C.A.C.W is supported by a Medical Research Council PhD studentship. G.M.F. is supported in part by a Medical Research Council New Investigator Award (MR/N000609/1) and a Tenovus Scotland research grant.

1. Collation of Small Molecule Kinase Inhibitor Libraries
<ol>
	<li>Collate inhibitor libraries from publicly accessible kinase inhibitor collections, e.g. Library of Integrated Network-based Cellular Signatures (LINCS) from Harvard Medical School, a targeted collection of 228 potent and selective kinase inhibitors (http://lincs.hms.harvard.edu), and the Protein Kinase Inhibitor Set (PKIS), a 376 compound collection assembled by industrial researchers (https://www.ebi.ac.uk/chembldb/extra/PKIS)13. Alternatively, assemble a bespoke kinase inhibitor collection from commercially available compounds. 
	NOTE: In our laboratory, we have assembled a collection of 72 potent and selective tool kinase inhibitors covering 51 primary target kinases, all of which are available from commercial sources.</li>
	<li>Assemble kinase inhibitors into 96-well plates for optimal high-throughput processing at multiple concentrations (use 1, 0.1 and 0.01 mM). Include in each plate control wells containing DMSO only and reference control inhibitors PD173074 (FGFRi) and Ruxolitinib (JAKi), which are known to promote na&#239;ve and primed pluripotent states respectively. Annotate in a spreadsheet or similar software.</li>
</ol>
2. mESC Culture Conditions and Procedures for Screen Preparation
<ol>
	<li>Prepare 0.1% gelatin-coated 10 cm dishes by adding 5 mL of 0.1% (w/v) gelatin to 10 cm plates and incubate at room temperature for 5 min. Aspirate gelatin and let plate dry for 2 min.</li>
	<li>Culture any standard mESC line at 37 &#176;C 5% CO2 on 0.1% gelatin-coated 10 cm dishes in standard mESC media (see Materials Table) containing 100 ng/mL GST-LIF, 10% Fetal Calf Serum and 5% Knockout Serum Replacement. Replace media every day and passage mESCs at around 80% confluency every second day at a dilution of 1:5-1:10.</li>
	<li>To passage mESCs, aspirate media and wash with 5 mL of phosphate-buffered saline (PBS) per plate.</li>
	<li>Add 1 mL trypsin-EDTA (0.05% Trypsin, 0.02% EDTA) per plate of mESCs and incubate at 37 &#176;C for 10 min.</li>
	<li>Resuspend trypsinized cells in 4 mL of mESC media and centrifuge at 1,200 rpm for 5 min.</li>
	<li>Thoroughly resuspend cell pellet in 5 mL of mESC media, pipetting up and down to generate a single cell suspension.</li>
	<li>Count cells by combining a 10 &#181;L cell suspension and 10 &#181;L of Trypan Blue (0.4%). Place into a cell counting chamber or use a hemocytometer and light microscope.</li>
	<li>Seed 3 x 103 mESCs into 0.1% gelatin coated 96 well plates, final volume 100 &#181;L of media, using a multichannel pipette.</li>
	<li>Apply kinase inhibitors at 1:100 dilution (1 &#181;L inhibitor added to 100 &#181;L media) using a multi-channel pipette. Gently pipette media to mix inhibitor and cell suspension, then allow cells to settle in a tissue culture hood for 1 h to ensure equal distribution across the plating surface. Culture cells for 48 h without changing the media. 
	NOTE: Stock plates of 1/0.1/0.01 mM will give a final inhibitor concentration of 10/1/0.1 &#181;M respectively. we recommend starting the screen at a final concentration of 1 &#181;M to ensure effective inhibition of primary target kinases whilst minimizing off-target effects.</li>
</ol>
3. Kinase Inhibitor Screening Analysis
<ol>
	<li>Wash 96 well mESC plates in 200 &#181;L PBS using multi-channel aspirator and pipettes.</li>
	<li>Make cell extracts in 150 &#181;L of lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 (v/v), 0.5% sodium deoxycholate (w/v), 10 mM &#946;-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM NaF, 2 mM Na3VO4, Complete Protease Inhibitor Cocktail Tablets).</li>
	<li>Clarify extracts by centrifugation at 1,500 x g for 30 min in V-bottomed 96-well plates.</li>
	<li>Immobilize 100 &#181;L of supernatants onto a nitrocellulose membrane using a 96-well vacuum dot blot manifold.</li>
	<li>Dry the membrane, and stain with 40 mL of Ponceau S to ensure consistent transfer.</li>
	<li>Wash membrane with TBST and block in TBST/3% (w/v) milk.</li>
	<li>Incubate in Nanog and Dnmt3b antibodies at a dilution of 1:1,000 (v/v) in TBST/3% (w/v) milk powder overnight.</li>
	<li>Wash membranes in 3 x 10 min in TBST and incubate in 30 mL of secondary antibodies at a dilution of 1:10,000 (v/v) in TBST/3% (w/v) milk.</li>
	<li>Develop using a digital immunoblotting imaging system. 
	NOTE: If possible, use quantitative fluorescence immunoblotting to detect Nanog and Dnmt3b. However, the background signal for Dnmt3b by fluorescence immunoblotting is too high. Therefore, 800 nm fluorescence anti-rabbit (Nanog) and anti-mouse-HRP (Dnmt3b) secondary antibodies are used.</li>
</ol>
4. Analysis of Screen Data and Validation of Inhibitors as Bona Fide Pluripotency Regulators
NOTE: This is a critical step to ensure that only bona fide pluripotency modulators are identified. It is essential to triage inhibitors based on both Nanog:Dnmt3b ratio and overall signal, to ensure that kinase inhibitors which adversely affect mESC survival are not selected for further analysis.
<ol>
	<li>Quantify Nanog and Dnmt3b signal using immunoblotting analysis software, and import values into a spreadsheet or similar software. Calculate Nanog:Dnmt3b ratio for all samples compared to DMSO control. Also, determine total Nanog and Dnmt3b signal to ensure that kinase inhibitors do not affect Nanog:Dnmt3b ratio by altering mESC viability.</li>
	<li>Use ranked Nanog:Dnmt3b ratios to identify inhibitors which promote na&#239;ve pluripotency (Nanog:Dnmt3b ratio higher than control) and primed pluripotency (Nanog:Dnmt3b ratio lower than control). Set cut-off at 2x deviation from control values, and filter out kinase inhibitors which produce a total Nanog+Dnmt3b signal of less that 50% of the DMSO control value to generate a high confidence list of kinase inhibitors which stabilise na&#239;ve (high Nanog:Dnmt3b ratio) and primed (low Nanog:Dnmt3b ratio) pluripotent states.</li>
	<li>Validate kinase inhibitors by seeding mESCs at 2 x 105 cells per 6 well in 2 mL media, and add kinase inhibitors at 1 &#181;M for 48 h, without changing media.</li>
	<li>Lyse mESCs in lysis buffer and analyze Nanog and Dnmt3b expression by conventional SDS-PAGE immunoblotting.</li>
	<li>Further validate hits by SDS-PAGE immunoblotting for na&#239;ve pluripotency markers Klf2 and Klf4, and Oct4 to ensure that pluripotency is maintained following 48 h inhibitor treatment.</li>
</ol>

<ol>
	<li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292 (5819), 154-156 (1981).</li><li>Nichols, J., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+and+primed+pluripotent+states.">Naive and primed pluripotent states.</a> Cell Stem Cell. 4 (6), 487-492 (2009).</li><li>Boroviak, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-Specific+Profiling+Delineates+the+Emergence+and+Progression+of+Naive+Pluripotency+in+Mammalian+Embryogenesis.">Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis.</a> Developmental Cell. 35 (3), 366-382 (2015).</li><li>Tesar, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+cell+lines+from+mouse+epiblast+share+defining+features+with+human+embryonic+stem+cells.">New cell lines from mouse epiblast share defining features with human embryonic stem cells.</a> Nature. 448 (7150), 196-199 (2007).</li><li>Brons, I. G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+pluripotent+epiblast+stem+cells+from+mammalian+embryos.">Derivation of pluripotent epiblast stem cells from mammalian embryos.</a> Nature. 448 (7150), 191-195 (2007).</li><li>Festuccia, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esrrb+is+a+direct+Nanog+target+gene+that+can+substitute+for+Nanog+function+in+pluripotent+cells.">Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.</a> Cell Stem Cell. 11 (4), 477-490 (2012).</li><li>Ficz, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signaling+inhibition+in+ESCs+drives+rapid+genome-wide+demethylation+to+the+epigenetic+ground+state+of+pluripotency.">FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.</a> Cell Stem Cell. 13 (3), 351-359 (2013).</li><li>Guo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klf4+reverts+developmentally+programmed+restriction+of+ground+state+pluripotency.">Klf4 reverts developmentally programmed restriction of ground state pluripotency.</a> Development. 136 (7), 1063-1069 (2009).</li><li>Chambers, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanog+safeguards+pluripotency+and+mediates+germline+development.">Nanog safeguards pluripotency and mediates germline development.</a> Nature. 450 (7173), 1230-1234 (2007).</li><li>Findlay, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+domains+of+Sos1/Grb2+are+finely+tuned+for+cooperative+control+of+embryonic+stem+cell+fate.">Interaction domains of Sos1/Grb2 are finely tuned for cooperative control of embryonic stem cell fate.</a> Cell. 152 (5), 1008-1020 (2013).</li><li>Niwa, H., Burdon, T., Chambers, I., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+pluripotent+embryonic+stem+cells+is+mediated+via+activation+of+STAT3.">Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.</a> Genes Dev. 12 (13), 2048-2060 (1998).</li><li>Kunath, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+stimulation+of+the+Erk1/2+signalling+cascade+triggers+transition+of+pluripotent+embryonic+stem+cells+from+self-renewal+to+lineage+commitment.">FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.</a> Development. 134 (16), 2895-2902 (2007).</li><li>Elkins, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+characterization+of+the+Published+Kinase+Inhibitor+Set.">Comprehensive characterization of the Published Kinase Inhibitor Set.</a> Nature Biotechnology. 34 (1), 95-103 (2016).</li><li>Williams, C. A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erk5+Is+a+Key+Regulator+of+Naive-Primed+Transition+and+Embryonic+Stem+Cell+Identity.">Erk5 Is a Key Regulator of Naive-Primed Transition and Embryonic Stem Cell Identity.</a> Cell Reports. 16 (7), 1820-1828 (2016).</li><li>Cao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+human+fibroblasts+into+functional+cardiomyocytes+by+small+molecules.">Conversion of human fibroblasts into functional cardiomyocytes by small molecules.</a> Science. 352 (6290), (2016).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+reprogramming+of+fibroblasts+into+neural+stem+cells+by+signaling-directed+transcriptional+activation.">Pharmacological reprogramming of fibroblasts into neural stem cells by signaling-directed transcriptional activation.</a> Cell Stem Cell. 18 (5), 653-667 (2016).</li></ol>

The authors declare no conflicts of interest arising from this study.

Here we demonstrate a widely accessible methodology to probe the role of kinase signalling pathways in regulating na&#239;ve-primed pluripotent transition. This addresses a key question in the ESC field. Although high-throughput genomics and transcriptomics approaches are routinely used to identify key transcriptional regulators of na&#239;ve and primed pluripotent states, elucidating pluripotency signalling networks has proven to be challenging. We now provide a flexible strategy employing chemical inhibitors to identify kinases that control na&#239;ve-primed pluripotent transition in mESCs. This relies on simple equipment, reagents and materials that are available to most laboratories that study cell signalling and ESC biology. Critical to the success of this platform is our development of a robust assay to quantify the transition between na&#239;ve and primed pluripotent states. Establishing this assay required significant iterative modifications to cell plating density, time of inhibitor incubation and immunoblotting conditions.
Small molecule approaches are gaining traction for rational use in therapeutic ESC applications15,16. A major strength of small molecule screening is that tool compounds are designed and/or modified for efficient cellular uptake. Transfection efficiency is not limiting, and use of hazardous delivery systems such as lentivirus is not necessary. Furthermore, inhibitors frequently inhibit multiple isoforms within a kinase family (e.g. Fgfr1-4, Jak1-3), which overcomes functional redundancy that hinders genetic/genomic interference techniques such as RNAi and CRISPR/Cas9. As more potent and selective tool compounds become available from both academic and pharmaceutical drug discovery programmes, understudied and poorly understood kinases will be pushed to the forefront of ESC research. We therefore propose that this high-throughput screening approach will open up new avenues of research into core ESC regulatory networks.
One minor limitation of this technology is that even the most potent and selective small molecule inhibitors engage and inhibit multiple unrelated kinases in vivo. However, increasingly comprehensive kinase inhibitor profiling data allows functional targets of kinase inhibitors to be readily identified (http://lincs.hms.harvard.edu/kinomescan; http://www.kinase-screen.mrc.ac.uk/kinase-inhibitors). Finally, in principle our platform can be applied to interrogate any small molecule collection and/or cellular assay for which high-quality immunoblotting antibodies are available. This will allow study of multiple regulatory systems in pluripotency regulation and facilitate identification of small molecule inhibitors which modify diverse cellular processes. Specifically, we envisage that application of emerging small molecule collections such as the epigenetic probes being developed by the Structural Genomics Consortium (http://www.thesgc.org/chemical-probes/epigenetics) has the potential to uncover further novel regulators of pluripotent transitions.

Embryonic stem cells (ESCs) have the capacity to self-renew or differentiate into any cell type in the adult body, a phenomenon known as pluripotency1. Recent evidence indicates that developmentally distinct pluripotent states exist, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency2. Na&#239;ve ESCs represent a state of development similar to that found in the preimplantation embryo3. In contrast, primed pluripotent ESCs are poised to exit pluripotency and differentiate into specialized embryonic lineages4,5.
Na&#239;ve and primed pluripotent states are marked by distinct gene regulatory networks. Na&#239;ve pluripotency is characterized by expression of key pluripotency transcription factors such as Nanog, Krueppel-like transcription factors (Klfs), Rex12 and Esrrb6. In mouse ESCs (mESCs), primed pluripotency is characterized by reduced expression of na&#239;ve markers and a specific gene expression signature which includes the de novo DNA methyltransferase Dnmt3b7. In vivo, primed pluripotent post implantation epiblast stem cells (EpiSCs)4,5 additionally express the Epiblast marker Fgf5 and markers of lineage priming such as Brachyury8.
mESCs provide a tractable model to probe mechanisms that control na&#239;ve-primed pluripotent transitions in vitro. When cultured in leukemia inhibitory factor (LIF) and fetal bovine serum (FBS), mESCs undergo dynamic transition between na&#239;ve and primed pluripotent states9,10. LIF-Jak-Stat3 signaling functions to promote a na&#239;ve gene regulatory network11, whilst autocrine signaling via the fibroblast growth factor 4 (Fgf4) Erk1/2 pathway drives transition to the primed state12. However, it remains a challenge to systematically evaluate the role of protein kinases in specifying distinct pluripotent states.
Here, we describe a quantitative and scalable platform by which to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition. We use simple mESC culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated ability to stabilize na&#239;ve pluripotency. Furthermore, we discuss potential extended applications for this technology, including for screening of other small molecule collections such as emerging inhibitor libraries targeting epigenetic regulators.

<table><tbody><tr><td>&lt;strong&gt;mESC media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CCE mESCs</td><td>ATCC</td><td>ATCC&amp;nbsp;SCRC-1023</td><td></td></tr><tr><td>DMEM Media</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030081</td><td>Final: 2 mM</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td>Final: 50 U/ml</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M6250</td><td>Final: 0.1 mM</td></tr><tr><td>Leukemia Inhibitory Factor (GST fusion)</td><td>MRC-PPU reagents and services</td><td></td><td>Final: 100 ng/mL</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Thermo</td><td>PMC9484</td><td>Final: 100 ng/mL</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco</td><td>11140050</td><td>Final: 0.1 mM</td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360070</td><td>Final: 1 mM</td></tr><tr><td>KnockOut Serum Replacement</td><td>Invitrogen</td><td>10828-028</td><td>Final: 5%</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>Fisher</td><td>10703464</td><td>Final: 10%</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;TC materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo</td><td>25300054&amp;nbsp;</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>1890</td><td></td></tr><tr><td>Nunc MicroWell 96-Well Microplates</td><td>Thermo</td><td>156545</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190136</td><td></td></tr><tr><td>V-bottom 96 well plates</td><td>Greiner Bio-one</td><td>651261</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Lysis Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris buffer</td><td>VWR</td><td>103157P</td><td></td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>97061</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E4884</td><td></td></tr><tr><td>1% NP-40</td><td>Sigma</td><td>9016-45-9</td><td></td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma</td><td>D6750-100g</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma</td><td>G9422</td><td></td></tr><tr><td>Sodium Pyrophosphate</td><td>Sigma</td><td>13472-36-1</td><td></td></tr><tr><td>Sodium Fluoride</td><td>Sigma</td><td>450022</td><td></td></tr><tr><td>Sodium Orthovanadate</td><td>Sigma</td><td>450243</td><td></td></tr><tr><td>Roche Complete Protease Inhibitor Cocktail Tablets</td><td>Sigma</td><td>CO-RO</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tween</td><td>Sigma</td><td>P1379-1L</td><td></td></tr><tr><td>Milk Powder</td><td>Marvel</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Western Blotting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Protran BA 85 Nitrocellulose 0.45 &amp;micor;M&amp;nbsp; Pore size (20 cm x 20 cm Sheets) (25 Sheet)</td><td>GE</td><td>10401191</td><td></td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P7170-1L</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Laboratory Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Minifold I System</td><td>GE</td><td>10447850</td><td></td></tr><tr><td>ChemiDoc imager</td><td>BioRad</td><td>1708280&amp;nbsp;</td><td></td></tr><tr><td>LiCor Odyssey Imager</td><td>LiCor</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse Nanog</td><td>Reprocell</td><td>RCAB001P</td><td></td></tr><tr><td>Anti-Dnmt3b</td><td>Imgenex</td><td>IMG-184A</td><td></td></tr><tr><td>IRDye 800CW Donkey anti-Rabbit</td><td>Licor</td><td>926-32213</td><td></td></tr><tr><td>Anti-mouse-HRP</td><td>Cell Signalling Technology</td><td>7076S</td><td></td></tr><tr><td>Anti-Klf2</td><td>Millipore</td><td>09-820</td><td></td></tr><tr><td>Anti-Klf4</td><td>R&amp;amp;D Systems</td><td>AF3158</td><td></td></tr><tr><td>Anti-Oct4</td><td>Santa Cruz</td><td>sc-5279</td><td></td></tr></tbody></table>

a simple method to identify kinases that regulate embryonic stem cell pluripotency by high-throughput inhibitor screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Using the procedure presented here (Figure 1), we screen the LINCS library of 228 potent and selective kinase inhibitors to identify those which modulate mESC pluripotency. The library is prepared at a concentration of 0.1 mM for a 1:100 dilution and final screening concentration of 1 &#181;M in mESCs. 48 h later, mESCs were lysed and extracts prepared for quantitative dot blot analysis of Nanog and Dnmt3b expression (Figure 2, top). Results from other screening concentrations are not represented here for brevity. Nanog:Dnmt3b ratio is determined for each kinase inhibitor and compounds ranked and a 2x cut-off applied (Figure 2, bottom). Total Nanog and Dnmt3b signal relative to control is overlaid onto inhibitor ranking and subjected to a 0.5x cut-off, to allow triaging of inhibitors which show significant mESC toxicity. Select kinase inhibitors which stabilize the na&#239;ve state are validated using conventional Nanog/Dnmt3b immunoblotting (Figure 3). The primary kinase targets of these inhibitors are presented in Table 1. We also demonstrate the applicability of this screen to identify inhibitors which stabilize the primed state14.
<img alt="Figure 1" src="/files/ftp_upload/55515/55515fig1.jpg" /> 
Figure 1: Workflow for screening kinase inhibitors that modulate na&#239;ve-primed transition. mESC were treated with a 228 compound kinase inhibitor library at a final concentration of 1 &#181;M. Lysates were prepared and transferred onto nitrocellulose membranes for immuno dot-blot analysis, and Nanog and Dnmt3b levels determined (Figure adapted from Williams et al.14). <a href="http://cloudflare.jove.com/files/ftp_upload/55515/55515fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55515/55515fig2.jpg" /> 
Figure 2: Na&#239;ve-primed pluripotency screen analysis and inhibitor identification. (Top) Representative Nanog and Dnmt3b immuno dot-blot images. (Bottom) Nanog and Dnmt3b values for each kinase inhibitor were determined relative to the DMSO control, and Nanog:Dnmt3b ratios and total relative Nanog+Dnmt3b signal determined and inhibitors ranked in comparison to DMSO control. Inhibitors found to alter Nanog:Dnmt3b ratio beyond a 2-fold threshold above or below DMSO control were identified as drivers of na&#239;ve or primed pluripotency. A threshold of 2x below DMSO control was set to triage inhibitors which compromise mESC survival. Inhibitors selected for further validation are highlighted. (Figure adapted from Williams et al.14). <a href="http://cloudflare.jove.com/files/ftp_upload/55515/55515fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55515/55515fig3.jpg" /> 
Figure 3: Validation of selected hit compounds identified. mESCs were treated with either 1 &#181;M AZD4547 (a selective FGFR inhibitor) or the indicated inhibitors identified from the screen in Figure 2. Nanog:Dnmt3b levels determined by standard SDS-PAGE and immunoblot analysis. Numerical values of relative Nanog:Dnmt3b and Nanog+Dnmt3b are indicated in the table below, and inhibitors which stabilize the na&#239;ve pluripotent state highlighted in green. <a href="http://cloudflare.jove.com/files/ftp_upload/55515/55515fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td colspan="2">Primary Targets</td>
			<td>Other Targets</td>
		</tr>
		<tr>
			<td>AV-951</td>
			<td colspan="2">VEGFR</td>
			<td></td>
		</tr>
		<tr>
			<td>HG-6-64-01</td>
			<td colspan="2">ABL, BRAF, RET, CSF1R, EGFR, EPHA8, FGFR, FLT3, KIT, LOK, MAP4K1, p38b, MUSK,PDGFR, TAOK3, TNNI3K&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>WH-4-023</td>
			<td colspan="2">Lck</td>
			<td>Src</td>
		</tr>
		<tr>
			<td>R406</td>
			<td colspan="2">Syk</td>
			<td></td>
		</tr>
		<tr>
			<td>GW-572016</td>
			<td colspan="2">HER2</td>
			<td>EGFR</td>
		</tr>
		<tr>
			<td>KIN001-244</td>
			<td colspan="2">PDK1</td>
			<td></td>
		</tr>
		<tr>
			<td>WZ-4-145</td>
			<td colspan="2">CSF1R, DDR1, EGFR, TIE1, PDGFR2</td>
			<td></td>
		</tr>
		<tr>
			<td>WZ-7043</td>
			<td colspan="2">CSF1R, DDR1, FGFR and TAO1</td>
			<td></td>
		</tr>
		<tr>
			<td>GW786034</td>
			<td colspan="2">VEGFR1</td>
			<td>VEGFR2, VEGFR3&#160;</td>
		</tr>
		<tr>
			<td>AZD-1480</td>
			<td colspan="2">JAK2</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Selected hit compounds and their primary kinase target(s).

Watch this Scientific Journal Video about A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening at JoVE.com

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

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Research

JoVE Journal

Developmental Biology

6.4K Views.  University of Dundee.  Hier presenteren we een kwantitatief en schaalbaar protocol voor het uitvoeren van gerichte kleine molecuulschermen voor kinase-regulatoren van de naïeve-primed pluripotente transitie. 

Video: A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Biochemistry

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Biology

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Immunology and Infection

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

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