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

Abstract

ABSTRACT. 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 semiquantitative 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.

SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis

“…Accordingly, SOX2 overexpression reduced the level of OCT4 and NANOG in human ESCs (Adachi et al, 2010). In line with this data, we detected downregulation of OCT4 gene expression in pluripotent embryonal carcinoma stem cells NT2/D1 with constitutive SOX2 overexpression (Drakulic et al, 2012). We also demonstrate that transition from proliferation to retinoic acid (RA) induced neural differentiation of NT2/D1 cells coincides with complete OCT4 down-regulation (Stevanović et al, 2017).…”

“…Constitutive Sox2 expression kept NPCs in a precursor state and inhibited neuronal differentiation, while expression of a dominant-interfering form of Sox2 led to exit from cell cycle, delamination of NPCs from the ventricular zone, loss of expression of progenitor markers and initiation of neuronal differentiation (Graham et al, 2003). We showed that constitutive SOX2 overexpression altered expression of neuronal markers and reduced number of mature MAP2 (Microtubule Associated Protein 2) positive neurons upon RA induced neural differentiation of NT2/D1 cells (Drakulic et al, 2012;Klajn et al, 2014). Schematic illustration of stepwise neuronal differentiation process (left) and glial differentiation process (right).…”

“…We also demonstrate that transition from proliferation to retinoic acid (RA) induced neural differentiation of NT2/D1 cells coincides with complete OCT4 down-regulation (Stevanović et al, 2017). However, SOX2 overexpressing NT2/D1 cells retain ability to differentiate (Drakulic et al, 2012;Klajn et al, 2014) even in the presence of elevated SOX2 expression after 21 days of treatment with RA (Drakulic et al, 2012).…”

All-trans retinoic acid influences viability, migration and adhesion of U251 glioblastoma cells

“…One μg of the total RNA was reversely transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer's protocol. SOX3 (SRY (sex determining region Y)-box 3) was amplified from the synthesized cDNAs with primers: 5'-CAC GGG TCC TCC GGG TTG CGA GGG GCG GAC C-3' (forward) and 5'-TGG GGA ACA AGG GTG GAC GAG C-3' (reverse); glial fibrillary acidic protein (GFAP) with primers: 5' -GCA GAG ATG ATG GAG CTC AAT GAC C-3' (forward) and 5'-GTT TCA TCC TGG AGC TTC TGC CTC A -3'(reverse) [20] and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with primers 5'-GCC TCA AGA TCA TCA GCA ATG C-3' (forward) and 5'-CCA CGA TAC CAA AGT TGT CAT GG-3' (reverse) [21]. The expression of GAPDH was used to normalize the levels of the total RNA used in the assays.…”

RC-6 ribonuclease induces caspase activation, cellular senescence and neuron-like morphology in NT2 embryonal carcinoma cells

“…Therefore, NT2 cells are often used as cell models for cancer therapy and neuron differentiation studies (5)(6)(7). Currently, cisplatin, fisetin and nucleoside drugs are applied to NT2 cell treatment.…”