High-efficiency, high-throughput transfection of pluripotent human embryonic stem cells

Abstract

DNA transfer into human embryonic stem cells (hESC), derived from the inner cell mass of a four day old embryo and able to differentiate into derivatives of all 3 germ layers, is limited by their general resistance to common methods used to introduce exogenous DNA.

Here we describe a transfection method using the Nucleofector 96-well Shuttle System that can be used with hESC to express plasmid-encoded protein or to inhibit protein production with siRNAs in a reliable, efficient, and high-throughput manner. Nucleofection was found to yield a large fraction of transfected cells with minimal effect on cell viability or loss of pluripotency marker.

Introduction

Human embryonic stem cells (hESC) are unique in their ability to continuously self renew, while maintaining the potential to differentiate into any cell in the adult body (Thomson et al, 1998). These characteristics give hESC the ability to be useful in many different aspects of basic and clinical research, including use as an ex vivo source for cellular transplantation, cells for screening potential new drugs and their toxicity, and as an in vitro modeling system for human development and disease. The difficulty in producing genetically modified hESC lines has hindered the development of some of the most promising applications of hESC research. Successful genetic manipulation will allow for the generation of reporter cell lines and the ability to develop differentiation strategies based on the expression of certain growth factors, as well as provide for the isolation of specific populations based on marker expression.

The most common strategy for introducing exogenous DNA into cultured cells is chemical transfection. In this process, DNA is introduced into the cells via liposomes or cationic polymers. While this process is relatively inexpensive and the vectors used have very few limitations, there are two major disadvantages. The first is that, while many different chemical transfection strategies have been attempted to genetically modify hESC, the resulting efficiencies have generally been very poor, often significantly less than 1% (reviewed in Moore et al., 2005). An additional problem is that, even when the exogenous DNA is successfully introduced into the cell, integration into the genome is poor, making isolation of stable cell lines inefficient.

The low efficiency of transfection combined with the inefficient integration of the construct into the genome, makes the average efficiency for generating stable clones in hESC around 1 in 10-5 (Eiges et al., 2001).

To date, the bulk of successful genetic modification of hESC has been done with lentiviruses (reviewed in Moore et al., 2005). Lentiviruses are advantageous in several ways, most important is their ability to integrate into the genome and produce stably expressing cell lines. Lentiviruses have been shown to transduce many cell types (including primary and non-dividing cells) at high efficiencies, including hESC (Ma et al., 2003). Unfortunately, the use of lentiviruses has two major disadvantages. First, viral constructs larger than 11 kb package poorly, leading to a lower than normal titer, which reduces the efficiency of hESC transduction, effectively limiting the size of DNA that can be transferred. The other concern is safety-since these viruses integrate randomly, they could activate oncogenes in the transduced cells, making them significantly less safe for future use in human patients.

A third strategy for obtaining genetically modified cells is the use of electroporation. In this method, DNA passes through the cell membrane after an electrical current is used to induce pore formation in the membrane. While a disadvantage of this technique is reduced cell survival after electroporation, this technique has been shown to be successful in hESC (Zwaka and Thomson, 2003; Costa et al., 2005). Here we show that the Amaxa Nucleofector 96-well Shuttle System can efficiently deliver exogenous DNA via Nucleofection and that the viability of the cells after transfection remains high.

Although, as is the case in chemical transfections, the introduced DNA is only rarely integrated into the genome, this method allows for the generation of a large number of transfectants, increasing the chance that successful genomic integration will occur.

Materials and methods

Cell culture Undifferentiated hESC (H9, K306 and RG8) were cultured in the feeder-free system described by Ludwig et al. (2006).

Briefly, cells were passaged every 7 days at a splitting density of 1:6 to 1:10, so that the resulting cultures were 80-90% confluent on the day of passage. Once the cells were loosened from the culture dish with 1 U/ml Dispase (BD Biosciences) and washed with DMEM/ F12, they were gently scraped to completely remove the colonies from the plate and break them into pieces. These pieces were transferred to hESC-qualified Matrigel (BD Biosciences) coated plates and mTeSR Medium (Stem Cell Technologies). Starting 24 hours after passaging, the medium was replenished daily.

Nucleofection Undifferentiated hESC were washed twice with PBS and removed from the culture dish by 5 minute incubation with Accutase (Stem Cell Technologies) at 37°C. After Accutase dissociation, the cells were counted and pelleted at 115xG for 3 minutes.

In order to transfect 1 well in a 96-well plate, 200,000 cells were resuspended in 20 µl Human Stem Cell (H9) Nucleofector Solution (Lonza) with the supplement added. These cells were then transferred to 1 well of the Nucleocuvette Plate and 400 ng of pmaxFP- Green Plasmid (Lonza) was added. The plate was then electroporated with program 96-CB-150 on the Nucleofector 96-well Shuttle System. After transfection, cells were resuspended in 80 µl of preequilibrated mTeSR Medium, transferred to Matrigel (BD Biosciences) coated 96-well plates and grown at 37°C.

siRNA Nucleofection Transfections were done as described above, with the exception that siRNA targeting GFP (Lonza) was added to the transfection mixture at concentrations of 200, 300 and 600 ng/µl.

FACS 24 hours post-transfection, the cells were washed 2 times with PBS and removed from the culture dish using Accutase (Stem Cell Technologies). Cells were then resuspended in 250 µl BSA/ PBS/Azide buffer as described in Moore et al. (2005). Samples that were analyzed for SSEA4 expression were blocked for 15 minutes in DMEM/F12 containing 10% FBS (Invitrogen), followed by incubation with SSEA4-PE for 30 minutes. After washing with DMEM/F12, cells were resuspended in 250 µl BSA/PBS/Azide buffer. All samples were analysed on a BD FACSCalibur (Becton Dickinson), and the CellQuest Software program was used for data acquisition and analysis. All values are reported as mean ± standard error of the mean.

Viability To determine the percentage of viable cells after transfection, the Vialight Plus Kit from Lonza was used according to the manufacturer’s protocol. Briefly, 24 hours post-transfection, cells were lysed in 50 µl of Cell Lysis Reagent. After incubation for 15 minutes at room temperature, the cell lysates were transferred to a luminometer plate and mixed with 100 µl ATP Monitoring Reagent Plus (AMR Plus). After 2 minutes of incubation at room temperature in the dark, the plate was read on a luminometer. All values are reported as mean ± standard error of the mean.

Immunofluorescence 24 hours post Nucleofection, cells in the 96-well plates were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and permeabilized and blocked in 4% normal goat serum with 0.1% Triton X-100. Primary antibodies used were Oct4 (Millipore; at 0.5 µg/ml) and SSEA4 (Millipore; at 1 µg/ml). After washing with PBS, the cells were incubated with Alexa Fluor Conjugated Secondary Antibodies (Molecular Probes) at 1: 500 and visualized by fluorescence microscopy.

Results

Efficient and high viability transfection of H9 hESC To test the efficiency of the Nucleofector 96-well Shuttle System, a single cell suspension of 200,000 H9 undifferentiated hESC were transfected with 400 ng pmaxFP-Green Plasmid using program 96-CB-150.

After 6 hours, GFP positive cells were visible by fluorescence microscopy (Figure 1A) and 24 hours post-transfection, cells were analyzed by FACS and, as seen in Figure 1B, 67.7 ± 3.0% of the cells expressed GFP. As a control, an equal number of untransfected cells were also plated and these cells had no significant GFP expression (p value <0.001). In order to determine the percentage of cells that were viable after transfection, the ViaLight Plus Kit from Lonza was used.

Figure 1: Transfection of H9 is efficient and results in a high viability. A: Transfected H9, phase contrast image (left panel) and GFP fluorescent image (right panel). B: FACS analysis demonstrates that the efficiency of transfection is 67.7 ± 3.0%. C: Quantification of cell viability shows that, after transfection, 56.7 ± 2.9% of the cells remain viable. The negative controls consist of cells that did not receive the electroporation pulse. (** - P value <0.01).

Figure 1C shows that the percentage of cells that remained viable after the transfection was 56.7 ± 2.9%, while the viability of the non-electroporated control cells was 98.7 ± 3.3%; a statistically significant difference (p value <0.001). Although transfection does reduce cell viability, the remaining viable cells are sufficient for most purposes and once transfected can easily be expanded (data not shown).

Transfected H9 hESC remain pluripotent The ability of hESC to maintain their pluripotency after transfection is key to developing a successful protocol. In order to ensure that the H9 cells remained pluripotent after transfection, immunofluorescence with Oct4 and SSEA4 (two commonly used pluripotent markers) was tested. As seen in Figure 2A, the majority of the transfected cells were SSEA4 positive (red) and Oct4 positive (purple). To quantify the percentage of hESC that remained pluripotent, the percentage of SSEA4/GFP double positive cells were analysed by FACS (Figure 2B). Non-transfected control cells were 78.7 ± 2.6% SSEA4 positive, while the transfected cells were 80.9 ± 4.3% SSEA4 positive (Figure 2B, blue bars). In addition, the FACS analysis showed that 57.6 ± 2.7% of the transfected cells were double positive for GFP and SSEA4. The majority of the transfected cells retain their pluripotency (only 7.6 ± 0.87% of the GFP positive cells are not SSEA4 positive, data not shown) and can be easily separated from the differentiated cells, allowing a pure culture of transfected, pluripotent cells to be propagated.

Figure 2: Transfected H9 express markers of pluripotency. A: Fluorescence image of GFP-transfected cells (green) that express Oct4 (purple) and SSEA4 (red). B: FACS analysis for markers of pluripotency demonstrate that control and transfected cells expressed similar amounts of SSEA4 (blue bars), while 57.6 ± 2.7% of the transfected cells also express GFP (purple bars).

Transfection of siRNA To demonstrate that the Amaxa 96-well Shuttle System was also capable of efficiently delivering siRNAs for gene knock-down, the pmaxFP-Green Plasmid was co-transfected with a siRNA construct that targeted GFP. The amount of siRNA needed to effectively knock-down GFP expression was determined by titrating various amounts of siRNA with the pmaxFP-Green Vector (siRNA mass to pmaxFP-Green mass ratios of 3:1, 1.5:1, and 0.75:1). Visual inspection by fluorescence microscopy showed that GFP expression was clearly reduced when 3 times the amount of GFP-targeting siRNA was co-transfected (Figure 3A). FACS analysis showed that an siRNA to DNA ratio of 3:1 reduced the percentage of cells expressing GFP from 56.6 ± 3.1% to 16.0 ± 0.37%, p value <0.01 (Figure 3B). A ratio of siRNA to DNA of 1.5:1 and 0.75:1 also reduced the percentage of GFP positive cells to 38.7 ± 0.3% (p value <0.01), and 46.37 ± 0.5% (p value <0.05), respectively (Figure 3B).

Figure 3: Co-transfection with siRNA results in GFP knock-down. A: Co-transfection of an siRNA that targets GFP at a 3:1 mass ratio to pmaxFP-Green results in reduced GFP expression (left panel). Transfection with pmaxFP-Green only (right panel). B: Quantification of GFP knock-down by FACS analysis demonstrates that ratios of 3:1, 1.5:1, and 0.75:1 all result in significant GFP reduction (p values <0.01, 0.01 and 0.05, respectively).

Figure 4: Multiple cell lines are efficiently transfected. In addition to the H9 cell line, two other cell lines were tested and were also efficiently transfected with the percentage of GFP expressing cells at higher than 50%.

Transfection efficiency in multiple hESC lines To ensure that the transfection efficiency seen with the H9 was not specific to that cell line, two additional hESC lines were tested. These lines were RG8, a newly derived cell line generated from a collaboration between Reprogenetics, Inc. and our facility (Moore et al., in preparation) and the HS306 hESC line derived at the Karolinska Institute in Sweden.

Figure 4 shows the percentage of cells that were GFP positive by FACS analysis 24 hours post-transfection (as described above). The RG8 and HS306 lines demonstrated 55.8 ± 4.8% and 53.6 ± 2.6% GFP expressing cells, respectively.

Summary and conclusions

The ability to easily and efficiently genetically modify hESC will provide scientists with additional tools to understand basic human development, track cells transplanted into animal models, and understand the protein signalling pathways that direct differentiation.

Although there are several methods that have been shown to be effective to generate transgenic hESC lines, each of the other methods have their own disadvantages. Here we establish that the Nucleofector 96-well Shuttle System can be used to generate genetically modified hESC lines.

The biggest advantage of this system is the high transfection efficiency that can be achieved with a small number of cells. We were able to routinely transfect multiple hESC lines at efficiencies of at least 50% and sometimes as high as 70%. Similarly, siRNA Nucleofection proved to be effective on a co-transfected plasmid.

Since the culture of hESC is time consuming and expensive, it is important to note that the number of cells necessary to achieve these high transfection efficiencies was only 200,000 and that the viability of the transfected cells was often above 55%, demonstrating the utility of the 96-well Shuttle System. Since the 96-well Shuttle System uses a 96-well format, this method may be easily scaled up to perform many Nucleofection Procedures in parallel for multiple replicates of multiple groups or for screening large numbers of plasmids or siRNAs.

Another advantage of this system is that the amount of time needed for gene expression to be detected is much shorter than other methods, typically only 4-6 hours. While we did not test for stable recombinants, it is expected that the combination of high efficiency and viability would enhance the usual random integration process.

Experiments in which hESC were transfected with pmaxFP-Green Plasmid resulted in a high percentage of green cells after just 6 hours, so selection of recombinants could begin quickly after Nucleofection.

In order for genetically modified hESC to be usable in most applications, the modified cells must retain their pluripotency. We used immunofluorescence and FACS analysis to validate the pluripotency of the modified cells with two commonly used markers, Oct4 and SSEA4. While a small decrease in pluripotency was observed, this decrease would not be enough to prevent the establishment of stable, pluripotent lines upon selection and may be expected considering the cells were dissociated into single cells before transfection, a process that reduces pluripotency (Amit et al., 2000).

Overall, we have demonstrated that Nucleofection is an efficient process for introducing nucleic acids into hESC with minimal effects on viability and without affecting pluripotency. Due to the low cell numbers required for Nucleofection with the 96-well Shuttle System and the 96-well format, this should prove to be a valuable method for studying stem cell biology.

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By Jennifer C. Moore¹, Kristin Atze², Alana Toro-Ramos¹, Chris Ricupero¹, Ronald P. Hart¹1, Rick I Cohen¹; ¹Stem Cell Research Center, Rutgers the State University of New Jersey, Room D251, 604 Allison Drive, Piscataway, NJ 08854, USA; ²Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany