Cell Journal (Jan 2010)
Gene Expression Profiling of NCAM NCAM-L1 N-Cadherin-in Ninjurin-1 and Ninjurin-2 during the Course of Differentiation of Murine Neural Stem Cells
Abstract
Objective: To evaluate and compare the gene expression profiles of Ninjurin-1 and Ninjurin-2 with the expressions of L1 family of cell adhesion molecules (NCAM-L1), neuralcell adhesion molecules (NCAM), and N-cadherin during the course of neural differentiationof mouse neural stem cells (NSCs).Materials and Methods: Briefly, for neural stem cell isolation, the frontal part of an adultmouse brain was minced in phosphate buffered saline (PBS) and digested by an enzymesolution which contained hyaluronidase and trypsin. Isolated cells were culturedin medium supplemented by epidermal growth factor (EGF) and basic fibroblast growthfactor (bFGF). After seven days, primary neurospheres appeared in culture medium. Aftertransfer to poly-L-ornithine coated dishes that contained culture medium supplementedwith 1% fetal bovine serum (FBS), the primary neurospheres differentiated into neural-likeand neuroglial-like cells. Differentiated cells were examined by morphological, immunocytochemical,and molecular evaluations.Results: Our results showed that isolated cells from the preventricular area of mouseadult brain proliferated in medium which contained EGF and bFGF, and expansion of thecells continued until passage 14 without losing morphological and neurogenesis capacity.Multiple passaging confirmed the stemness nature of the isolated cells. The isolatedNSCs were able to differentiate into neural-like and neuroglial-like cells after transfer topoly-L-ornithine coated dishes that contained culture medium supplemented with 1%FBS. Molecular studies for NCAM, NCAM-L1, and N-Cadherin genes, as well as immunocytochemicalanalysis for NCAM-L1 and NCAM proved the differentiation. Our dataalso revealed, for the first time, gene expression profiling of Ninurin-1 and Ninjurin-2, twonovel cell adhesion molecules (CAMs), during the course of differentiation of neural stemcells.Conclusion: The induction of neural differentiation in mouse NSCs initiates the expressionof NCAM-L1, NCAM, and N-cadherin which can be the proof of complete neural differentiation.In addition, our results indicate that the expression of Ninjurin-1 increases afterneural induction much like the expressions of NCAM-L1, NCAM, and N-cadherin. Thisdata suggests the probable importance of Ninjurin-1 in both the morphology and functionof neural cells. The data in this study also reveals that the expression of Ninjurin-2, in contrastwith Ninjurin-1, initiates and continues until the differentiation termination point. Thissuggests that although Ninjurin-1 and Ninjurin-2 share conserved hydrophobic regions fortheir transmembrane domains, their roles in nerve cells are probably different.
