Abstract
The differentiation potential of pluripotent embryonic stem cells (ESCs) can be manipulated via serum and medium conditions for direct cellular development or to maintain a naïve ground state. The self-renewal state of ESCs can thus be induced by adding inhibitors of mitogen activated protein kinase (MAPK) and glycogen synthase kinase-3 (Gsk3), known as 2 inhibitors (2i) treatment. We have used a shotgun proteomics approach to investigate differences in protein expressions between 2i- and serum-grown mESCs. The results indicated that 164 proteins were significantly upregulated and 107 proteins downregulated in 2i-grown cells compared to serum. Protein pathways in 2i-grown cells with the highest enrichment were associated with glycolysis and gluconeogenesis. Protein pathways related to organ development were downregulated in 2i-grown cells. In serum-grown ESCs, protein pathways involved in integrin and focal adhesion, and signaling proteins involved in the actin cytoskeleton regulation were enriched. We observed a number of nuclear proteins which were mostly involved in self-renewal maintenance and were expressed at higher levels in 2i compared to serum - Dnmt1, Map2k1, Parp1, Xpo4, Eif3g, Smarca4/Brg1 and Smarcc1/Baf155. Collectively, the results provided an insight into the key protein pathways used by ESCs in the ground state or metastable conditions through 2i or serum culture medium, respectively.
Highlights
Pluripotent embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos
As expected, cellular morphology and homogeneity of pluripotency-associated gene expression differed between the two growth conditions which was in line with previous report1. 2i ESCs were morphologically uniform and homogeneously expressed pluripotency-associated genes while serum ESCs were heterogeneous for both
The results provided an insight into the key protein pathways utilized by ESCs in the ground state or metastable condition through 2i or serum culture medium, respectively
Summary
Pluripotent embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos These cells have a remarkable capacity to form differentiated cell types in culture, contingent upon extracellular signals. Distinct transcriptome and epigenome profiles have been identified for ESCs grown in serum as opposed to a medium that contains inhibitors of MAPK and glycogen synthase kinase-3 (Gsk3), known as 2 inhibitors (2i) treatment[7], suggesting that specific signaling pathways are required to support ESCs self-renewal. The key intracellular signaling pathways utilized by pluripotent ESCs that initiate differentiation or maintain a ground state remain to be identified at the proteome level. We compared our proteomic findings to the previously reported transcriptome profile of 2i-grown cells in order to investigate whether additional post-translational modification pathways might contribute to ESC self-renewal capability
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