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Published on: July 9, 2019
Knockdown of Oct-4 or Sox-2 attenuates neurogenesis of mouse embryonic stem cells
Stephen Chen1, Andre B H Choo, Wang Nai-Dy
1Stem Cell Group, Bioprocessing Technology Institute, Centros, Singapore.
Abstract:
We employed a stromal-derived inducing activity (SDIA) model of neurogenesis to investigate the effects of targeted knockdown of Oct-4 and Sox-2 by short interfering RNAs (siRNAs) in mouse embryonic stem (mES) cells. Quantitative real-time PCR showed 40-90% knockdown of specific transcripts with cognate Oct-4 or Sox-2 siRNA transfection compared to FAM-labeled negative control (FAM) siRNA or mock transfection and was confirmed at the protein level by western blot analyses. Upon differentiation using PA6 SDIA co-cultures, neurogenesis is significantly diminished in Oct-4 or Sox-2-targeted mES cells. It was observed that 45 +/- 12%, 65 +/- 13%, and 90 +/- 8% of the colonies were stained with neuron-specific beta-tubulin III in Oct-4, Sox-2, and FAM siRNA transfected mES cells, respectively, with similar results observed using neural inducing factors collected from the surface of PA6. Together, our results extend observations for a role of Oct-4 in SDIA and implicate a similar role for Sox-2.
Insights
Targeted knockdown of Oct-4 and Sox-2 genes in mouse embryonic stem cells significantly reduced neurogenesis during stromal-derived inducing activity (SDIA) differentiation. This implicates both Oct-4 and Sox-2 in neural development.
Area of Science:
- Stem Cell Biology
- Developmental Neuroscience
Background:
- Oct-4 is a key transcription factor in maintaining pluripotency of embryonic stem cells.
- Stromal-derived inducing activity (SDIA) is a method used to induce neural differentiation in mouse embryonic stem cells (mESCs).
Purpose of the Study:
- To investigate the role of Oct-4 and Sox-2 in SDIA-mediated neurogenesis.
- To determine the effect of targeted knockdown of Oct-4 and Sox-2 on neural differentiation of mESCs.
Main Methods:
- Utilized short interfering RNAs (siRNAs) for targeted knockdown of Oct-4 and Sox-2 in mESCs.
- Employed quantitative real-time PCR and western blot analysis to confirm gene and protein knockdown.
- Assessed neurogenesis by co-culturing mESCs with PA6 stromal cells and quantifying neuron-specific beta-tubulin III staining.
Main Results:
- Oct-4 or Sox-2 siRNA transfection resulted in 40-90% knockdown of respective transcripts.
- Neurogenesis was significantly diminished in mESCs with targeted Oct-4 or Sox-2 knockdown.
- Neuron-specific beta-tubulin III staining was observed in 45% (Oct-4), 65% (Sox-2), and 90% (control) of colonies.
Conclusions:
- Oct-4 plays a role in SDIA-mediated neurogenesis.
- Sox-2 also plays a significant role in SDIA-mediated neurogenesis.
- These findings highlight the importance of Oct-4 and Sox-2 in neural differentiation pathways.
