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 JoVE Biology

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

1, 1, 1

1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology

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    Summary

    This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

    Date Published: 4/07/2008, Issue 14; doi: 10.3791/734

    Cite this Article

    Welstead, G. G., Brambrink, T., Jaenisch, R. Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4. J. Vis. Exp. (14), e734, doi:10.3791/734 (2008).

    Abstract

    Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

    Protocol

    Step 1:

    Thaw out 293 cells and plate for lentivirus production.

    1. Prepare 9 ml of DMEM+ medium in a 15-ml tube.
    2. Remove a vial of frozen 293 stocks from the liquid nitrogen tank and put the vial in a 37°C water bath until most (but not all) cells are thawed.
    3. Remove cells from freezing vial and place into the 15ml tube from step 1.
    4. Centrifuge at 180g for 5 min, and then discard the supernatant.
    5. Resuspend the cells with 10 ml of DMEM+ medium, and transfer to a gelatin-coated 100-mm dish. Incubate the cells in a 37°C, 5% CO2 incubator until they are 80–90% confluent.
    6. Passage of the 293 cells: wash cells with PBS, aspirate the PBS, and add 4 ml per 10-cm dish of 0.05% trypsin/0.53 mM EDTA, and incubate for 1 min at 37°C.
    7. Detach cells from dishes by tapping, resuspend with 10 ml DMEM+ medium, and transfer to a 15-ml tube. Centrifuge at 180g for 5 min, and aspirate the supernatant.
    8. Add appropriate volume of DMEM+ medium, and break up the cells into a single cell suspension by pipetting up and down several times.
    9. Count the number of cells and adjust the concentration to 8x105 cells per ml with FP medium.
    10. Seed cells at 8x106 cells (10 ml) per 100-mm culture dish, and incubate overnight at 37°C, 5% CO2.

    Step 2:

    Transfect 293 cells with lentivirus plasmids and harvest virus

    1. For each 10-cm plate, use 10 µg of FUW-tetO-cDNA (Oct4, Sox2, Klf4 or c-Myc) lentivirus backbone with 2.5ug of pMD-G and 7.5 µg of pPax2.
    2. While aliquoting the three plasmids into a tube, deliver 30µl of Fugene 6 transfection reagent into a second tube containing 500µl of DMEM without serum and mix gently by finger tapping and incubate for 5 min at room temperature.
    3. Add the DNA mix drop-by-drop into the Fugene 6/DMEM-containing tube, mix gently by finger tapping and incubate for 20 min.
    4. Add the DNA/Fugene 6 complex dropwise into the dish of 293’s, and incubate overnight at 37°C, 5% CO2.
    5. Next morning: Aspirate the transfection reagent–containing medium, add 5 ml of fresh ES cell medium, and return the cells to the incubator.
    6. At 48 hours and 72 hours after transfection, collect the medium from the 293’s by using a 10-ml sterile disposable syringe, filtering it through a 0.45- µm pore size cellulose acetate filter, and transferring into a 15-ml tube.
    7. Concentrate the virus supernatant by ultracentrifugation. Resuspend the virus pellet in desired volume and make amixture of equal parts of the medium containing Oct-3/4-, Sox2-, Klf4- and c-Myc-lentiviruses.
    8. While making the virus, prepare Oct4-neo/rtTA or Oct4-GFP/rtTA MEFs (passage <3) to 90% confluency in 10-cm dishes (2x106 cells per dish)
    9. Aspirate the culture medium and wash with 10 ml of PBS.
    10. Discard PBS, add 1 ml per dish of 0.05% trypsin/0.53 mM EDTA, and incubate at 37°C for 10 min.
    11. Add 9 ml of the culture medium, suspend the cells to a single cell, and transfer to a 15-ml tube.
    12. Count the number of cells, and adjust the concentration to 8x104 cells per ml. Transfer 10 ml of cell suspension (8x105 cells) to a 10-cm dish coated with gelatin. Incubate the dish overnight at 37°C, 5% CO2.
    13. Aspirate the medium from a fibroblast dish, and add 10 ml of the virus-containing medium. Incubate the cells from 4 h to overnight at 37°C, 5% CO2.
    14. After 24 aspirate the medium from a fibroblast dish, and add 10 ml of fresh ES cell medium.
    15. Replace regular ES cell medium with medium containing Doxycycline to initiate the expression of the four genes; in other words, initiate reprogramming.
    16. If using an Oct4-neo reporter, then initiate drug section anytime between 6 and 9 days.
    17. Change the medium every day until the colonies become big enough to be picked up. Colonies should first become visible approximately a week after the initiation of reprogramming. They should become large enough to be picked up around day 20.

    Step 3:

    Picking up the iPS colonies

    The Day before Picking:

    1. Seed the required number of gelatin-coated 24-well plates with γ-irradiated DR4 MEFs

    Picking Clones on Day 1:

    1. Feed the colonies on the plates 1-2 hours before picking.
    2. Add 15 μl of PBS to the wells of a V-bottomed 96-well dish.
    3. Wash the dish containing the colonies to be picked once with PBS and add 10ml of PBS to the dish.
    4. Pick the colonies with the small 5μl pipet tips (set the P20 pipetman on 4 μl). Transfer colonies to a well in the 96-well dish.
    5. Add 20 μl of trypsin to each well using the multi-channel (12) pipetor. Pipet up and down 3 times. Incubate at 37°C for 4 minutes.
    6. Add 100 μl of ES medium to the trypsinized colonies. Pipet up and down 10 times. Transfer 6 clones at a time from the 96-well dish to a 24-well dish using a tip at every other position on the 12 place pipetor.
    7. Grow the cells on the 24-well plate in a 37°C, 5% CO2 incubator until the cells reach 80–90% confluency, feeding daily with ES cell medium. Cells will probably be ready to expand in 3-7 days.

    Step 4

    1. Expansion of iPS cells
      1. Aspirate the medium, and wash the cells with 1 ml of PBS.
      2. Remove PBS completely, add 0.1 ml of 0.25% trypsin/1 mM EDTA and incubate at 37°C for 10 min.
      3. Add 0.4 ml of the ES medium and suspend the cells by pipetting up and down to single cell suspension.
      4. Transfer the cell suspension to a well of 6-well plate, add 1.5 ml ES cell medium, and incubate in a 37°C, 5% CO2 incubator until cells reach 80–90% confluency in 6-well plates. At this point, prepare frozen stock of the cells, as follows.
    2. Preparation of freeze stock
      1. Aspirate the medium, and wash the cells with 2 ml of PBS.
      2. Remove PBS completely, add 0.5 ml of 0.25% trypsin/1 mM EDTA and incubate at 37°C for 10 min.
      3. Add 2 ml of the ES medium and suspend the cells by pipetting up and down to single cell suspension.
      4. Transfer the cell suspension to a 15-ml tube, count the number of cells and spin down the cells.
      5. Discard the supernatant, resuspend the cells with ES medium to the concentration at 2x106 cells per ml.
      6. Prepare 2 ES cell freezing medium (20% DMSO in ES medium) and aliquot it at 0.5 ml per vial.
      7. Transfer 0.5 ml of the cell suspension to freeze vials and mix gently.
      8. Put the vials in a cell-freezing container and keep it at -80°C overnight; transfer to liquid nitrogen the next day for long-term storage.

    Discussion

    In this video, we demonstrate how to use an inducible, lentiviral system to generate GFP-positive, pluripotent iPS cells from MEFs derived from Oct4-GFP/R26-M2rtTA and Nanog-GFP/R26-M2rtTA mice. This procedure is a useful method for generating iPS cells because it allows for the control of transgene expression during the reprogramming process. Although the use of viruses in the generation of iPS cells impedes the use of these cells in a clinical setting, this procedure does enable us to answer some of the major biological questions that underline the yet defined reprogramming process.

    Disclosures

    Materials

    Name Type Company Catalog Number Comments
    Doxycycline hyclate Reagent Sigma-Aldrich D9891-100G

    References

    1. Takahashi, K., Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126, 663-676 (2006).
    2. Wernig, M., et al. et al . In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 448, 318-324 (2007).
    3. Okita, K., Ichisaka, T., Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature. 448, 313-317 (2007).
    4. Maherali, N., et al. Global epigenetic remodeling in directly reprogrammed fibroblasts. Cell Stem Cell. 1, 55-70 (2007).
    5. Brambrink, T., et al. Sequential expression of pluripotency markers during direct reprogramming of somatic cells. Cell Stem Cell. 2, 151-159 (2008).

    Comments

    18 Comments

    Did you change new irradiate MEF cell plate between cells were reprogramming?
    Reply

    Posted by: AnonymousMay 29, 2008, 3:44 PM

    Do you consider feasible to use MEF "inactivated" with mitomycin in order to use as feather??
    Reply

    Posted by: AnonymousAugust 20, 2008, 12:01 AM

    Do you consider feasible to use MEF "inactivated" with mitomycin as feeder??
    Reply

    Posted by: AnonymousAugust 20, 2008, 12:05 AM

    While I haven't done this I can't see why this wouldn't work.
    Reply

    Posted by: AnonymousNovember 20, 2008, 9:08 AM

    Hello I don't know how you deal with the cells during the time before ips colonies appear. Because during such a long time the MEFs should expand a lot. Do you passage them every few days or have other ways to deal with it. Thank you a lot!
    Reply

    Posted by: AnonymousNovember 20, 2008, 5:23 AM

    You can go either way. If you passage the cells, the MEFs will expand, but will be outcompeted by the faster growing iPS cells and tranformed cells. If you don't passage the cells, the MEFs will stop proliferating due to contact inhibition and only cells that can overcome contact inhibition (iPS and transformed cells) will be able to expand once the plate is confluent.
    Reply

    Posted by: AnonymousNovember 20, 2008, 9:14 AM

    Beautiful work, guys!Inspires me to try to reproduce it and to build upon it.is there a chance to get FUW-M²rtTA lentivectors anywhere?Good luck with what you do!Luda
    Reply

    Posted by: AnonymousDecember 10, 2008, 9:09 PM

    hello, My name is Ashutosh Dhingra. I am a graduate student of Neuroscience at University of tuebingen, germany.  I want to ask that do you know where these 4 genes for the transcription factors are integrating. Is there any interaction between these 4 genes? if yes, in what way? please reply at ashutoshdhingra@gmail.com   Thanks you very much. Regards, Ashutosh
    Reply

    Posted by: AnonymousDecember 14, 2008, 10:59 AM

    Great visual illustration! What are the lentiviral titers or MOI's for infection of each of the four genes? What kind of reprogramming efficiency do you achieve? Cheers, Dhruv
    Reply

    Posted by: AnonymousDecember 15, 2008, 12:33 PM

    Great demo. As a couple have inquired, how can I get the vectors that are required for the generation of the iPS?   Thanks. Cathy
    Reply

    Posted by: AnonymousMarch 1, 2009, 2:50 PM

    Do you use the same DNA concentrations and packaging systems for your FUW-Tet-OKSM policistronic construct?
    Reply

    Posted by: Alexander K.August 21, 2009, 10:19 AM

    Yes
    Reply

    Posted by: AnonymousJune 11, 2010, 8:43 AM

    Great. My question is: so, after picking up, these iPS cells were cultured in ES medium but without addition of Doxycycline, is it? Is that means these four genes expression is only necessary for initiation of reprogramming?
    Reply

    Posted by: AnonymousDecember 21, 2009, 12:40 AM

    Hello,
    Why dŒs everybody produce the virus using fugene/lipofectamine. Why not using the cheap calcium phosphate? Is there a difference in the virus titer or another reason?
    Thanks!
    Irene
    Reply

    Posted by: AnonymousJune 11, 2010, 8:00 AM

    You can use any method that gives you good transfection in your hands. If you are getting good transfection with calcium phosphate then by all means use it.
    Reply

    Posted by: AnonymousJune 11, 2010, 8:45 AM

    You used all these trascriptation factors. But how can you get these factors out of the ovocyte and how do you proof that they are absent ?
    Also I am really interested in your reply to Ashutosh.

    Thank you a lot !
    Cynthia
    Reply

    Posted by: AnonymousAugust 18, 2010, 4:15 PM

    why are the cells expanded as single cells (before attaching to surface) - I thought that ES cells/iPS cells should be passaged as cell clumps??? Thank you so much for answering this question - Im so confused!
    Reply

    Posted by: AnonymousOctober 22, 2010, 8:59 AM

    What is the concentration of doxycycline used during induction?
    Reply

    Posted by: AnonymousAugust 25, 2011, 8:12 PM

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