Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents
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Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.
KeywordsTransfection Mouse Embryonic stem cells ES cells siRNA DNA Plasmid
For the last decades mouse embryonic stem (mES) cells have been considered to be one of the most interesting model systems for basic cellular and molecular studies and developmental research. The ability to efficiently transfer DNA or small interfering RNA (siRNA) is one of the key technologies used for studying the role of single genes in these cells. However, mES cells grow in tight clusters and are therefore quite inefficiently transfected using chemical reagents. In addition, we have found that when mES cells are cultured in the serum-free 2i medium (containing two inhibitors, against the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) and the glycogen synthase kinase 3 (GSK3), respectively ), chemical transfection is even more challenging as compared to when cells are cultured in standard culture media . Replacement of serum by targeted inhibition of relevant signaling pathways reduces cellular heterogeneity as well as transcriptome and epigenome differences in mES cell cultures [3 , 4 ]. In 2i medium PD0325901 inhibits the autocrine activation of the ERK1/2 pathway by fibroblast growth factor-4 (FGF4), shown to be instrumental for ES cell differentiation [5 ]. The GSK3 inhibition impairs Tcf3 activity, which represses the core pluripotency network in mES cells [6 ]. The cell cultures exhibit a more dense and uniform morphology, with negligible signs of spontaneous differentiation as compared to cells cultured in serum-containing media .
There are many vastly different transfection techniques available [7 ]. Virus-mediated transfections, or so-called biological transfection methods, are suitable for stable transfections due to the integration of transfected DNA into the host cell genome. However, although viral methods are usually highly efficient they suffer from limitations such as time-consuming production of vectors, mutagenesis, cytotoxicity, and DNA size restriction [8 ]. Physical transfection methods are the means of diverse physical tools to deliver nucleic acids into host cells. Despite being effective, these methods are both complicated and time-consuming (e.g. micro injections) or may cause massive cell death (e.g. electroporation and nucleofection). En masse they also require expensive in-house equipment. Instead, much effort has been put into the development of chemical methods, such as calcium phosphates, cationic lipids, cationic polymers, dendrimers, polyethylenimine, peptides and nanoparticle-based compounds [7 ]. Although generally less efficient in delivering DNA, these non-viral vectors have higher genetic material carrying capacity, are easier to produce and cause low cytotoxicity. However, many challenges remain, such as increasing transfection efficacy while maintaining or reducing cytotoxicity, as well as improving penetration of cells that are difficult to transfect.
Here we present a side-by-side comparison of commercially available, non-viral transfection reagents for plasmid DNA or siRNA delivery using the manufacturers’ instructions. For DNA and RNA delivery we selected some of the most commonly used reagents on the market, both liposomal and non-liposomal polymer based. In addition we have also used a positively charged polymer embedded into a porous carrier nanoparticle. One of the unique challenges when transfecting mES cells is that these cells often grow in dense multilayered colonies, leading to lower transfection rates in the cells that are positioned in the middle of the colonies. Hence, in the present study we have transfected both adherent colonies as well as cells dispersed into single cell suspensions. We here present an optimized, rapid, single-cell suspension transfection protocol, which results in unparalleled efficacy while maintaining low post-transfection toxicity.
Material and Methods
Embryonic Stem Cell Culturing
Feeder-independent E14 and R1 mES cells were cultured in 2i medium , a serum-free N2B27 medium supplemented with MEK inhibitor PD0325901 (1 μM) and GSK3 inhibitor CHIR99021 (3 μM) (both from Selleckchem), and 1000 U/ml LIF (Millipore). TrypLE™ Express (Life Technologies) was used to passage the cells. To ensure good adherence 2 % ES-qualified FBS was supplemented to the 2i media when seeding the cells, as previously described .
Transfections with Lipofectamine® 2000 (herein named L2K), Lipofectamine® 3000 (L3K, Life Technologies), TurboFect™ (GE Healthcare Dharmacon), FuGENE® HD, ViaFect™ (Promega), TransIT-2020®, TransIT-X2®, TransIT-TKO®, TransIT-siQUEST® (Mirus Bio), Xfect™ mESC (Clontech), X-tremeGENE™ HP, X-tremeGENE™ 9 (Roche), Nanofectin, Nanofectamin (PAA, now part of GE Healthcare), JetPrime™ (Polyplus transfection) were performed according to the manufacturer’s instructions within the recommended reagent/DNA or reagent/siRNA ratio range. For adherent plasmid-DNA transfections cells were plated at constant density 2 x 105 cells/well in 12 well-plates one day prior to transfection. For the single cell suspension; 5 x 105 cells/well were transfected and plated in 12 well-plates. The pmaxGFP (AMAXA Biosystems), pCMV β-galactosidase, or pCAGGs heparanase vectors were introduced at a final concentration of 1.5 μg DNA/well. For the adherent siRNA transfections; cells were plated at a density 5 x 104 cells/well in 24 well plates one day prior to transfection with either scrambled siRNA or siRNA against Oct4 at a final concentration of 50 or 100 nM/well. For the siRNA single cell suspension transfections; cells were transfected and plated at a density 5 x 104 cells/well in a 24 well plates. (sense 5′-AGGUGUUCAGCCAGACCACTT-3′, antisense 5′-GUGGUCUGGCUGAACACCUTT-3′).
24 h post-transfection, cells were washed once with PBS and then trypsinized using TrypLE™ Express for 5 min at 37 °C. Cell suspensions were analysed in a Becton Dickinson FACSCalibur flow cytometer for FL1 (GFP) fluorescence to determine the percentage of reporter expressing cells. Background fluorescence and autofluorescence were determined using non-transfected cells as control. Analysis was carried out in the freeware Flowing Software (http://www.flowingsoftware.com).
Cells were harvested in lysis buffer as previously described [9 ]. Total protein concentration was measured using BCA Protein Assay kit (Pierce). Samples were run on 10 % SDS PAGE gels, and subsequently transferred to an Immobilon-FL membrane (Millipore). The membranes were blotted with anti-Oct3/4 (POUF5F1, cl. 7F9.2 (Millipore) and anti-β actin ab8229 (Abcam). The membranes were then incubated with Alexa Fluor anti-rabbit 680 and IRDye 800CW anti-mouse. Immunosignals were imaged using an Odyssey fluorescent imaging scanner (LI-COR Biosciences).
Β-Galactosidase Activity Assay
Twenty-four hours post-transfection cells were harvested in lysis buffer and mixed with ONPG buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 50 mM 2-mercaptoethanol, 1.2 mM ONPG) in a microtiter plate and incubated at 37 °C for 30 min. Absorbance was read at 420 nm in a microplate photometer reader (with Multiscan® PLUS, Labsystems).
Twenty four and 48 h post-transfection total RNA was extracted and purified with the GenElute™ mammalian total RNA miniprep kit (Sigma-Aldrich) according to the manufacturer’s instructions. First-strand cDNA was produced according to the manufacturer’s protocol with iScript™ cDNA synthesis kit (Bio-Rad) using 1 μg RNA. Quantitative real-time PCR was performed according to the manufacturer’s instructions using the SsoFast™ EvaGreen™ supermix on the Miniopticon™ Real-Time PCR Detection System (Bio-Rad). The average C(t) value for each gene was normalized against 18S and the comparative C t value (fold change) was calculated using 2−ΔΔC(t). Primers used were: 18S (forward: AGTCCCTGCCCTTTGTACACA, reverse: GATCCGAGGGCCTCACTAAAC).
Oct4 (forward:GATGCTGTGAGCCCAAG-GCAAG, reverse: GGCTCCTGATCAACAGCATCAC),
Dab2 (forward: TGAAGCAGACAGCCAGAACA, reverse: CAACAGACAAGGATTTGATAGGG).
Efficiency of siRNA Delivery
Oct3/4 down-regulation is known to lead to differentiation of mES cells . Hence, to determine the efficacy of the siRNA mediated Oct3/4 knockdown, expression of the early endoderm differentiation gene Dab2 was assessed in the same qPCR samples as used for assessing Oct3/4 levels 48 h post-transfection (Fig. 1a). As expected, the samples with the best Oct3/4 knockdown showed the highest increase in Dab2 mRNA levels 48 h post-transfection. Western blotting for Oct3/4 protein expression after 72 h of transfection further confirmed the high knockdown efficacy obtained with the four most efficacious siRNA delivery reagents as assessed by level of RNA interference and cell survival (Fig. 1b). Trypan blue exclusion assay was then used to assess the toxicity of these four reagents 24 and 48 h post-transfection with the scrambled siRNA. The results showed no significant differences between the various reagents when using scrambled siRNA independent of time-point although Nanofectin appeared slightly more toxic to the cells (Fig. 1c). Cells transfected with Oct4 siRNA showed reduced viability as compared to cells transfected with scrambled siRNA and survival decreased over time suggesting that the Oct4 knockdown in combination with transfection negatively affects cells survival.
Efficiency of DNA-Plasmid Delivery in Adherent Cells
Efficiency of DNA-Plasmid Delivery in Single Cell Suspension
The five most efficient reagents from the initial screenings were then used for transfecting cells with a β-galactosidase (β-gal) reporter construct. The β-gal activity results concurred with the results presented above, both as to the efficiency of the different reagents but also that transfection rate of single cells exceeds that of adherent cultures (Fig. 3).
Fast and Efficient DNA-Plasmid and siRNA Delivery in Suspended mES Cells
Finally, to validate if short-time single cell suspension transfection works equally well for siRNA delivery, similar set-up, as presented above, was tested using L2K and TransIT-X2 with 100 nM Oct3/4 siRNA. Oct3/4 expression was markedly reduced 24 h post-transfection with 5, 15 and 30 min incubation (Fig. 4g). In fact, the level of Oct3/4 expression was clearly lower than after similar transfections in adherent 2i media cultures (Fig. 1a). Hence, as expected, these novel transfection conditions seem to work equally well for both plasmid-DNA as well as for siRNA delivery.
Over the years various biological, chemical, and physical strategies for introducing nucleic acids into cells have been developed. Today there are numerous commercially available products optimized for certain cell or nucleic acid types. Stem cells and primary cell cultures are often difficult to transfect. Consequently, working with these cells often means accepting low transfection efficiencies or laborious optimizations. Instead many studies are carried out in easily transfectable cell lines, such as HEK293 or HeLa, despite being poor cell models. Multilayered mES cell colonies give poor transfection efficiency due to the lack of homogenous cellular access for transfection reagents and nucleic acid complexes. As we have previously reported this quandary is even more troublesome when growing the cells in 2i media . Moreover, post-transfection intrusion on cellular proteostasis (e.g. due to expression of plasmid DNA-encoded genes) and subsequent decrease in proliferation rates result in cultures being promptly overtaken by the less affected non-transfected cells. Although the latter is a common concern it becomes more critical for mES cells due to their excessive proliferation rates and alternate day sub-culturing needs.
In the present study we evaluated a panel of commonly used non-viral commercially available transfection reagents with regard to their applicability to deliver plasmid DNA and siRNA into mES cells cultured in 2i media. TransIT-X2 was found to be the most efficient reagent for siRNA transfection of adherent mouse ES cells cultured in 2i medium. When transfecting adherent cultures with DNA plasmid, the Xfect mESC non-liposomal polymer was the only reagent that generated more than 50 % transfection rate. However, L2K followed by Nanofectamin were the most efficient reagents when transfecting dissociated single cell suspensions, suggesting that lipofection is the method of choice for this experimental approach. Although Xfect mES reagents at first glance seemed equally efficient as L2K, the reagent was very toxic to cells in suspension. Suspension transfection is not mentioned in the manufacturer’s instructions for the Xfect mESC reagent. However, the traditional Xfect transfection reagent has been shown to work for suspension cell cultures [10 ].
It is likely that the inefficient transfection of cells cultured in 2i medium is partly due to the densely packed cell cultures making the transfection agent less prone to access all the cells. Enzymatic dissociation induces shaving of membrane-associated factors, including glycoproteins with highly sulfated glycosaminoglycan (GAGs) polysaccharide chains [11 ]. The large number of sulfate groups renders the chains anionic, which allows interaction with a wide variety of positively charged substrates. Previous studies have shown that cell surface GAGs interact with transfection reagent polymers and liposomes and inhibit cation-mediated gene transfer [12 ]. Hence, the increase in transfection efficiency and transgene expression observed after single cell suspension transfections is likely a result of a combination of the homogenous cellular accessibility for the transfection complexes in addition to a temporary removal of GAGs from the plasma membrane in response to trypsinization.
In an effort to fine-tune and reduce toxicity we optimized the use of L2K for plasmid-DNA delivery in single mES cell suspension. When optimizing transfection – whether for stem cells or other cell types – various cell-related factors such as culture health, passage numbers, and culture confluence need to be taken into consideration. However, those variables will be highly research group- and cell lab-dependent. Instead, we focused on parameters such as ratios of nucleic acid to transfection reagent and incubation time. We show that a DNA/reagent ratio of 1:2 rendered the best efficacy without significantly increasing toxicity. Also, by reducing incubation times to just 5 to 10 min we could maintain a high transfection efficacy but significantly increase cell viability. In fact, completely omitting the suspension incubation, and instead seeding the cells directly after adding the reagent and DNA-complexes, yielded only a slight decrease in transfection efficacy but a viability comparable to those obtained in adherent culture transfections (data not shown). Furthermore, similar results were obtained using R1 mES cells showing that the high efficiency and low toxicity is independent of cell line. Similar approaches have previously been reported for other cell types [13 – 15 ]. Although the focuses in those studies were on siRNA delivery, the efficiency for plasmid-DNA delivery was also tested. In concordance with our results these studies reported that a 10–15 min incubation of trypsinized cells with transfection complexes using L2K increases transfection efficiency 2–9 fold and that incubation times longer than that decrease cell viability. Consequently, this rapid transfection approach, which is both more time-efficient and gentle than the standard protocol, seems generally applicable to numerous cell types. In conclusion, the preferred method for plasmid DNA delivery of mESCs, cultured in 2i medium, is by transfecting the cells in suspension using L2K at a DNA:L2K ratio of 1:2 for 5–10 min. This gives a transfection rate of >80 % with a survival of >75 %. Similarly, the most efficient method for siRNA mediated knockdown is by transfecting cells with TransIT-X2 or L2K for 5–30 min, which mediates a > 90 % knockdown of Oct3/4. In addition, transfecting trypsinized cells effectively shortens the experimental time by one day as a result of not having to plate the cells 24 h prior to transfection as is required when transfecting adherent cells.
In the past, cationic lipid and polymer-based transfection had yet to reach the levels observed with viral transduction. With the presented method we have shown that this is no longer the case and that high-efficient transfections are achievable using non-viral methods. The optimized methods presented herein offers solutions to the current need for efficient and fast transfection in order to simplify and enhance studies of individual genes in pluripotent stem cells. Moreover, the method of transfecting trypsinized cells would be very advantageous in large scale and high-throughput transfection applications.
The following individuals and provided reagents: Dr. Birgitta Tomkinson, Uppsala University (pCMV-beta-gal), Dr. Jin-Ping Li, Uppsala University (pCAGGS-heparanase and heparanase antibody). This work was supported by the Faculty of Medicine, Uppsala University.
Compliance with Ethical Standards
Conflict of Interest
Dr. Annerén is an employee of GE Healthcare Bio-Sciences AB; the other authors do not have anything to disclose. No competing financial interests exist.
- 2.Tamm, C., Pijuan Galitó, S., & Annerén, C. (2013). A comparative study of protocols for mouse embryonic stem cell culturing. PloS One, 10, e81556.Google Scholar
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