-do, 443-270, South Korea.
authors.forename: Kyuhee
authors.initials: K
authors.lastname: Park
authors.forename: Yeonju
authors.initials: Y
authors.lastname: Nam
authors.forename: Yongmun
authors.initials: Y
authors.lastname: Choi
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D058953:Neural Stem Cells / Q000166:cytology*; D016548:Prosencephalon / Q000166:cytology / Q000196:embryology*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose / Q000737:chemistry; D014404:Tubulin / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D012685:Sepharose; D014404:Tubulin; C488416:Tubb3 protein, rat; C510879:neurogenin-1, rat