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SOX2, a  universal marker of pluripotent stem cells, is a transcription factor that  helps control embryonic development in vertebrates; its expression persists in  neural stem/progenitor cells into adulthood. Considering the critical role of  the SOX2 transcription factor in the regulation of genes required for  self-renewal and pluripotency of stem cells, we developed and characterized SOX2-overexpressing  NT2/D1 cell clones. Using Southern blot and semi-quantitative RT-PCR, we  confirmed integration and expression of exogenous SOX2 in three NT2/D1  cell clones. Overexpression of the SOX2 gene was detected in two of  these clones. SOX2 overexpression in NT2/D1 cell clones resulted in  altered expression of key pluripotency genes OCT4 and NANOG.  Furthermore, SOX2-overexpressing NT2/D1 cell clones entered into  retinoic acid-dependent neural differentiation, even when there was elevated SOX2 expression. After 21 days of induction by retinoic acid, expression of  neural markers (neuroD1 and synaptophysin) was higher in induced cell  clones than in induced parental cells. The cell clone with SOX2 overexpression  had an approximately 1.3-fold higher growth rate compared to parental cells. SOX2 overexpression did not increase the population of cells undergoing  apoptosis. Taken together, we developed two SOX2-overexpressing cell  clones, with constitutive SOX2 expression after three weeks of retinoic  acid treatment. SOX2 overexpression resulted in altered expression of  pluripotency-related genes, increased proliferation, and altered expression of  neural markers after three weeks of retinoic acid treatment.

Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones