flow cytometry analysis of transfected mouse pluripotent stem cells &#40;mpscs&#41;

Comparative Analysis of Forward and Reverse Transfection Methods on mPSCs

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.

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

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

Our work generally focuses on using synthetic genetic devices to manipulate stem cell fate and behavior. By controlling cellular states, we aim to advance the development of next generation cellular therapies. Recently, a deeper understanding of the relationship between the dosage of a fate determining gene and the cell's lineage has been established.This was facilitated by the development of genetic controllers that are able to titrate a given gene precisely. Classic molecular biology techniques such as transfection or lentiviral transduction for simple gene over expression will always be a mainstay. However, our field has recently adopted synthetic devices that incorporate linear and non-linear feedback in order to obtain more complex outputs from delivered DNA.Gene dosage has a key role in determining the outcome of gene expression. When delivering multiple exogenous genes, a key experimental factor is how much of each gene will give the optimal or desired outcome. Our protocol allows researchers to rapidly scan across dosages in a single treatment, looking for the desired ratio of DNA devices.

Flow Cytometry Analysis of Transfected Mouse Pluripotent Stem Cells &#40;mPSCs&#41;

After transfection of mPSCs, aspirate the media from the six-well plate. Then add 200 microliters of trypsin into each well. Swirl and incubate for 30 seconds at 37 degrees Celsius.Tap the side of the plate and add 200 microliters of ice cold FACS buffer. Using a P200 pipette, mix the contents of each well to dislodge cells and make single cell solutions. Transfer 200 microliters from each well to the appropriate well on the 96-well plate Centrifuge the plate at 300 G for five minutes.After centrifugation, resuspend the pellets in 150 microliters of FACS buffer before running the sample on a flow cytometer. Compared to reverse transfections, forward transections showed a significantly lower proportion of cells positive for the marker. Interestingly, forward Transfect did not deliver a significantly lower amount of DNA on average to the population of cells.In reverse transections, incubation time significantly influenced the percentage of reporter positive cells. However, the average reporter expression in positive cells was not affected by incubation time. For both poly and co-transfection, a forward transfection significantly decreased the number of cells present at the chosen endpoint compared to reverse transfections.In the case of reverse poly-transections, higher transfection efficiency and a broader dynamic range of well-represented DNA ratios were observed.

flow-cytometry-analysis-transfected-mouse-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

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.

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.

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

Preparation of Mouse Pluripotent Stem Cells &#40;mPSCs&#41; for Transfection

To prepare a gelatin coated 24 well plate add 200 microliters of the 0.2%gelatin solution to each well. Gently shake the plate to evenly spread the solution and allow the wells to coat at room temperature. After 15 minutes, using a vacuum aspirator carefully remove any leftover gelatin from the wells.Add 0.5 milliliters of freshly prepared NBIL media to each well. And place the plate in a tissue culture incubator to prewarm. Aliquot 30 milliliters of wash media into a 50 milliliter conical tube.Aspirate the media from the tissue culture dish of mESCs and add required amount of cell detachment medium. After three to five minutes, pipette up and down 15 to 20 times using a P1, 000 pipette. Observe the cells under a microscope.To ensure a single cell suspension transfer the cell suspension into a 50 milliliter tube containing wash media. Centrifuge the suspension at 300G for five minutes at room temperature and aspirate the wash media. Resuspend the pellet in approximately 300 microliters of wash media.

Reverse and Forward Transfection of Mouse Pluripotent Stem Cells &#40;mPSCs&#41;

To begin, aliquot 200 microliters of NBIL media into 1.5 milliliter tubes. Add 26 microliters of the optimum transfection reagent mixture to the DNA optimum mixture. Pipe at the reagents vigorously approximately 10 times.Transfer the required volume of mPSC suspensions to the 200 microliters of NBIL tubes. Then add lipofectamine 2000 and DNA mixture to the tubes and mix well by pipetting. After five minutes, centrifuge the mixture at 300 G for five minutes and remove the supernatant, leaving approximately 50 microliters in the tube.Re suspend the pellet in 100 microliters of NBIL media. Transfer the cell suspension to a gelatin-coated 24-well plate and place plate in the incubator. Transfer the required volume of mPSC suspension to seed 25, 000 cells per well of a gelatin coated 24-well plate and place the plate in the incubator.One hour prior to transfection. Replace the media from the wells with 0.5 milliliters of NBIL media. After incubation, dropwise, add lipofectamine 2000 and DNA mixture to the wells.Place the plate in the incubator for 48 hours.