Untangling the Web of Protein Kinase C Mediated Regulation of Naïve Vs Primed State of Pluripotency

Abstract

Introduction‐Pluripotency of stem cells is associated with the dynamicity of the embryo’s developmental stage from which they are derived. Stem cells derived from the pre‐implantation embryo, called embryonic stem cells (ESCs), are naïve in nature, whereas post‐implantation embryo‐derived stem cells, termed epiblast stem cells (EpiSCs), represent the primed state of pluripotency. Maintaining these stem cells in a naïve state is an essential arena of stem cell research since naïve ESCs are capable of clonal propagation and germline transmission, making them a crucial research tool in organogenesis and disease modeling. Inhibition of PKC (PKCi) signaling was found to maintain an undifferentiated stage of pluripotency in both mouse and rat. Interestingly, when used along with a cocktail of inhibitors, the same inhibitor facilitated the derivation of naïve state pluripotency in human ESC. These findings lead us to a hypothesis that PKC signaling inhibition alone has a role in maintaining the naïve state of pluripotency. Hence, it has intrigued us to explore the complex network of mechanisms exerted by PKC signaling in dictating the transition of naïve to primed state of pluripotency.Methodology‐In that pursuit, we first observed the morphology of the mouse ESCs (mESCs) by phase‐contrast microscopy across passages and performed MTT assay to optimize the concentration used for further assay. Quantitative Real‐time PCR and western blotting were performed to determine the expression of transcription factors associated with naïve and primed states of pluripotency. Other techniques, including FACS, TiO2 phospho‐enrichment followed by Mass‐spectrometric (MS) analysis, Immunofluorescence assay, Immunoprecipitation assay, Nuclear‐cytoplasmic protein extraction and others were exploited to study the mechanism.Results‐We observed that PKCi could maintain naïve state of pluripotency in mESC even after prolonged culturing by reducing the expression of a critical primed transcription factor. The inhibition of phosphorylation of the PKCζ isoform is responsible for naïve state maintenance. Cell cycle analysis showed that more cells are in the G1 phase in the PKCi maintained condition than in the cells maintained in the presence of Leukemia Inhibitory Factor (LIF). Also, cell proliferation marker CDK1 expression was less in PKCi maintained cells, which probably is negatively regulated by the transcription factor KLF4. Increased nuclear expression of KLF4 was observed in PKCi maintained ESCs than that of ESCs cultured with LIF. Beside these transcription factor‐mediated regulations, PKCi exerts an epigenetic control over the ESCs absent in LIF maintained ESCs. PKCi mediated loss in phosphorylation of the epigenetic factor contributes to naïve state maintenance. Further, our data indicates towards PKCζ mediated NF‐κβ pathway activation, thereby dictating the transition of naïve to primed state of pluripotency.Conclusions‐PKCi maintained mESCs could maintain the naïve state of pluripotency. We have unraveled a multifaceted functionality of PKC signaling in the transition of naïve to primed state of pluripotency, which extends to both transcriptional and epigenetic regulation.