The authors would like to acknowledge the many contributions to the field that were not cited in this work due to space limitations, as well as the funding agencies that provided this opportunity.&#160;The authors acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR), which supported this work. K.M. is the recipient of a CGS-M scholarship from NSERC and a Killam Doctoral Scholarship from the University of British Columbia. N.S. is the recipient of a Michael Smith Health Research BC Scholar Award.

1. Preparation of reagents for mPSC culture
<ol>
	<li>Prepare N2 supplement.
	<ol>
		<li>In non-sterile conditions, prepare the following stock solutions (step 1.1.1.1) in a chemical safety fume hood. Add the solid powder of each chemical to a pre-weighed 50 mL conical tube. Weigh each tube after adding the powder and add an appropriate amount of solvent to achieve the concentrations below. For one batch of media, prepare the minimum amount listed. 
		NOTE: The following reagents are hazardous and should be handled in accordance with local chemical safety guidelines. Ensure proper PPE when handling, and only work with the solid powder forms of these chemicals in a chemical fume hood to prevent inhalation.
		<ol>
			<li>Minimum of 0.05 mL of sodium selenite in water at 0.518 mg/mL, 0.5 mL of putrescine in water at 160 mg/mL, 0.165 mL of progesterone in 100% ethanol at 0.6 mg/mL (see Table of Materials).</li>
			<li>Store solutions at -20 &#176;C for up to 2 years.</li>
		</ol>
		</li>
		<li>In a biosafety cabinet (BSC), add the following to 58.035 mL of DMEM-F12.
		<ol>
			<li>0.05 mL of a 0.518 mg/mL sodium selenite solution, 0.5 mL of a 160 mg/mL putrescine solution, 0.165 mL of a 0.6 mg/mL progesterone solution.</li>
			<li>Filter sterilize the above mixture with a 0.22 &#181;m filter.</li>
		</ol>
		</li>
		<li>Still in the BSC, prepare a 100 mg/mL solution of apotransferrin.
		<ol>
			<li>Add 5 mL of DMEM-F12 to 500 mg of apotransferrin (see Table of Materials). Slowly resuspend and wash the container, avoiding bubbles.</li>
			<li>After resuspending and removing as much as possible with 5 mL, add it to the selenite/putrescine/progesterone solution. Take more of the previous solution and wash the apotransferrin bottle, getting as much of it out as possible.</li>
		</ol>
		</li>
		<li>Add 5 mL of 7.5% BSA fraction V (see Table of Materials) to the solution.</li>
		<li>Mix and aliquot 6.875 mL per tube. Store aliquots at -80 &#176;C for up to 1 year.</li>
		<li>When using the above N2 aliquots to make N2B27 media, thaw the desired aliquot and add 3.125 mL of a 4 mg/mL insulin solution (see Table of Materials) to the 6.875 N2 for 10 mL of 100x N2.</li>
	</ol>
	</li>
	<li>Prepare N2B27 basal media.
	<ol>
		<li>Thaw N2 and B27 supplements in a 4 &#176;C fridge for a few hours or overnight.</li>
		<li>In a BSC, mix 484.5 mL of DMEM-F12 and 484.5 mL of Neurobasal media (see Table of Materials) in a sterile 1 L bottle.</li>
		<li>Add 10 mL of B27 and 10 mL of N2 supplement to the media.</li>
		<li>Add 1 mL of 55 mM beta-mercaptoethanol (see Table of Materials). Add 10 mL of 100x glutamax. Mix vigorously by swirling the bottle.</li>
		<li>Aliquot the desired volume per aliquot (typically 100 mL) and store at -80 &#176;C for up to 1 year. Store in use after thawing at 4 &#176;C for up to 3 weeks.</li>
	</ol>
	</li>
	<li>Prepare NBiL media.
	<ol>
		<li>Thaw a 100 mL aliquot of N2B27 basal media from -80 &#176;C in the 4 &#176;C fridge.</li>
		<li>In a BSC, add LIF (see Table of Materials) to a final concentration of 10 &#181;g/L.</li>
		<li>Add CHIR99021 (3 &#181;M final) and PD0325901 (1 &#181;M final) (see Table of Materials). Mix well with a 50 mL serological pipette. Store at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare 0.2% gelatin solution.
	<ol>
		<li>Transfer 500 mL of double-distilled H2O into a 1 L glass bottle.</li>
		<li>Measure out 1 g of gelatin (see Table of Materials) and add it to the water. Mix by shaking the bottle until mostly dissolved.</li>
		<li>Autoclave the gelatin and water mixture at 15 psi, 121 &#176;C for 35 min. Once cooled, filter sterilize it with a 0.22 &#181;m filter in a BSC. Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare wash media.
	<ol>
		<li>In a BSC, mix 160 &#181;L of 7.5% BSA per 10 mL of DMEM-F12.</li>
		<li>Scale the above and prepare in large quantities for ease of future use (e.g. 8 mL of 7.5% BSA to 500 mL of DMEM-F12). Store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of reagents for mPSC poly-transfection
NOTE: The following values are provided for a single well of a 24-well plate. Values can be scaled accordingly. The sequences of all the DNA/plasmids are detailed in Supplementary File 1.
<ol>
	<li>Calculate the volume of DNA solution needed per treatment.
	<ol>
		<li>Prepare 500 ng of DNA per well/condition.
		<ol>
			<li>If transfecting a single plasmid with a DNA solution of 100 ng/&#181;L, prepare one tube with 5 &#181;L of DNA.</li>
			<li>If transfecting two plasmids at 100 ng/&#181;L each, prepare two tubes with 2.5 &#181;L in each, one for each plasmid.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In a BSC, perform the following: For each 500 ng transfection treatment (see Table of Materials), aliquot 25 &#181;L of OptiMEM into a 1.5 mL tube.
	<ol>
		<li>Scale this accordingly - for the example above, prepare two tubes, each with 250 ng of DNA (2.5 &#181;L) and 12.5 &#181;L of OptiMEM.</li>
	</ol>
	</li>
	<li>Add the required volume of DNA for that treatment to the OptiMEM in the tube.</li>
	<li>For each tube with 500 ng DNA and OptiMEM, create a matching tube with another 25 &#181;L of OptiMEM and 1 &#181;L of cationic lipid-based transfection reagent.
	<ol>
		<li>Again, these values must be scaled accordingly. For the above example, prepare two tubes, each with 12.5 &#181;L of OptiMEM and 0.5 &#181;L of transfection reagent. 
		&#8203;NOTE: When scaling values, use the mass of DNA added, not the volume required for that amount of DNA. Reactions with transfection reagent and DNA can be scaled (e.g., if 5 wells are to be transfected with the same DNA). Keep the ratios of reagents the same, but scale accordingly (e.g., 1000 ng DNA: 50 &#181;L OptiMEM: 2 &#181;L transfection reagent: 50 &#181;L OptiMEM).</li>
	</ol>
	</li>
</ol>
3. Preparation of mPSCs for transfection
NOTE: For reverse transfection, prepare the culture vessel and passage the mPSCs directly before adding the transfection reagents. For forward transfection, passage and plate the cells 12-18 h prior to transfection to allow the cells to adhere to the plate.
<ol>
	<li>Prepare a 0.2% gelatin-coated cell culture vessel.
	<ol>
		<li>Plan the number of wells needed for the transfection (one well of a 24-well plate per treatment/group).</li>
		<li>Add 200 &#181;L of the 0.2% gelatin solution to each of the desired wells in the 24-well plate.</li>
		<li>Gently shake the plate to evenly spread the solution, ensuring it covers the entire bottom of the well.</li>
		<li>Allow the wells to coat for a minimum of 15 min at room temperature.</li>
		<li>Using a vacuum aspirator, carefully remove any leftover gelatin from the wells (tilt the plate to ensure removal of all liquid without scraping off the gelatin layer).</li>
		<li>Add 0.5 mL of NBiL media (step 1.3) to each well and leave the plate in a tissue culture incubator to prewarm.</li>
	</ol>
	</li>
	<li>Passage mPSCs.
	<ol>
		<li>Aliquot 30 mL of wash media (step 1.5) into a 50 mL conical tube.</li>
		<li>Aspirate the old media from the tissue culture dish of mESCs. 
		&#8203;NOTE: Several aspiration techniques are permissible. Whatever technique is chosen, ensure that cells do not remain dry for extended periods (approximately 30 s maximum).</li>
		<li>Add the required amount of commercially available cell detatchment medium (1.5 mL for a 10 cm dish, scale accordingly, see Table of Materials).</li>
		<li>Wait for 3-5 min before pipetting up and down 15-20 times with a P1000 pipette to obtain a single-cell suspension. View cells under a microscope to ensure a single-cell suspension.</li>
		<li>Transfer the cells in the detatchment medium into the 50 mL tube containing wash media.</li>
		<li>Centrifuge at 300 x g for 5 min at room temperature. Aspirate wash media, ensuring no residual liquid remains in the tube.</li>
		<li>Resuspend the pellet in a small volume of wash media (~300 &#181;L). Count the cell suspension using a hemocytometer to obtain the cell density.</li>
	</ol>
	</li>
</ol>
4. Reverse transfection of mPSCs
<ol>
	<li>Prepare poly-transfection reagents according to step 2.</li>
	<li>Prepare the cell culture vessel and passage mPSCs according to step 3.</li>
	<li>Aliquot 200 &#181;L of NBiL media to 1.5 mL tubes, one for each treatment.</li>
	<li>Add 26 &#181;L of the OptiMEM:transfection reagent mixture to the DNA:OptiMEM mixture. Mix the reagents quickly and vigorously by pipetting approximately 10 times. Allow the mixture to incubate at room temperature for 5 min.</li>
	<li>Based on cell density, transfer the required volume of cells from the 300 &#181;L cell suspension to the 200 &#181;L NBiL tubes to obtain 25,000 cells per tube.</li>
	<li>After 5 min of incubation of the Lipofectamine 2000 (L2K, transfection reagent) and DNA mixture, add it to the 1.5 mL tube of cells and mix well by pipetting. Allow the cells to incubate with the reagent for 5 min at room temperature.</li>
	<li>Centrifuge at 300 x g for 5 min at room temperature. Pipette out the liquid, leaving approximately 50 &#181;L.</li>
	<li>Resuspend the cell pellet with 100 &#181;L of NBiL media. Transfer the cells to the plate and place the plate in the incubator. Change the media 24 h later with fresh NBiL media.</li>
</ol>
5. Forward transfection of mPSCs
<ol>
	<li>12-18 h prior to transfection, prepare the cell culture vessel and passage mPSCs according to step 3.</li>
	<li>Based on cell density, transfer the required volume of cells from the 300 &#181;L cell mixture to seed 25,000 cells per well. Place the plate in the incubator.</li>
	<li>1 h prior to transfection, aspirate the media from all wells and replace it with 0.5 mL of NBiL media. Place the plate in the incubator.</li>
	<li>At the time of transfection, prepare mPSC poly-transfection reagents according to step 2.</li>
	<li>Add 26 &#181;L of the OptiMEM:transfection reagent mixture to the DNA:OptiMEM mixture. Mix the reagents quickly and vigorously by pipetting approximately 10 times. Allow the combined mixture to incubate at room temperature for 5 min.</li>
	<li>Add the combined mixture to the wells dropwise. Place the plate in the incubator. Change the media 24 h later.</li>
</ol>
6. Flow cytometry
<ol>
	<li>Aspirate the media from the transfected 24-well plate 48 h after transfection.</li>
	<li>Add 200 &#181;L of trypsin to each well, swirl, and incubate for 30 s at 37 &#176;C. Tap the sides of the plate and add 200 &#181;L of ice-cold FACS buffer (2% FBS in PBS).</li>
	<li>Open and label a V-bottom 96-well plate, starting from A1. Mix the contents of each well on the transfected plate to dislodge cells and make single-cell solutions with a P200 pipette. Transfer 200 &#181;L from each well to the appropriate well on the 96-well plate.</li>
	<li>Add 15 mL of FACS buffer to a 50 mL reagent reservoir. Centrifuge the plate at 300 x g for 5 min at room temperature. Discard the supernatant by inverting the 96-well plate into the sink with a fast and smooth movement.</li>
	<li>Use a multichannel pipette to resuspend the pellets on the 96-well plate in 150 &#181;L of FACS buffer from the reagent reservoir. Repeat steps 6.4-6.5 twice. Place the 96-well plate in an ice-filled box protected from light.</li>
	<li>Run the samples through a flow cytometer to obtain the population distributions of the fluorescent reporters.
	<ol>
		<li>Ensure the cytometer is appropriate in terms of laser and filter combinations for the experiment to be performed (e.g., do not use an infrared reporter if it is not possible to excite or detect in the far-red spectrum).</li>
		<li>Include additional samples for standardization beads to allow for MEFL conversion of data during analysis. Ensure that enough cells are collected per sample for appropriate statistical analysis9.</li>
	</ol>
	</li>
	<li>Convert fluorescent units from the raw cytometer files and analyze the data using any of several methods, such as Python- or MATLAB-based pipelines or commercially available software9,13.</li>
</ol>

Comparative Analysis of Forward and Reverse Transfection Methods on mPSCs

<ol>
	<li>Shakiba, N., Jones, R. D., Weiss, R., Del Vecchio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-aware+synthetic+biology+by+controller+design:+Engineering+the+mammalian+cell.">Context-aware synthetic biology by controller design: Engineering the mammalian cell.</a> Cell Systems. 12 (6), 561-592 (2021).</li><li>Fus-Kujawa, A., 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=An+overview+of+methods+and+tools+for+transfection+of+eukaryotic+cells+in+vitro.">An overview of methods and tools for transfection of eukaryotic cells in vitro.</a> Frontiers in Bioengineering and Biotechnology. 9, (2021).</li><li>FAU, K. T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and Bioanalytical Chemistry. 397 (8), 3173-3178 (2010).</li><li>Tamm, C., Galit&#243;, S. P., Anner&#233;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+protocols+for+mouse+embryonic+stem+cell+culturing.">A comparative study of protocols for mouse embryonic stem cell culturing.</a> PLoS One. 8 (12), 81156 (2013).</li><li>Morgani, S., Nichols, J., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+of+pluripotency:+in+vitro+adaptations+of+a+continuum+of+in+vivo+states.">The many faces of pluripotency: in vitro adaptations of a continuum of in vivo states.</a> BMC Developmental Biology. 17 (1), (2017).</li><li>Mulas, 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=Defined+conditions+for+propagation+and+manipulation+of+mouse+embryonic+stem+cells.">Defined conditions for propagation and manipulation of mouse embryonic stem cells.</a> Development. 146 (6), 173146 (2019).</li><li>Tamm, C., Kadekar, S., Pijuan-Galit&#243;, S., Anner&#233;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+efficient+transfection+of+mouse+embryonic+stem+cells+using+non-viral+reagents.">Fast and efficient transfection of mouse embryonic stem cells using non-viral reagents.</a> Stem Cell Reviews and Reports. 12 (5), 584-591 (2016).</li><li>Chong, Z. X., Yeap, S. K., Ho, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+types,+methods+and+strategies:+A+technical+review.">Transfection types, methods and strategies: A technical review.</a> PeerJ. 9, 11165 (2021).</li><li>Gam, J. J., DiAndreth, B., Jones, R. D., Huh, J., Weiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+&amp;#34;poly-transfection&amp;#34;+method+for+rapid,+one-pot+characterization+and+optimization+of+genetic+systems.">A &amp;#34;poly-transfection&amp;#34; method for rapid, one-pot characterization and optimization of genetic systems.</a> Nucleic Acids Research. 47 (18), 106 (2019).</li><li>Jones, R. D., 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=An+endoribonuclease-based+feedforward+controller+for+decoupling+resource-limited+genetic+modules+in+mammalian+cells.">An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells.</a> Nature Communications. 11 (1), 5690 (2020).</li><li>Wauford, N., Jones, R., Van De Mark, C., Weiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+development+of+cell+state+identification+circuits+with+poly-transfection.">Rapid development of cell state identification circuits with poly-transfection.</a> Journal of Visualized Experiments. (192), e64793 (2023).</li><li>Hori, Y., Kantak, C., Murray, R. M., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+extract+based+optimization+of+biomolecular+circuits+with+droplet+microfluidics.">Cell-free extract based optimization of biomolecular circuits with droplet microfluidics.</a> Lab on a Chip. 17 (18), 3037-3042 (2017).</li><li>Teague, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoflow:+A+python+toolbox+for+flow+cytometry.">Cytoflow: A python toolbox for flow cytometry.</a> bioRxiv. , (2023).</li><li>Qin, J. Y., 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=Systematic+comparison+of+constitutive+promoters+and+the+doxycycline-inducible+promoter.">Systematic comparison of constitutive promoters and the doxycycline-inducible promoter.</a> PLoS One. 5 (5), 0010611 (2010).</li></ol>

The authors report no conflicts of interest.

A key reason for the widespread adoption of transfection protocols is their reproducibility and accessibility; however, these protocols do require optimization across experimental contexts. Not mentioned above is the standard testing required when attempting to transfect a new cell line for the first time. First, the choice of transfection reagent is key, as commercially available reagents are not one-size-fits-all and will vary in the efficiency of NA delivery viability across cell types. Additionally, finding the ideal...

Delivery of DNA and RNA into mammalian cells serves as a core pillar of biomedical research1. A common method for introducing exogenous nucleic acids (NA) into mammalian cells is through transient transfection2,3. This technique relies on mixing NA with commercially available transfection reagents capable of delivering them into the recipient cells. Typically, NA is delivered via forward transfection, where cells adhering to a two-dimensional surface receive the transfection complex. While forward transfection for the most common established cell lines is robust and protocols are well-published, more niche cell types with non-monolayer morphologies do not transfect easily, limiting the amount of NA that can be delivered and the number of cells that receive it.
Pluripotent stem cells (PSCs) serve as an attractive model for understanding development and as a tool for regenerative medicine, given their ability to divide indefinitely and produce any bodily cell type. For mouse PSCs (mPSCs), routine in vitro culture conditions with 2 inhibitors and LIF&#160;(2i/LIF) maintain a dome-like colony morphology, directly limiting the number of cells exposed to a forward transfection4,5,6. To address this, a reverse transfection can be performed: cells are added to a dish containing media and transfection reagent, rather than adding transfection reagent to adherent cells7. While this increases the number of cells exposed to the reagent, it also requires the cells to be passaged and transfected concurrently.
Moving beyond simple single-NA transfections, researchers often aim to deliver several NA constructs into a population of cells in vitro. This is typically achieved through a co-transfection, where the NAs are mixed at a given ratio (1:1, 9:1, etc.) and are then combined with the chosen transfection reagent8. This yields a mix of NAs and reagent that preserves the original ratio of NAs to one another - while cells in the treatment may receive different amounts of this mix, they all receive the same ratio9. While this is advantageous when the desired ratio of parts is known, determining this ratio ahead of time can be labor-intensive, with each ratio constituting a different condition. One alternative is to perform a &#34;poly-transfection,&#34; where individual NAs are mixed with the transfection reagent independently from one another9. By combining transfection complexes containing individual NAs (rather than combining NAs before creating the complexes), researchers can explore a wide array of NA stoichiometries in a single transfection experiment9. This is particularly valuable in cases where the products of several NAs are expected to interact with one another, such as with inducible transcription systems or systems with feedback built in1,10,11. However, to do so effectively, a high transfection efficiency is needed. Indeed, as the number of unique transfected NAs increases, the probability of a given cell receiving all of the desired NAs decreases exponentially9, 12.
The following report describes a reverse transfection protocol for mPSCs using a cationic lipid-based transfection reagent, in which cells are exposed to the reagent-NA mix for a maximum of 5 min to maximize viability and minimize the time outside of typical culture conditions. Comparing this protocol to the standard forward transfection of these cells demonstrates a higher transfection efficiency and an increase in the total number of surviving transfected cells. By combining this reverse transfection with a three-plasmid poly-transfection involving simple fluorescent reporters, an expanded potential to screen NA ratios with high transfection efficiency is demonstrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase&#160;</td>
			<td>MilliporeSigma</td>
			<td>SCR005</td>
			<td></td>
		</tr>
		<tr>
			<td>Apotransferrin&#160;</td>
			<td>MilliporeSigma</td>
			<td>T1147-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA fraction V (7.5%)</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021&#160;</td>
			<td>MilliporeSigma</td>
			<td>SML1046-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>MilliporeSigma</td>
			<td>D6421-24X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry standardization beads</td>
			<td>Spherotech</td>
			<td>URCP-38-2K</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin&#160;</td>
			<td>MilliporeSigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Gibco</td>
			<td>12585-0014</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM&#160;</td>
			<td>Invitrogen</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901&#160;</td>
			<td>MilliporeSigma</td>
			<td>PZ0162-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>MilliporeSigma</td>
			<td>P8783</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>MilliporeSigma</td>
			<td>P6780</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Recombinant mLIF&#160;</td>
			<td>BioTechne</td>
			<td>8878-LF-500/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite&#160;</td>
			<td>MilliporeSigma</td>
			<td>S5261-25G</td>
			<td>Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized mouse embryonic stem cell based reverse poly-transfection technique for rapid exploration of nucleic acid ratios

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Both forward and reverse transfections rely on the interaction between the cell membrane and incoming transfection reagent-DNA complexes, allowing the delivery of NA to the recipient cells. Where these techniques differ is the state of the cell upon delivery - while DNA is typically delivered to adherent cell monolayers in traditional forward transfection, reverse transfection instead relies on having the reagent-DNA complex meet the cells while in a single-cell suspension. This difference can be particularly crucial in ...

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Author Spotlight: Advancements in Synthetic Genetic Devices for Stem Cell Fate Manipulation and Cellular Therapy Development

The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical ...

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Research

JoVE Journal

Developmental Biology

952 Views.  University of British Columbia. The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

Video: Author Spotlight: Advancements in Synthetic Genetic Devices for Stem Cell Fate Manipulation and Cellular Therapy Development

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

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 ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

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 ...

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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 ...

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

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Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

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

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Isolation of Murine Embryonic Hemogenic Endothelial Cells

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

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

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