Efficient, high-throughput transfection of human embryonic stem cells

Abstract

Introduction: Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications.

Methods: We investigated the ability of two commercially available electroporation systems, the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency.

Results: Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference.

Conclusions: Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC.

Figure 1

Transfection of H9 is efficient and the resulting cells have a high viability. (a) Comparison of the Neon and Shuttle^® Systems. Both systems can result in the efficient transfection of hESC. While the Neon system had a higher transfection efficiency, it also had increased variability between multiple transfections (36.2 ± 10.3% vs. 21.2 ± 0.8%). b) Transfected H9, phase contrast image (left panel) and GFP fluorescent image (right panel). (c) Quantification of Viability and Efficiency. FACS analysis demonstrates that after electroporation 73.5 ± 3.2% of the cells remain viable (compared to the 91.6 ± 2.7% viable in control reactions; P < 0.001) and that the transfection efficiency is 74.2 ± 1.4% (P < 0.001). (d) Viability and Transfection Efficiency in High Through-put Formats. All 96 wells of a shuttle plate were electroporated and the viability and efficiency of the first and last columns were compared. FACS analysis demonstrates that there is no change in viability between the first and last columns (61.8 ± 0.04% vs. 61.9 ± 0.03%, P > 0.05), however there is a small but significant decrease in transfection efficiency (41.0 ± 1.2% vs. 36.5 ± 1.3%, P < 0.05).

Figure 2

Transfected H9 express markers of pluripotency. (a) Fluorescence image of GFP transfected cells (green) that express Oct4 (red). (b) Fluorescence image of GFP transfected cells (green) that express SSEA4 (red). (c) FACS analysis for markers of pluripotency 24 hours after electroporation demonstrate that control and transfected cells expressed similar amounts of SSEA4 (blue bars; 74.4 ± 4.3% vs. 72.8 ± 4.4%; P > 0.5). In addition, of the GFP expressing cells 79.9 ± 4.7% also express SSEA4 (green bars).

Figure 3

Co-transfection with siRNA can suppress GFP expression. (a) Co-transfection of a siRNA that targets GFP at a 3:1 mass ratio to pMax-GFP results in reduced GFP expression (left panel). Transfection with pMax-GFP only (right panel). (b) Quantification of GFP knock-down by FACS analysis demonstrates that siRNA to vector DNA ratios of 3:1,1.5:1 and 0.75:1 result in GFP reductions of 71.75%, 31.7% and 18.0% (P < 0.01, 0.01 and 0.05, respectively).

Figure 4

Multiple hESC lines are transfected efficiently. In addition to the H9 cell line, two hESC cell lines, RNJ8 and HS306, were also efficiently transfected, with resulting percentages of GFP-expressing cells greater than 50%.

Figure 5

Stable hESC cell line expressing red fluorescent protein. RNJ9 cells transfected with EF1α-RFP/PGK-Neo were selected with 50 μg/ml G418 for two weeks after transfection and then grown for 10 passages. (a) Fluorescence images of dapi (blue), SSEA4 (green; in fixed cells SSEA4 staining appears punctuate due to the disruption of the cell membrane by paraformaldehyde, and RFP (red). (b) Fluorescence images of dapi (blue), Oct4 (green), and RFP (red).