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 "naïve" and "primed" pluripotency. The mechanisms that control naïve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify naïve 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 naïve-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.


Introduction
Embryonic stem cells (ESCs) have the capacity to self-renew or differentiate into any cell type in the adult body, a phenomenon known as pluripotency 1 . Recent evidence indicates that developmentally distinct pluripotent states exist, termed "naïve" and "primed" pluripotency 2 . Naïve ESCs represent a state of development similar to that found in the preimplantation embryo 3 . In contrast, primed pluripotent ESCs are poised to exit pluripotency and differentiate into specialized embryonic lineages 4,5 . Naïve and primed pluripotent states are marked by distinct gene regulatory networks. Naïve pluripotency is characterized by expression of key pluripotency transcription factors such as Nanog, Krueppel-like transcription factors (Klfs), Rex1 2 and Esrrb 6 . In mouse ESCs (mESCs), primed pluripotency is characterized by reduced expression of naïve markers and a specific gene expression signature which includes the de novo DNA methyltransferase Dnmt3b 7 . 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 Brachyury 8 .
mESCs provide a tractable model to probe mechanisms that control naïve-primed pluripotent transitions in vitro. When cultured in leukemia inhibitory factor (LIF) and fetal bovine serum (FBS), mESCs undergo dynamic transition between naïve and primed pluripotent states 9,10 . LIF-Jak-Stat3 signaling functions to promote a naïve gene regulatory network 11 , whilst autocrine signaling via the fibroblast growth factor 4 (Fgf4) Erk1/2 pathway drives transition to the primed state 12 . 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 naïveprimed 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 naïve 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.  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. 2. Assemble kinase inhibitors into 96-well plates for optimal high-throughput processing at multiple concentrations (use 1, 0.1 and 0.01 mM).

Collation of Small Molecule Kinase Inhibitor Libraries
Include in each plate control wells containing DMSO only and reference control inhibitors PD173074 (FGFRi) and Ruxolitinib (JAKi), which are known to promote naïve and primed pluripotent states respectively. Annotate in a spreadsheet or similar software.

mESC Culture Conditions and Procedures for Screen Preparation
1. 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. 2. Culture any standard mESC line at 37 °C 5% CO 2 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. 3. To passage mESCs, aspirate media and wash with 5 mL of phosphate-buffered saline (PBS) per plate. 4. Add 1 mL trypsin-EDTA (0.05% Trypsin, 0.02% EDTA) per plate of mESCs and incubate at 37 °C for 10 min. 5. Resuspend trypsinized cells in 4 mL of mESC media and centrifuge at 1,200 rpm for 5 min. 6. Thoroughly resuspend cell pellet in 5 mL of mESC media, pipetting up and down to generate a single cell suspension. 7. Count cells by combining a 10 µL cell suspension and 10 µL of Trypan Blue (0.4%). Place into a cell counting chamber or use a hemocytometer and light microscope. 8. Seed 3 x 10 3 mESCs into 0.1% gelatin coated 96 well plates, final volume 100 µL of media, using a multichannel pipette.
9. Apply kinase inhibitors at 1:100 dilution (1 µL inhibitor added to 100 µL 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 µM respectively. we recommend starting the screen at a final concentration of 1 µM to ensure effective inhibition of primary target kinases whilst minimizing off-target effects.

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.

Representative Results
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 µM 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 naïve 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 state 14 .

Discussion
Here we demonstrate a widely accessible methodology to probe the role of kinase signalling pathways in regulating naïve-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 naïve 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 naïve-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 naïve 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 applications 15,16 . 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 Copyright © 2017 Creative Commons Attribution 3.0 License May 2017 | 123 | e55515 | Page 6 of 6 developed by the Structural Genomics Consortium (http://www.thesgc.org/chemical-probes/epigenetics) has the potential to uncover further novel regulators of pluripotent transitions.

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