Nature provides us with two distinct states of cell pluripotency: naïve and primed. The naïve state is the initial pluripotent state observed in embryonic development which is represented by embryonic stem (ES) cells. The primed state is a slightly differentiated state of ES cells marked by epigenetic and gene expressions alterations; primed pluripotent cells are thusly termed epiblast stem cells (EpiSC)¹,². While this transition has been observed in embryo development through in vivo cross-sectional studies, the exact mechanism of this transition and dependency on certain signalling events remains elusive. For decades it was challenging to observe blastocyst implantation in real-time due to accessibility to the embryo in utero. Now, with the advances in 3D culturing techniques ³,⁴, in vitro conditions of implantation may be mimicked to induce this transition for real time observations.

The main objective of my project is to investigate the transition of naïve to primed pluripotent stem cells at the morphological and gene expression level in vitro via the described 3D culturing technique. Key targets of interest include the initiation of the MAPK/ERK pathway⁵,⁶,⁷,⁸, which has been demonstrated as a crucial peri-implantation event for ES cell to EpiSC transition. It has been proposed that FGF4 plays the leading initiative role in activating this pathway⁸, thus an emphasis on decoding the expression, localization, and activation of this protein may be key in understanding this transition. A genetic approach will be used to create double knockout mice models, produced via the novel CRISPR/Cas9n ⁹,¹⁰, to investigate the necessity of FGF4 and its downstream activation elements in the context of cell morphology. Protein and mRNA expression related to the MAPK/ERK cascade will be analyzed by in situ immunostaining, and hybridization with follow-up sequencing for greater understanding of the mechanism of transition in a multi-cellular organism.

The importance of these findings will better help understand the necessary factors and preparation attributing to successful and controlled cell differentiation. While current stem cell techniques used to differentiate human induced pluripotent stem cells (iPSC) have progressed in the last few years¹¹,¹², we have yet to achieve efficient and controllable differentiation of iPSC into desirable cell types for regenerative medicine purposes. Since EpiSC act as the gateway cell type for differentiation from iPSC, understanding this transition could lead to more refined techniques for specific cell type differentiation for regenerative medicine therapies. Additionally, this process may provide a possible related retro-model for understanding epithelial-mesenchymal transition observed in malignant cancer cells¹³; this model may be used for better understanding the mechanisms of malignant tumors and how might they may be sequestered. It is therefore highly promising that investigating the role of FGF4 in the transition of naïve to primed pluripotency will unlock greater understanding for embryonic development and provide beneficial avenues for regenerative and possibly oncological medicine.

References

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2. Nichols, J. & Smith, A. Pluripotency in the embryo and in culture. Cold Spring Harb. Perspect. Biol. 4, a008128 (2012).

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6. Posfai, E., Tam, O. H. & Rossant, J. Mechanisms of pluripotency in vivo and in vitro. Curr. Top. Dev. Biol. 107, 1–37 (Elsevier Inc., 2014).

7. Wray, J., Kalkan, T. & Smith, A. G. The ground state of pluripotency. Biochem. Soc. Trans. 38, 1027–32 (2010).

8. Lanner, F. et al. Heparan sulfation-dependent fibroblast growth factor signaling maintains embryonic stem cells primed for differentiation in a heterogeneous state. Stem Cells 28, 191–200 (2010).

9. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–9 (2013).

10. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–9 (2013).

11. Löhle, M. et al. Differentiation efficiency of induced pluripotent stem cells depends on the number of reprogramming factors. Stem Cells 30, 570–9 (2012).

12. Lam, A. Q. et al. Rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers. J. Am. Soc. Nephrol. 25, 1211–25 (2014).

13. Larue, L. & Bellacosa, A. Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3’ kinase/AKT pathways. Oncogene 24, 7443–54 (2005).