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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mESC media</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCE mESCs</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">ATCC&#160;SCRC-1023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM Media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutamine</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25030081</td>
			<td>Final: 2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td>Final: 50U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M6250</td>
			<td>Final: 0.1mM</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Leukemia Inhibitory Factor (GST fusion)</td>
			<td style="width:71px;">MRC-PPU reagents and services</td>
			<td style="width:119px;"></td>
			<td>Final: 100ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leukemia Inhibitory Factor</td>
			<td style="width:71px;">Thermo</td>
			<td style="width:119px;">PMC9484</td>
			<td>Final: 100ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Non-Essential Amino Acids</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11140050</td>
			<td>Final: 0.1mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Pyruvate</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11360070</td>
			<td>Final: 1mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KnockOut Serum Replacement</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">10828-028</td>
			<td>Final: 5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Defined Fetal Bovine Serum</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">10703464</td>
			<td>Final: 10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TC materials</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:71px;">Thermo</td>
			<td style="width:119px;">25300054&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin from porcine skin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">1890</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc MicroWell 96-Well Microplates</td>
			<td style="width:71px;">Thermo</td>
			<td style="width:119px;">156545</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DPBS, no calcium, no magnesium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">14190136</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">V-bottom 96 well plates</td>
			<td style="width:71px;">Greiner Bio-one</td>
			<td style="width:119px;">651261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysis Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris buffer</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">103157P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Chloride</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">97061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1% NP-40</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">9016-45-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Deoxycholate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D6750-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#946;-glycerophosphate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G9422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Pyrophosphate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">13472-36-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Fluoride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">450022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Orthovanadate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">450243</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Roche Complete Protease Inhibitor Cocktail Tablets</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CO-RO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemicals</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Milk Powder</td>
			<td style="width:71px;">Marvel</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Western Blotting</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protran BA 85 Nitrocellulose 0.45uM&#160; Pore size (20 X 20cm Sheets) (25 Sheet)</td>
			<td style="width:71px;">GE</td>
			<td style="width:119px;">10401191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ponceau S</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P7170-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laboratory Equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Minifold I System</td>
			<td style="width:71px;">GE</td>
			<td style="width:119px;">10447850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ChemiDoc imager</td>
			<td style="width:71px;">BioRad</td>
			<td style="width:119px;">1708280&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LiCor Odyssey Imager</td>
			<td style="width:71px;">LiCor</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibodies</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-mouse Nanog</td>
			<td style="width:71px;">Reprocell</td>
			<td style="width:119px;">RCAB001P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Dnmt3b</td>
			<td>Imgenex</td>
			<td>IMG-184A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IRDye 800CW Donkey anti-Rabbit</td>
			<td style="width:71px;">Licor</td>
			<td style="width:119px;">926-32213</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Anti-mouse-HRP</td>
			<td style="width:71px;">Cell Signalling Technology</td>
			<td>7076S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Klf2</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">09-820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Klf4</td>
			<td style="width:71px;">R&#38;D Systems</td>
			<td style="width:119px;">AF3158</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Oct4</td>
			<td style="width:71px;">Santa Cruz</td>
			<td style="width:119px;">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 ...

a-simple-method-to-identify-kinases-that-regulate-embryonic-stem-cell

Research

JoVE Journal

Developmental Biology

6.4K Views.  University of Dundee. Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition.

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

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...