Journal of Bioscience and Bioengineering VOL. 119 No. 5, 614e616, 2015 www.elsevier.com/locate/jbiosc
TECHNICAL NOTE
Magnetically labeled feeder system for mouse pluripotent stem cell culture Masanobu Horie,x Akira Ito, Takeshi Maki, Yoshinori Kawabe, and Masamichi Kamihira* Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Received 16 August 2014; accepted 23 October 2014 Available online 20 November 2014
We report here a magnetically labeled feeder system for mouse embryonic stem/induced pluripotent stem (ES/iPS) cells. Magnetic attraction of feeder cells labeled with magnetite nanoparticles significantly increased ES/iPS colonyforming efficiency. Magnetic labeling of feeder cells also facilitated separation of ES/iPS cells from feeder cells. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pluripotent stem cells; Feeder cells; E-cadherin; Cell separation; Magnetite nanoparticles]
Pluripotent stem cells, including mouse embryonic stem (ES) cells (1,2) and induced pluripotent stem (iPS) cells (3,4), have gained attention for their potential applications in biological research. For pluripotent stem cells, a robust culture system to maintain their self-renewal and differentiation potential is crucial. The undifferentiated state of mouse ES/iPS cells is generally maintained by culture on mitotically inactivated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of anti-differentiation factors such as leukemia inhibitory factor (LIF). MEFs are primary cells derived from mouse fetuses and can be cultured for several passages before senescence. To replace MEF feeders, various culture methods have been developed for ES/iPS cells (5e7). Based on the hypothesis that close contact between feeders and ES/iPS cells may enhance the performance of feeder cells in ES/iPS cell culture, we previously demonstrated an improvement in the feeder performance of the STO mouse stromal cell line by introduction of the E-cadherin gene (8). Furthermore, STO/EL cells established by the introduction of E-cadherin and LIF genes maintain undifferentiated growth of ES/iPS cells without LIF addition (9). In the present study, we employed functional magnetite nanoparticles and a magnetic force to enhance the physical interaction between ES/iPS cells and feeder cells. In this method, feeder cells were magnetically labeled with magnetite cationic liposomes (MCLs) (10) and then seeded with ES/iPS cells, allowing attraction of the cells to the culture surface by a magnetic force. Additionally, the MCL-labeled feeder cells were magnetically removed from the co-culture system, resulting in negative isolation of the ES/iPS cells. A mouse ES cell line, 129sv (Chemicon, Pittsburgh, PA, USA), and an iPS cell line, iPS-MEF-Ng-20D-17 (4) (Riken BioResource Center, Tsukuba, Japan), were cultured on MEF feeder cells treated with mitomycin C (MMC; Wako Pure Chemical Industries, Osaka, Japan) for 2 h. The cells were cultured in ES/iPS medium consisting of Knockout Dulbecco’s modified Eagle’s medium (DMEM)
* Corresponding author. Tel.: þ81 92 802 2743; fax: þ81 92 802 2793. E-mail address: [email protected] (M. Kamihira). x Present address: Division of Biochemical Engineering, Radioisotope Research Center, Kyoto University; Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
(Invitrogen, Carlsbad, CA, USA) supplemented with 4 mM L-glutamine (Wako Pure Chemical Industries), non-essential amino acid (NEAA; Invitrogen), 100 mM 2-mercaptoethanol (Millipore, Billerica, MA, USA), 100 U/mL penicillin G potassium (Wako Pure Chemical Industries), 50 mg/mL streptomycin sulfate (Wako Pure Chemical Industries), 15% knockout serum replacement (Invitrogen), and 1000 U/mL LIF (ESGRO; Millipore). MEFs were isolated from the fetuses of BALB/c mice at embryonic day 14 and cultured in DMEM (SigmaeAldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Miami, FL, USA) and 4 mM L-glutamine. STO cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin G potassium, 50 mg/mL streptomycin sulfate, and NEAA. The cells were cultured at 37 C in a 5% CO2 incubator. MCLs were prepared from colloidal magnetite nanoparticles (Fe3O4; average particle size: 10 nm; Toda Kogyo, Hiroshima, Japan) and a liquid mixture consisting of N-(a trimethylammonioacetyl)didodecyl-D-glutamate chloride, dilauroylphosphatidylcholine, and dioleoylphosphatidyl-ethanolamine at a molar ratio of 1:2:2 as described previously (10). For magnetic labeling of feeder cells, MCLs were added to cell cultures at a net magnetite concentration of 100 pg/cell. After 24 h of incubation, the cells were washed twice with phosphate-buffered saline (PBS) and then exposed to medium containing 10 mg/mL MMC for 2 h at 37 C in a CO2 incubator. ES/iPS cells (1 105 cells/well) were seeded with the mitotically inactivated feeder cells (1 105 cells/well) in 24-well culture plates (Thermo Fisher Scientific, Waltham, MA, USA). A cylindrical neodymium magnet [diameter: 30 mm; magnetic induction: 0.4 T (11)] was then placed under the wells to apply a vertical magnetic force. The cells were cultured for 10 days. The culture medium was changed daily, and the cells were passaged every 2e3 days. For alkaline phosphatase (AP) staining, 1 104 ES/iPS cells separated from the feeder cells were reseeded on MEF feeders. After 3 days of culture, AP staining was performed as described previously (9). Briefly, the cells were washed with PBS, fixed in 0.25% glutaraldehyde for 5 min at room temperature, and then exposed to a solution containing naphthol AS-MX phosphate (Sigma) as the substrate and Fast Violet B Salt (Sigma) as the coupler for 20 min at 37 C in a CO2 incubator. AP-positive colonies were counted in five fields of
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.10.020
VOL. 119, 2015 view of three individual wells per sample. The colony-forming efficiency was determined by the following equation: colony-forming efficiency () ¼ (the number of AP-positive colonies)/(the number of initially seeded cells). In the experiments for magnetic cell separation, ES/iPS cells and MCL-labeled feeder cells were pre-stained with a red fluorescent probe, (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR, Molecular Probes, Eugene, OR, USA), and a green fluorescent probe, 5-chloromethylfluoroscein diacetate (CMFDA, Molecular Probes), respectively. After 2 days of co-culture, the cells were harvested for magnetic cell separation or pre-plating. For magnetic separation, the co-cultured cells were dispersed into single cells by digestion with trypsin, resuspended in 1 mL of medium, and transferred to sterile 1.5-mL polypropylene tubes. A magnetic force was applied to the tube for 3 min using a 0.4 T magnet, and ES/iPS cells and feeder cells in the supernatant or cell pellet where the magnet was placed were counted using a hemocytometer under a fluorescence microscope (Olympus, Tokyo, Japan). For pre-plating, the cells were plated onto gelatin-coated dishes for 30 min to allow the feeder cells to attach onto the culture surface, and ES cells and feeder cells in the supernatant were counted using a hemocytometer under a fluorescence microscope. Removal of feeder cells and recovery of ES cells were measured by the following equations: Removal of feeder cells(%) ¼ [(the number of feeder cells before separation e the number of feeder cells after separation)/(the number of feeder cells before separation)] 100 (1)
TECHNICAL NOTE Recovery of ES/iPS cells (%) ¼ [(the number of ES/iPS cells after separation)/(the number of ES/iPS cells before separation)] 100
615
(2)
All data are expressed as the mean standard deviation (SD). Statistical comparisons were performed using the ManneWhitney rank sum test. Values of P < 0.05 were considered as statistically significant. The animal experiment was performed according to the guideline of the Ethics Committee for Animal Experiment, Kyushu University. The culture scheme of ES/iPS cells using the magnetically labeled feeder system is illustrated in Fig. 1A. In this method, MCLs were used to induce uptake of magnetite nanoparticles into cells via electrostatic interactions between MCLs and the cell membrane. For magnetic labeling of feeder cells, 100 pg/cell of MCLs were added to the culture medium. The uptake of MCLs into STO cells reached a maximum at 24 h after MCL addition, and the amount was 29 pg of magnetite/cell (12). After MMC treatment of MCLlabeled feeder cells, ES/iPS cells were seeded with the feeder cells and attracted to the culture surface by applying a magnetic force. The cells were then cultured for 10 days. For quantitative evaluation of the undifferentiated state, the AP-positive colony-forming efficiency of ES/iPS cells was measured (Fig. 1B and C). Consistent with our previous study (8), STO feeders were inferior to MEF feeders in terms of colony-forming efficiency (Fig. 1B and C). Additionally, in the absence of LIF, the colony-forming efficiency was considerably low for ES/iPS cells co-cultured with STO feeders, whereas LIFoverexpressing STO cells [STO/L cells (9)] increased the colony-
FIG. 1. Magnetically labeled feeder system to maintain undifferentiated growth of ES/iPS cells. (A) Schematic diagram of culture and isolation of ES/iPS cells using magnetic force. (B, C) ES (B) or iPS (C) cells were co-cultured with MCL-labeled feeder cells in the absence (open columns) or presence (closed columns) of a magnet. The experiments were performed in triplicate, and the data are presented as the mean SD. *P < 0.05.
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HORIE ET AL.
J. BIOSCI. BIOENG., plating to remove feeder cells (termed as pre-plating), which is based on the differential adhesion properties of ES/iPS cells and feeder cells on a tissue culture surface. In this study, three cycles of plating and re-plating were required to achieve 90% removal of feeder cells from co-cultures with ES cells (Fig. 2A). The pre-plating process is accompanied by a reduction in the recovery yield of ES/ iPS cells with each pre-plating cycle. As shown in Fig. 2B, the recovery yield of ES cells after three cycles of pre-plating was approximately 20%. Conversely, a single magnetic separation achieved more than 90% removal of feeder cells (Fig. 2A) with approximately 80% recovery of ES/iPS cells (Fig. 2B). These data indicate that the magnetic separation of MCL-labeled feeder cells represents a single-step, high-yield method. In conclusion, we demonstrated that MCL-labeled feeder cells can be used for efficient maintenance of ES/iPS cells and as an efficient tool to facilitate their purification. It is worth noting that the magnetic attraction culture using STO/EL cells without LIF addition exhibited high performance comparable to that of MEF feeders with LIF addition. This result suggests that the magnetic feeder system using the STO/EL cell line can replace the MEF feeder system that involves laborious and time-consuming processes requiring the sacrifice of mice. We believe that the magnetic feeder system provides a contribution to medical and biological research using mouse ES/iPS cells. References
FIG. 2. Magnetic separation of ES/iPS cells from feeder cells. (A) Percentage removal of feeder cells from co-cultures with ES/iPS cells by pre-plating separation (numbers indicate each cycle using ES cells) or magnetic separation. Data represent the mean SD of three experiments. (B) Recovery yield of ES/iPS cells separated from feeder cells by pre-plating separation (numbers indicate each cycle using ES cells) or magnetic separation. Data represent the mean SD of three experiments.
forming efficiency and STO/EL cells further increased the colonyforming efficiency (9). Using magnetic attraction, the colonyforming efficiency was significantly increased under each co-culture condition (P < 0.05). For STO/EL feeders with magnetic attraction, we achieved a high colony-forming efficiency comparable to that of MEF feeders with LIF addition. These results suggest that the magnetic attraction of MCL-labeled feeder cells effectively supported the undifferentiated state of ES/iPS cells. While the magnetic attraction may be quite useful for ES/iPS culture, the mechanism remains yet to be elucidated. Currently, effects of the cellecell interaction between ES/iPS cells and feeder cells under magnetic force are being investigated. In this method, the MCL-labeled feeder cells were magnetically removed from the co-cultures (Fig. 1A), resulting in negative isolation of the ES/iPS cells. Generally, ES/iPS cells are enriched by
1. Evans, M. J. and Kaufman, M. H.: Establishment in culture of pluripotential cells from mouse embryos, Nature, 292, 154e156 (1981). 2. Martin, G. R.: Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, Proc. Natl. Acad. Sci. USA, 78, 7634e7638 (1981). 3. Takahashi, K. and Yamanaka, S.: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell, 126, 663e676 (2006). 4. Okita, K., Ichisaka, T., and Yamanaka, S.: Generation of germline-competent induced pluripotent stem cells, Nature, 448, 313e317 (2007). 5. Shi, Y. T., Huang, Y. Z., Tang, F., and Chu, J. X.: Mouse embryonic stem cellderived feeder cells support the growth of their own mouse embryonic stem cells, Cell Biol. Int., 30, 1041e1047 (2006). 6. Takahashi, K., Narita, M., Yokura, M., Ichisaka, T., and Yamanaka, S.: Human induced pluripotent stem cells on autologous feeders, PLoS One, 4, e8067 (2009). 7. Zou, C., Chou, B. K., Dowey, S. N., Tsang, K., Huang, X., Liu, C. F., Smith, C., Yen, J., Mali, P., Zhang, Y. A., Cheng, L., and Ye, Z.: Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines, Stem Cells Dev., 21, 2298e2311 (2012). 8. Horie, M., Ito, A., Kiyohara, T., Kawabe, Y., and Kamihira, M.: E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells, J. Biosci. Bioeng., 110, 582e587 (2010). 9. Horie, M., Ito, A., Kawabe, Y., and Kamihira, M.: A genetically engineered STO feeder system expressing E-cadherin and leukemia inhibitory factor for mouse pluripotent stem cell culture, J. Bioprocess Biotech., S3, 001 (2013). 10. Shinkai, M., Yanase, M., Honda, H., Wakabayashi, T., Yoshida, J., and Kobayashi, T.: Intracellular hyperthermia for cancer using magnetite cationic liposomes: in vitro study, Jpn. J. Cancer Res., 87, 1179e1183 (1996). 11. Ito, A., Hayashida, M., Honda, H., Hata, K., Kagami, H., Ueda, M., and Kobayashi, T.: Construction and harvest of multilayered keratinocyte sheets using magnetite nanoparticles and magnetic force, Tissue Eng., 10, 873e880 (2004). 12. Ito, A., Jitsunobu, H., Kawabe, Y., Ijima, H., and Kamihira, M.: Magnetic separation of cells in coculture systems using magnetite cationic liposomes, Tissue Eng. Part C Methods, 15, 413e423 (2009).
TECHNICAL NOTE
Magnetically labeled feeder system for mouse pluripotent stem cell culture Masanobu Horie,x Akira Ito, Takeshi Maki, Yoshinori Kawabe, and Masamichi Kamihira* Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Received 16 August 2014; accepted 23 October 2014 Available online 20 November 2014
We report here a magnetically labeled feeder system for mouse embryonic stem/induced pluripotent stem (ES/iPS) cells. Magnetic attraction of feeder cells labeled with magnetite nanoparticles significantly increased ES/iPS colonyforming efficiency. Magnetic labeling of feeder cells also facilitated separation of ES/iPS cells from feeder cells. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pluripotent stem cells; Feeder cells; E-cadherin; Cell separation; Magnetite nanoparticles]
Pluripotent stem cells, including mouse embryonic stem (ES) cells (1,2) and induced pluripotent stem (iPS) cells (3,4), have gained attention for their potential applications in biological research. For pluripotent stem cells, a robust culture system to maintain their self-renewal and differentiation potential is crucial. The undifferentiated state of mouse ES/iPS cells is generally maintained by culture on mitotically inactivated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of anti-differentiation factors such as leukemia inhibitory factor (LIF). MEFs are primary cells derived from mouse fetuses and can be cultured for several passages before senescence. To replace MEF feeders, various culture methods have been developed for ES/iPS cells (5e7). Based on the hypothesis that close contact between feeders and ES/iPS cells may enhance the performance of feeder cells in ES/iPS cell culture, we previously demonstrated an improvement in the feeder performance of the STO mouse stromal cell line by introduction of the E-cadherin gene (8). Furthermore, STO/EL cells established by the introduction of E-cadherin and LIF genes maintain undifferentiated growth of ES/iPS cells without LIF addition (9). In the present study, we employed functional magnetite nanoparticles and a magnetic force to enhance the physical interaction between ES/iPS cells and feeder cells. In this method, feeder cells were magnetically labeled with magnetite cationic liposomes (MCLs) (10) and then seeded with ES/iPS cells, allowing attraction of the cells to the culture surface by a magnetic force. Additionally, the MCL-labeled feeder cells were magnetically removed from the co-culture system, resulting in negative isolation of the ES/iPS cells. A mouse ES cell line, 129sv (Chemicon, Pittsburgh, PA, USA), and an iPS cell line, iPS-MEF-Ng-20D-17 (4) (Riken BioResource Center, Tsukuba, Japan), were cultured on MEF feeder cells treated with mitomycin C (MMC; Wako Pure Chemical Industries, Osaka, Japan) for 2 h. The cells were cultured in ES/iPS medium consisting of Knockout Dulbecco’s modified Eagle’s medium (DMEM)
* Corresponding author. Tel.: þ81 92 802 2743; fax: þ81 92 802 2793. E-mail address: [email protected] (M. Kamihira). x Present address: Division of Biochemical Engineering, Radioisotope Research Center, Kyoto University; Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
(Invitrogen, Carlsbad, CA, USA) supplemented with 4 mM L-glutamine (Wako Pure Chemical Industries), non-essential amino acid (NEAA; Invitrogen), 100 mM 2-mercaptoethanol (Millipore, Billerica, MA, USA), 100 U/mL penicillin G potassium (Wako Pure Chemical Industries), 50 mg/mL streptomycin sulfate (Wako Pure Chemical Industries), 15% knockout serum replacement (Invitrogen), and 1000 U/mL LIF (ESGRO; Millipore). MEFs were isolated from the fetuses of BALB/c mice at embryonic day 14 and cultured in DMEM (SigmaeAldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Miami, FL, USA) and 4 mM L-glutamine. STO cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin G potassium, 50 mg/mL streptomycin sulfate, and NEAA. The cells were cultured at 37 C in a 5% CO2 incubator. MCLs were prepared from colloidal magnetite nanoparticles (Fe3O4; average particle size: 10 nm; Toda Kogyo, Hiroshima, Japan) and a liquid mixture consisting of N-(a trimethylammonioacetyl)didodecyl-D-glutamate chloride, dilauroylphosphatidylcholine, and dioleoylphosphatidyl-ethanolamine at a molar ratio of 1:2:2 as described previously (10). For magnetic labeling of feeder cells, MCLs were added to cell cultures at a net magnetite concentration of 100 pg/cell. After 24 h of incubation, the cells were washed twice with phosphate-buffered saline (PBS) and then exposed to medium containing 10 mg/mL MMC for 2 h at 37 C in a CO2 incubator. ES/iPS cells (1 105 cells/well) were seeded with the mitotically inactivated feeder cells (1 105 cells/well) in 24-well culture plates (Thermo Fisher Scientific, Waltham, MA, USA). A cylindrical neodymium magnet [diameter: 30 mm; magnetic induction: 0.4 T (11)] was then placed under the wells to apply a vertical magnetic force. The cells were cultured for 10 days. The culture medium was changed daily, and the cells were passaged every 2e3 days. For alkaline phosphatase (AP) staining, 1 104 ES/iPS cells separated from the feeder cells were reseeded on MEF feeders. After 3 days of culture, AP staining was performed as described previously (9). Briefly, the cells were washed with PBS, fixed in 0.25% glutaraldehyde for 5 min at room temperature, and then exposed to a solution containing naphthol AS-MX phosphate (Sigma) as the substrate and Fast Violet B Salt (Sigma) as the coupler for 20 min at 37 C in a CO2 incubator. AP-positive colonies were counted in five fields of
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.10.020
VOL. 119, 2015 view of three individual wells per sample. The colony-forming efficiency was determined by the following equation: colony-forming efficiency () ¼ (the number of AP-positive colonies)/(the number of initially seeded cells). In the experiments for magnetic cell separation, ES/iPS cells and MCL-labeled feeder cells were pre-stained with a red fluorescent probe, (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR, Molecular Probes, Eugene, OR, USA), and a green fluorescent probe, 5-chloromethylfluoroscein diacetate (CMFDA, Molecular Probes), respectively. After 2 days of co-culture, the cells were harvested for magnetic cell separation or pre-plating. For magnetic separation, the co-cultured cells were dispersed into single cells by digestion with trypsin, resuspended in 1 mL of medium, and transferred to sterile 1.5-mL polypropylene tubes. A magnetic force was applied to the tube for 3 min using a 0.4 T magnet, and ES/iPS cells and feeder cells in the supernatant or cell pellet where the magnet was placed were counted using a hemocytometer under a fluorescence microscope (Olympus, Tokyo, Japan). For pre-plating, the cells were plated onto gelatin-coated dishes for 30 min to allow the feeder cells to attach onto the culture surface, and ES cells and feeder cells in the supernatant were counted using a hemocytometer under a fluorescence microscope. Removal of feeder cells and recovery of ES cells were measured by the following equations: Removal of feeder cells(%) ¼ [(the number of feeder cells before separation e the number of feeder cells after separation)/(the number of feeder cells before separation)] 100 (1)
TECHNICAL NOTE Recovery of ES/iPS cells (%) ¼ [(the number of ES/iPS cells after separation)/(the number of ES/iPS cells before separation)] 100
615
(2)
All data are expressed as the mean standard deviation (SD). Statistical comparisons were performed using the ManneWhitney rank sum test. Values of P < 0.05 were considered as statistically significant. The animal experiment was performed according to the guideline of the Ethics Committee for Animal Experiment, Kyushu University. The culture scheme of ES/iPS cells using the magnetically labeled feeder system is illustrated in Fig. 1A. In this method, MCLs were used to induce uptake of magnetite nanoparticles into cells via electrostatic interactions between MCLs and the cell membrane. For magnetic labeling of feeder cells, 100 pg/cell of MCLs were added to the culture medium. The uptake of MCLs into STO cells reached a maximum at 24 h after MCL addition, and the amount was 29 pg of magnetite/cell (12). After MMC treatment of MCLlabeled feeder cells, ES/iPS cells were seeded with the feeder cells and attracted to the culture surface by applying a magnetic force. The cells were then cultured for 10 days. For quantitative evaluation of the undifferentiated state, the AP-positive colony-forming efficiency of ES/iPS cells was measured (Fig. 1B and C). Consistent with our previous study (8), STO feeders were inferior to MEF feeders in terms of colony-forming efficiency (Fig. 1B and C). Additionally, in the absence of LIF, the colony-forming efficiency was considerably low for ES/iPS cells co-cultured with STO feeders, whereas LIFoverexpressing STO cells [STO/L cells (9)] increased the colony-
FIG. 1. Magnetically labeled feeder system to maintain undifferentiated growth of ES/iPS cells. (A) Schematic diagram of culture and isolation of ES/iPS cells using magnetic force. (B, C) ES (B) or iPS (C) cells were co-cultured with MCL-labeled feeder cells in the absence (open columns) or presence (closed columns) of a magnet. The experiments were performed in triplicate, and the data are presented as the mean SD. *P < 0.05.
616
HORIE ET AL.
J. BIOSCI. BIOENG., plating to remove feeder cells (termed as pre-plating), which is based on the differential adhesion properties of ES/iPS cells and feeder cells on a tissue culture surface. In this study, three cycles of plating and re-plating were required to achieve 90% removal of feeder cells from co-cultures with ES cells (Fig. 2A). The pre-plating process is accompanied by a reduction in the recovery yield of ES/ iPS cells with each pre-plating cycle. As shown in Fig. 2B, the recovery yield of ES cells after three cycles of pre-plating was approximately 20%. Conversely, a single magnetic separation achieved more than 90% removal of feeder cells (Fig. 2A) with approximately 80% recovery of ES/iPS cells (Fig. 2B). These data indicate that the magnetic separation of MCL-labeled feeder cells represents a single-step, high-yield method. In conclusion, we demonstrated that MCL-labeled feeder cells can be used for efficient maintenance of ES/iPS cells and as an efficient tool to facilitate their purification. It is worth noting that the magnetic attraction culture using STO/EL cells without LIF addition exhibited high performance comparable to that of MEF feeders with LIF addition. This result suggests that the magnetic feeder system using the STO/EL cell line can replace the MEF feeder system that involves laborious and time-consuming processes requiring the sacrifice of mice. We believe that the magnetic feeder system provides a contribution to medical and biological research using mouse ES/iPS cells. References
FIG. 2. Magnetic separation of ES/iPS cells from feeder cells. (A) Percentage removal of feeder cells from co-cultures with ES/iPS cells by pre-plating separation (numbers indicate each cycle using ES cells) or magnetic separation. Data represent the mean SD of three experiments. (B) Recovery yield of ES/iPS cells separated from feeder cells by pre-plating separation (numbers indicate each cycle using ES cells) or magnetic separation. Data represent the mean SD of three experiments.
forming efficiency and STO/EL cells further increased the colonyforming efficiency (9). Using magnetic attraction, the colonyforming efficiency was significantly increased under each co-culture condition (P < 0.05). For STO/EL feeders with magnetic attraction, we achieved a high colony-forming efficiency comparable to that of MEF feeders with LIF addition. These results suggest that the magnetic attraction of MCL-labeled feeder cells effectively supported the undifferentiated state of ES/iPS cells. While the magnetic attraction may be quite useful for ES/iPS culture, the mechanism remains yet to be elucidated. Currently, effects of the cellecell interaction between ES/iPS cells and feeder cells under magnetic force are being investigated. In this method, the MCL-labeled feeder cells were magnetically removed from the co-cultures (Fig. 1A), resulting in negative isolation of the ES/iPS cells. Generally, ES/iPS cells are enriched by
1. Evans, M. J. and Kaufman, M. H.: Establishment in culture of pluripotential cells from mouse embryos, Nature, 292, 154e156 (1981). 2. Martin, G. R.: Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, Proc. Natl. Acad. Sci. USA, 78, 7634e7638 (1981). 3. Takahashi, K. and Yamanaka, S.: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell, 126, 663e676 (2006). 4. Okita, K., Ichisaka, T., and Yamanaka, S.: Generation of germline-competent induced pluripotent stem cells, Nature, 448, 313e317 (2007). 5. Shi, Y. T., Huang, Y. Z., Tang, F., and Chu, J. X.: Mouse embryonic stem cellderived feeder cells support the growth of their own mouse embryonic stem cells, Cell Biol. Int., 30, 1041e1047 (2006). 6. Takahashi, K., Narita, M., Yokura, M., Ichisaka, T., and Yamanaka, S.: Human induced pluripotent stem cells on autologous feeders, PLoS One, 4, e8067 (2009). 7. Zou, C., Chou, B. K., Dowey, S. N., Tsang, K., Huang, X., Liu, C. F., Smith, C., Yen, J., Mali, P., Zhang, Y. A., Cheng, L., and Ye, Z.: Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines, Stem Cells Dev., 21, 2298e2311 (2012). 8. Horie, M., Ito, A., Kiyohara, T., Kawabe, Y., and Kamihira, M.: E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells, J. Biosci. Bioeng., 110, 582e587 (2010). 9. Horie, M., Ito, A., Kawabe, Y., and Kamihira, M.: A genetically engineered STO feeder system expressing E-cadherin and leukemia inhibitory factor for mouse pluripotent stem cell culture, J. Bioprocess Biotech., S3, 001 (2013). 10. Shinkai, M., Yanase, M., Honda, H., Wakabayashi, T., Yoshida, J., and Kobayashi, T.: Intracellular hyperthermia for cancer using magnetite cationic liposomes: in vitro study, Jpn. J. Cancer Res., 87, 1179e1183 (1996). 11. Ito, A., Hayashida, M., Honda, H., Hata, K., Kagami, H., Ueda, M., and Kobayashi, T.: Construction and harvest of multilayered keratinocyte sheets using magnetite nanoparticles and magnetic force, Tissue Eng., 10, 873e880 (2004). 12. Ito, A., Jitsunobu, H., Kawabe, Y., Ijima, H., and Kamihira, M.: Magnetic separation of cells in coculture systems using magnetite cationic liposomes, Tissue Eng. Part C Methods, 15, 413e423 (2009).
