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Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Published on: May 30, 2012
Suppression of P-cadherin expression as a key regulatory element for embryonic stem cell stemness
Yuka Takeda1, Shuji Matsuguchi1, Sae Nozaki1
1Department of Biomedical Sciences, Graduate School of Science and Technology, Kwansei Gakuin University.
Abstract:
In embryonic stem (ES) cell colonies, a small subpopulation that changes cell shape and loses pluripotency often appears in two-dimensional (2D) cultures, even in the presence of a stemness factor. We have previously shown that membrane translocation of the syntaxin4, t-SNARE protein contributes to this phenomenon. Here, we show that ES cells in three-dimensional (3D) aggregates do not succumb to extruded syntaxin4 owing to suppressed expression of P-cadherin protein. While extracellular expression of syntaxin4 led to the striking upregulation of P-cadherin mRNA in both 2D and 3D-ES cells, morphological changes and appreciable expression of P-cadherin protein were detected only in 2D-ES cells. Importantly, the introduction of an expression cassette for P-cadherin practically reproduced the effects induced by extracellular syntaxin4, where the transgene product was clearly detected in 2D-, but not 3D-ES cells. An expression construct for P-cadherin-Venus harboring an in-frame insertion of the P2A sequence at the joint region gave fluorescent signals only in the cytoplasm of 2D-ES cells, demonstrating translational regulation of P-cadherin. These results provide the mechanistic insight into the uncontrollable differentiation in 2D-ES cells and shed light on the validity of the "embryoid body protocol commonly used for ES cell handling" for directional differentiation.Key words: differentiation, embryoid body, ES cells, P-cadherin, syntaxin4.
Insights
Embryonic stem (ES) cells in 3D cultures resist differentiation by suppressing P-cadherin expression, unlike 2D cultures. This study reveals syntaxin4 and P-cadherin
Area of Science:
- Stem Cell Biology
- Developmental Biology
- Cellular Differentiation
Background:
- Embryonic stem (ES) cells in 2D cultures can lose pluripotency and differentiate, even with stemness factors.
- Syntaxin4 membrane translocation was previously identified as a contributor to this differentiation.
- Three-dimensional (3D) ES cell aggregates (embryoid bodies) are often used for controlled differentiation.
Purpose of the Study:
- To investigate the mechanism preventing differentiation in 3D ES cell aggregates compared to 2D cultures.
- To elucidate the role of syntaxin4 and P-cadherin in ES cell pluripotency maintenance and differentiation.
- To understand the translational regulation of P-cadherin in different culture formats.
Main Methods:
- Comparison of ES cell behavior in 2D cultures versus 3D aggregates (embryoid bodies).
- Analysis of syntaxin4 and P-cadherin expression (mRNA and protein) under different culture conditions.
- Introduction of P-cadherin expression cassettes into ES cells and monitoring of transgene product localization.
Main Results:
- 3D ES cell aggregates suppress syntaxin4-induced differentiation, linked to suppressed P-cadherin expression.
- Extracellular syntaxin4 upregulates P-cadherin mRNA in both 2D and 3D ES cells, but protein expression and morphological changes occur only in 2D.
- P-cadherin introduction mimics syntaxin4 effects in 2D cells; its translational regulation is demonstrated by cytoplasmic localization in 2D-ES cells.
Conclusions:
- Suppressed P-cadherin expression is key to preventing differentiation in 3D ES cell aggregates.
- Translational regulation of P-cadherin underlies its differential expression and functional impact in 2D vs. 3D ES cell cultures.
- Findings support the utility of the embryoid body protocol for directed ES cell differentiation.
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