Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation

The role of L-proline and L-proline activated signalling in the regulation of pluripotency

© 2014 Dr. Boon Siang Nicholas TanThe study of pluripotent cell populations in the mouse embryo, and mouse pluripotent cells in culture, has revealed four identifiable pluripotent cell populations, or states, that comprise the pluripotent lineage. The four pluripotent populations are the epiblast precursor cells, epiblast, early primitive ectoderm and late primitive ectoderm in the embryo. They are represented in culture by ground state embryonic stem (ES) cells, primed ES cells, early primitive ectoderm-like (EPL) cells and epiblast stem cells (EpiSCs) respectively. Although these cell populations are relatively well characterised, the mechanisms that control progression between cell states remain poorly understood. Understanding the transition of primed ES cells to EPL cells, representing the progression of epiblast to early primitive ectoderm of the post-implantation embryo, is the focus in this thesis. EPL cells are derived from ES cells in response to factors within the conditioned medium MEDII. The ES to EPL cell transition provides a model to investigate the mechanisms that regulate pluripotent cell lineage progression. Amino acids have been shown to regulate cellular processes through modulation of intracellular cell signalling pathways. The amino acid L-proline has been identified as the bioactive factor of MEDII required for EPL cell formation, and ES cells cultured in L-proline-containing medium form EPL cells. EPL cell formation is accompanied by changes in cell morphology, gene expression and differentiation kinetics. In chapter 3, the main L-proline transporter on ES and EPL cells is determined to be the System A amino acid transporter 2 (SNAT2). Identification of SNAT2 was facilitated by radioactive uptake assays. SNAT2 uptake of L-proline can be inhibited by the addition of excess amounts of other SNAT2 substrates, such as alanine. ES cells cultured in L-proline and excess concentrations of SNAT2 substrates, but not other amino acids, were not able to form EPL cells. This suggests that L-proline uptake through SNAT2 is required for EPL cell formation. A requirement for L-proline uptake by SNAT2 in the regulation of pluripotent cells suggests a role for L-proline in activating intracellular pathways in the formation of EPL cells. In chapter 4, pharmacological inhibitors of cell signalling pathways were used to determine requirements of Src tyrosine kinases, p38 MAPK and Erk in the formation and maintenance of EPL cells. Exposure of ES cells to L-proline increased Src tyrosine kinase and p38 MAPK activity. Chemical inhibition of these pathways prevented the acquisition of many, but not all, features of EPL cells in culture. Although Erk1/2 signalling was not activated in response to L-proline addition, it was required within the cell for EPL cell formation. The involvement of multiple signalling pathways in EPL cell formation and maintenance supports the potential role of L-proline activated pathways in primitive ectoderm formation. Amino acids are critical for the developmental processes of the preimplantation mouse embryo to the blastocyst stage. However, regulatory roles of amino acids beyond the blastocyst stage, specifically in pluripotent lineage progression, are not well understood. In chapter 5, the expression of SNAT1 and SNAT2 transporters was determined in mouse embryos between the 2-cell stage and early post-implantation. Both transporters showed temporal dynamic expression patterns and differences in intracellular localisation within cell types. Changes in transporter expression likely reflect different amino acid needs at different stages of development. Dynamic SNAT2 expression was observed in the pluripotent lineage with up regulation in the epiblast prior to primitive ectoderm formation. Primed ES cells cultured in ground state conditions resulted in reduced Slc38a2 expression suggesting regulation of Slc38a2 in the transition of ground state to primed ES cells. SNAT1 and SNAT2 showed regulated expression in the trophectoderm (TE) lineage. SNAT1 was preferentially expressed on outer cells of the compacted morula that are fated to form TE, while SNAT2 was localised in the nucleus of TE and placenta cells. The unexpected nuclear localisation of SNAT2 suggests a novel role for an amino acid transporter in the nucleus. Localisation of SNAT1, in outer cells may indicate requirements for amino acids in TE formation. Collectively, data presented here support a role for amino acids and amino acid transporters in the progression of pluripotent states in vivo and in vitro. Understanding the role of amino acids in pluripotent cell transitions will significantly contribute to the optimisation of differentiation media and protocols used in regenerative medicine

The role of ERK1/2, Src Family Kinases and p38 MAPK in the maintenance of EPL cells.

<p><b>A-D.</b> Photomicrographs of mES cells cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (A), 1 μM PD0325901 (B) 10 μM PP2 (C) and 10 μM SB203580 (D). Cells were stained for alkaline phosphatase activity (red/purple stain). Scale bar = 200 μm. <b>E, F.</b> mES cells were cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (■), DMSO with 1 μM PD0325901 (E, □), DMSO with 10 μM PP2 (F, □) or DMSO with 10 μM SB203580 (G, □). RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i> and <i>Otx2</i> by real-time PCR. Expression was normalized to <i>β-actin</i>. Error bars represent SEM; n = 3. EPL cells cultured in MEDII with inhibitor were compared to EPL cells in MEDII with DMSO, ** <i>p</i> ≤ 0.05, or mES cells, # <i>p</i>≤ 0.05.</p

Inhibition of MEK1 prevents the formation of EPL cells in response to MEDII.

<p>A. mES cells were pre-treated with 1 μM PD0325901 for 60 minutes. 200 μM l-proline was added, as denoted, and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated ERK1 or ERK2. Total ERK1/2 was used as a loading control. The intensity of the pERK2 band was measured using Quantity One software (BioRad) and represented as a proportion of total ERK1/2. Error bars represent SEM; n = 4; * <i>p</i> ≤ 0.05 when compared to mES cells. B-C. mES cells were cultured in MEDII- and DMSO-containing medium (B) and MEDII- and 1μM PD0325901-contianing medium (C) for 3 days. Scale bar = 200 μm. D. MEDII- and DMSO-containing medium (■) and MEDII- and 1μM PD0325901-contianing medium (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 3. mES cells + MEDII + PD0325901 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05).</p

The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells

There is an increasing appreciation that amino acids can act as signaling molecules in the regulation of cellular processes through modulation of intracellular cell signaling pathways. In culture, embryonic stem (ES) cells can be differentiated to a second, pluripotent cell population, early primitive ectoderm-like cells in response to biological activities within the conditioned medium MEDII. The amino acid l-proline has been identified as a component of MEDII required for ES cell differentiation. Here, we define the primary l-proline transporter on ES and early primitive ectoderm-like cells as sodium-coupled neutral amino acid transporter 2 (SNAT2). SNAT2 uptake of l-proline can be inhibited by the addition of millimolar concentrations of other substrates. The addition of excess amino acids was used to regulate the uptake of l-proline by ES cells, and the effect on differentiation was analyzed. The ability of SNAT2 substrates, but not other amino acids, to prevent changes in morphology, gene expression, and differentiation kinetics suggested that l-proline uptake through SNAT2 was required for ES cell differentiation. These data reveal an unexpected role for amino acid uptake and the amino acid transporter SNAT2 in regulation of pluripotent cells in culture and provides a number of specific, inexpensive, and nontoxic culture additives with the potential to improve the quality of ES cell culture.Boon Siang Nicholas Tan, Ana Lonic, Michael B. Morris, Peter D. Rathjen and Joy Rathje

The regulation of progression of the pluripotent lineage in culture.

<p>The cell states represented in vitro, naïve mES cells, primed mES cells and EPL cells have been aligned with the expression of Nanog and Otx2 and with their deduced intracellular signaling activity. Inducers of lineage progression are shown in orange; Calcineurin exerts its effects through dephosphorylation of NFAT and promotes NFAT translocation to the nucleus.</p

Summary of inhibitors used in this study.

<p>Summary of inhibitors used in this study.</p

The role of p38 MAPK in the formation of EPL cells in response to MEDII.

<p><b>A.</b> mES cells were pre-treated with 10 μM SB203580 for 60 minutes. 200 μM l-proline was added, as denoted and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated pHspb2. Total Hspb2 was used as a loading control. <b>B, C.</b> mES cells were cultured medium supplemented with MEDII and DMSO (A) or MEDII and 10 μM SB203580 for 3 days. Scale bar = 200 μm. <b>D.</b> mES cells were cultured in medium supplemented with MEDII and DMSO (■) or MEDII and 10 μM SB203580 (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 4. mES cells + MEDII + SB203580 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05). <b>E.</b> mES cells were cultured in ES cell medium supplemented with MEDII, DMSO or MEDII and 10 μM 10 μM SB203580 for 3 days and formed into EBs. EBs were collected on days 2, 3 and 4, RNA was isolated and analyzed for expression of <i>T</i> and <i>Gapdh</i> by RT-PCR. n = 3.</p

The addition of l-proline to ES cells increases ROS.

<p><b>A-C</b>. MitoSox Red staining of mES incubated with 200 μM l-proline (B) or l-proline and ascorbic acid (C) and compared to untreated cells (A). Hoechst staining of the same fields of view are shown in Ai, Bi and Ci. D. ROS levels were measured as fluorescence using ROS-Glo™ H₂O₂ Assay (Promega) in cells that had been incubated with l-proline, 150 μM DHP or 1 mM GSH, as denoted. Results are shown as arbitrary fluorescent units. n = 4, *p≤0.05 when compared with mES cells. # p≤0.05 when compared with mES cells incubated with l-proline. E. The expression of <i>Dnmt3b</i> and <i>Otx2</i> was analysed in mES cells cultured in medium supplemented 200 μM l-proline or 200 μM l-proline and 150 μM DHP. Expression was normalized to β-actin and expressed relative to mES cells. Error bars represent SEM; n = 4. Comparisons were made to mES cells (*p ≤ 0.05) or mES cells cultured with 200 μM l-proline (# p ≤ 0.05).</p