Regulation of Self-renewal and Pluripotency by Sox2 in Human Embryonic Stem Cells

Fong H, Hohenstein KA, Donovan PJ
Source: Stem Cells
Publication Date: (2008)
Issue: 26(8): 1931-8
Research Area:
Stem Cells
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Nucleofector® I/II/2b
Human embryonic stem cells (H1, H9 lines) were nucleofected with siRNAs against beta-2-microglobulin (beta2M), Nanog, Oct4 and Sox2. Three additional siRNAs targeting Sox2 were also used to verify the phenotype and eliminate the possibility of off-tartget effects. In initial experiments, each siRNA was co-transfected with pmaxGFP vector in a ratio of 5:1 to identify transfected cells and determine morphology.
Human embryonic stem (hES) cells, derived from blastocysts, are capable of unlimited self-renewal and differentiation into all cell lineages of the body. Because of their pluripotent nature, hES cells are valuable tools for understanding human development and advancing the field of regenerative medicine. However, one key to harnessing the therapeutic power of hES cells for biomedical applications begins with determining how these cells maintain their pluripotent and undifferentiated state. Studies in mice have implicated three factors in regulating pluripotency in ES cells, Oct4, Nanog and Sox2. But significant differences in growth regulation between mES and hES cells have been identified suggesting a need to determine when and how factors work in hES cells. To date, the transcription factors Oct4 and Nanog have been identified as critical regulators of stem cell fate by functional studies in hES cells. To determine the role of Sox2 in maintaining hES cell pluripotency and self-renewal, we used RNA interference (RNAi) to specifically knockdown Sox2 gene expression. Reduction of Sox2 expression in hES cells results in loss of the undifferentiated stem cell state, as indicated by a change in cell morphology, altered stem cell marker expression, and increased expression of trophectoderm markers. In addition, knockdown of Sox2 results in reduced expression of several key stem cell factors including Oct4 and Nanog, linking these three factors together in a pluripotent regulatory network.