APPLICATION NOTE Page 1 Transfection of Mouse ES Cells and Mouse ips cells using the Stemfect 2.0 -mesc Transfection Reagent Authors: Amelia L. Cianci 1, Xun Cheng 1 and Kerry P. Mahon 1,2 1 Stemgent Inc., 10575 Roselle Street, San Diego, CA 92121 2 Correspondence: kerry.mahon@stemgent.com
APPLICATION NOTE Page 2 SUMMARY The Stemfect 2.0-mESC Transfection Reagent was used to transfect KH2 and R1 mouse embryonic stem (ES) cells as well as WP5 mouse induced pluripotent stem (ips) cells with a DNA plasmid encoding green fluorescent protein (GFP). Both control and transfected mouse ES and mouse ips cells were cultured on a feeder layer of mouse embryonic fibroblasts (MEFs). Transfected mouse ES and mouse ips cells retained healthy cell morphologies and maintained pluripotency, as determined by staining with the SSEA-1 antibody. Transfection efficiencies were 30±3%, 54±1%, and 38±2% for R1, KH2, and WP5 cells, respectively. When compared to leading commercially available transfection reagents, Stemfect 2.0 was 5 to 10 fold more efficient in transfecting both mouse ES and mouse ips cells. These data demonstrate the value of the Stemfect 2.0 for high efficiency transfection of mouse ES and mouse ips cells. INTRODUCTION Mouse development closely parallels human development but is more easily manipulated to create genetic variations for studying biological pathways and disease. As a result, the mouse model is an integral component in many fields of research. In particular, stem cell research has utilized mouse embryonic stem (mes) cells to study mechanisms of development, generate knockout and knockin mice, and investigate various differentiation pathways. Clearly, the ability to introduce genetic material into these cells is a valuable research tool. Induced pluripotent stem (ips) cells resemble embryonic stem (ES) cells genetically as well as phenotypically, but are derived from mature cell types that have been reprogrammed to an undifferentiated state through the introduction of exogenous transcription factors. This discovery was first reported using mouse embryonic fibroblasts (MEFs), but the technique has since been applied to human cells 1,2. ips cells can be utilized in the same ways as ES cells, with the added benefit of allowing the researchers to derive pluripotent cells from diseased cells 3. A recent report describing the use of ips cells to cure sickle cell anemia in mice highlights their enormous potential 4. One of the severe limitations to genetically modifying ES and ips cells is the lack of efficient and non-toxic delivery methods for DNA. Several nonviral gene delivery methods for mes cells such as electroporation, nucleofection, and liposomemediated delivery have been developed as alternatives to viral vector-based gene transfer. Electroporation not only requires large numbers of cells and DNA, but subjects the cells to extremely harsh experimental conditions, leading to low survival rates 5. Nucleofection has been found to achieve higher transfection efficiencies compared to electroporation, but it requires specialized equipment that can be cost-prohibitive and still induces high levels of apoptosis 6. A number of differently formulated lipid-based transfection reagents are available that have been used successfully in many cultured mammalian cell lines. Here we describe a polymer transfection reagent, (Cat. No. 00-0010), that is highly efficient in mes and mouse ips (mips) cells grown under typical cell culture conditions. Using a DNA plasmid encoding green fluorescent protein (GFP), we tested transfection efficiency in multiple mes and mips cells derived from MEFs. Each cell line transfected under these conditions achieved 5 to 10 fold greater number of GFP expressing cells when compared to leading reagents, while retaining pluripotency and maintaining high viability. RESULTS Transfection of mes Cells The transfection efficiency of the Stemfect 2.0-mESC Transfection Reagent in mes cells was tested on two cell lines, KH2 and R1, and compared with two leading commercially available transfection reagents ( Reagent L and Reagent F ) used according to the manufacturers instructions. Following transfection and overnight incubation, the cells were stained with the SSEA-1 antibody, a fluorescently labeled antibody directed against a marker for pluripotency in mes cells. For each transfection, this antibody bound the same percentage of cells observed in the
APPLICATION NOTE Page 3 control sample of untransfected cells, demonstrating that exposure to Stemfect 2.0 did not induce mes cell differentiation and did not impede mes cell proliferation. Transfection efficiency was calculated as the percent of cells that were positive for GFP expression in the SSEA-1 positive population (Figure 1). For KH2 mes cells, Stemfect 2.0 was highly efficient, achieving levels of 54±1%, while Reagent L and Reagent F were limited to 7.6±1 and 9.6±2%, respectively. For R1 mes cells, Stemfect 2.0 remained highly efficient, achieving 30±3% transfection efficiency compared to 1.7±0.1% for Reagent L and 2.0±0.2% for Reagent F. Stemfect 2.0 also resulted in the most viable cells after transfection (Figure 2). There was no significant difference in cell viability between the untransfected control sample and the sample transfected with Stemfect 2.0, while other leading transfection reagents were slightly lower. Figure 1. Comparison of transfection efficiency of three transfection reagents. Three transfection reagents (Stemfect 2.0, Reagent L, and Reagent F) were used to transfect three cell lines (KH2, R1, and WP5) under standard cell culture conditions. Cells were transfected with a plasmid encoding (GFP) and were stained with an SSEA-1 antibody. The transfection efficiency was calculated 24 hours post-transfection by dividing the number of cells positive for both GFP and SSEA-1 by the total SSEA-1 positive population.
APPLICATION NOTE Page 4 Figure 2. Comparison of cell viability using three transfection reagents. Cell viability was tested on KH2 and WP5 cell lines that were previously transfected using three transfection reagents (Stemfect 2.0, Reagent L, and Reagent F). Cells were transfected with a DNA plasmid encoding rtta and viability was assessed 24 hours post-transfection using the Guava ViaCount flow cytometry assay. Viability values are reported as relative to an untransfected control set of cells. Pluripotency Analysis of Transfected mes and mips Cells Cells transfected with a GFP plasmid were fixed with paraformaldehyde 24 hours post-transfection. The fixed cells were stained for SSEA-1 and imaged using a fluorescent microscope (Figure 3 and Figure 4). For both mes and mips cells, images confirm that colonies retained good morphology and pluripotency after transfection. Cells throughout the colony expressed GFP. These cells, along with the remainder of the colony, were positive for SSEA-1. This profile was seen in each colony throughout the well, indicating that the pluripotency of these cells was not compromised during the course of the experiment. Figure 3. Pluripotency analysis of KH2 mes cells transfected using Stemfect 2.0. KH2 cells were transfected with a GFP reporter plasmid and stained for the presence of the pluripotency marker SSEA-1 twenty-four hours post-transfection. A) brightfield image, B) GFP expression, C) SSEA-1 staining, D) merged GFP and SSEA-1 stained images.
APPLICATION NOTE Page 5 Figure 4. Pluripotency analysis of WP5 mips cells transfected using Stemfect 2.0. WP5 cells were transfected with a GFP reporter plasmid and stained for the presence of the pluripotency marker SSEA-1 twenty-four hours post-transfection. A) brightfield image, B) GFP expression, C) SSEA-1 staining, D) merged GFP and SSEA-1 stained images. Conclusions The efficiently transfects mes and mips and outperforms the leading commercially available transfection reagents by 5 to 10-fold. This reagent did not adversely affect cellular morphology or pluripotency EXPERIMENTAL PROCEDURES Preparation of Cells for Transfection MEFs were seeded at 5 x 10 4 cells per well on a 24- well gelatin coated plate in MEF Medium [500 ml high glucose DMEM supplemented with 50 ml FBS, 6 ml penicillin (10,000 U/ml)-streptomycin (10,000 g/ml), and 6 ml GlutaMAX -1]. The next day, the MEF Medium was aspirated and the cells were washed with 500 µl PBS. After aspirating the PBS, mes cells were plated at a density of 3 x 10 4 cells per well in mes-gm Medium [425 ml DMEM supplemented with 75 ml FBS (heat inactivated), 5 ml non-essential amino acids, 5 ml penicillin (10,000 U/ml)-streptomycin (10,000 g/ml), 5 ml GlutaMAX- 1, 500 µl 50 mm β-mercaptoethanol, and 250 µl LIF]. Cells were monitored and medium was changed as needed. The culture was observed to assess health and optimal density for transfection. The colony size generally ranged from 45 to 130 µm in diameter at the time of transfection. Transfection Procedure Cells were transfected following the protocol for when using a reporter plasmid, indicating that cells are healthy and retain their ES cell-like characteristics. Stemfect 2.0 has demonstrated utility on multiple mouse pluripotent cell lines and is currently the most effective reagent for transfecting mouse ES and ips cells. Stemfect 2.0 (reference Protocol: Stemfect 2.0-mES DNA Transfection Reagent online at www.stemgent.com/support/protocols). To transfect MEFs plated in one well of a 24-well plate, 3 µg of DNA and 0.9 µl of Stemfect 2.0 were each dissolved in 25 µl of Stemfect Buffer in two microcentrifuge tubes. The solutions were combined, vortexed, and incubated for 10 minutes. After incubation, 700 µl of mes-gm Medium was added. The medium was aspirated from the MEFs and 750 µl of the transfection medium was added to the well. Cells were incubated 4 hours, then medium was replaced with fresh mes-gm Medium. The plate was then incubated overnight. Flow Cytometry Flow Cytometry was performed using standard procedures (reference Protocol: Flow Cytometry online at www.stemgent.com/support/protocols). Brifely, cells were isolated and dissociated into a single cell suspension, then collected in a 15 ml conical tube. The cell suspension was centrifuged at
APPLICATION NOTE Page 6 300 x g for 5 minutes and the cell pellet was resuspended in PBS. After a second centrifugation the cell pellet was re-suspended in the leftover volume. 20 μl of the SSEA-1 antibody was added to the resuspended cells and incubated at 4 C for 60 minutes. The cells were then washed by adding PBS to bring the cell suspension to 4 ml and centrifuging at 300 x g for 5 minutes at 4 C. The cell pellet was then aspirated and agitated. The appropriate volume of PBS was added and the cells were analyzed by flow cytometry within 4 hours. ICC: Pluripotency Analysis ICC was performed using standard procedures (reference Protocol: Immunocytochemistry online at www.stemgent.com/support/protocols). The fixing solution used was 4% paraformaldehyde in PBS. The Anti-SSEA-1 antibody was diluted 1:100. Cell Viability Assay Assay was conducted according to the protocol included with the Guava ViaCount Reagent kit. REFERENCES 1. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861-872. 2. Takahashi, K. and Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676. 3. Park, I.H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M.W., Cowan, C., Hochedlinger, K., and Daley, G.Q. (2008) Diseasespecific induced pluripotent stem cells. Cell 134: 877-886. 4. Hanna, J., Wernig, M., Markoulaki, S., Sun, C.W., Meissner, A., Cassady, J.P., Beard, C., Brambrink, T., Wu, L.C., Townes, T.M., and Jaenisch, R. (2007) Treatment of sickle cell anemia mouse model with ips cells generated from autologous skin. Science 318: 1920-1923. 5. Tompers, D.M. and Labosky, P.A. (2004) Electroporation of murine embryonic stem cells: a step-by-step guide. Stem Cells 22: 243-249. 6. Lakshmipathy, U., Pelacho, B., Sudo, K., Linehan, J.L., Coucouvanis, E., Kaufman, D.S., and Verfaillie, C.M. (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells 22: 531-543. GlutaMAX is a trademark of Invitrogen Corporation Guava ViaCount is a trademark of Guava Technologies, Inc.