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X. Zhang, W. Li; Gene Regulation by Math5 in Neural Stem Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5768.
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Despite their potential in regenerative medicine, several critical issues of stem cells, including in vivo differentiation, are yet to be better defined. Transcriptional factor Math5 has been demonstrated to be one of the important intrinsic factors for retinal ganglion cell (RGC) development and could be potentially used to guide the differentiation of neural stem cells (NSC) toward RGCs. The purpose of this study is to elucidate the role of Math5 in NSC differentiation by investigating whether Math5 can regulate a gene network in NSCs, similar to the one in embryonic retina.
NSCs grew as neurospheres in a defined serum–free culture medium. Math5 coding sequence was subcloned into feline immunodeficiency virus (FIV) vector, followed by a bicistronic element of internal ribosomal entrance site (IRES) and neomycin phosphotransferase. NSCs were infected with FIV–Math5 or control vector and stably selected with G418. Gene expression was characterized by quantitative reverse transcription–PCR (qPCR) at mRNA level, which was normalized against GAPDH or ß–actin mRNA.
Math5 expression in G418–resistant NSCs was verified by qPCR. Math5–expressing NSCs grew in the defined medium as neurospheres with no detectable difference in morphology, as compared with controls. A number of genes have been previously reported to be regulated by Math5 in mouse embryonic retina. Our results revealed that Brn–3b, persyn/nynuclein–γ, sonic hedgehog, iroquois related homeobox 2 and growth associated protein 43 were upregulated by Math5, ranging from 2–fold to more than 100–fold increase. On the other hand, other reported Math5–responsive genes, such as ELAV–like 3/Hu C antigen, nel–like 2 homolog (Nell2) and microtubule–associated protein tau were minimally affected.
The effect of Math5 on NSC gene expression has been characterized. Several Math5–dependent genes were upregulated in Math5–expressing NSCs, while others were less responsive. This study will provide a clue for guided differentiation of NSCs with ex vivo gene therapy for eye diseases.
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