Name: generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4
Uploaded: 2008-04-07T10:00:00
Duration: 13 min 2 s
Description: Watch this Scientific Journal Video about Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 at JoVE.com

Step 1:
Thaw out 293 cells and plate for lentivirus production.
<ol>
<li>Prepare 9 ml of DMEM+ medium in a 15-ml tube.</li>
<li>Remove a vial of frozen 293 stocks from the liquid nitrogen tank and put the vial in a 37&deg;C water bath until most (but not all) cells are thawed.</li>
<li>Remove cells from freezing vial and place into the 15ml tube from step 1.</li>
<li>Centrifuge at 180g for 5 min, and then discard the supernatant.</li>
<li>Resuspend the cells with 10 ml of DMEM+ medium, and transfer to a gelatin-coated 100-mm dish. Incubate the cells in a 37&deg;C, 5% CO2 incubator until they are 80&ndash;90% confluent.</li>
<li>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&deg;C. </li>
<li>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.</li>
<li>Add appropriate volume of DMEM+ medium, and break up the cells into a single cell suspension by pipetting up and down several times. </li>
<li>Count the number of cells and adjust the concentration to 8x105 cells per ml with FP medium.</li>
<li>Seed cells at 8x106 cells (10 ml) per 100-mm culture dish, and incubate overnight at 37&deg;C, 5% CO2.</li>
</ol>
Step 2:
Transfect 293 cells with lentivirus plasmids and harvest virus
<ol>
<li>For each 10-cm plate, use 10 &micro;g of FUW-tetO-cDNA (Oct4, Sox2, Klf4 or c-Myc) lentivirus backbone with 2.5ug of pMD-G and 7.5 &micro;g of pPax2.</li>
<li>While aliquoting the three plasmids into a tube, deliver 30&micro;l of Fugene 6 transfection reagent into a second tube containing 500&micro;l of DMEM without serum and mix gently by finger tapping and incubate for 5 min at room temperature.</li>
<li>Add the DNA mix drop-by-drop into the Fugene 6/DMEM-containing tube, mix gently by finger tapping and incubate for 20 min.</li>
<li>Add the DNA/Fugene 6 complex dropwise into the dish of 293&rsquo;s, and incubate overnight at 37&deg;C, 5% CO2.</li>
<li>Next morning: Aspirate the transfection reagent&ndash;containing medium, add 5 ml of fresh ES cell medium, and return the cells to the incubator.</li>
<li>At 48 hours and 72 hours after transfection, collect the medium from the 293&rsquo;s by using a 10-ml sterile disposable syringe, filtering it through a 0.45- &micro;m pore size cellulose acetate filter, and transferring into a 15-ml tube.</li>
<li>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.</li>
<li>While making the virus, prepare Oct4-neo/rtTA or Oct4-GFP/rtTA MEFs (passage &lt;3) to 90% confluency in 10-cm dishes (2x106 cells per dish)</li>
<li>Aspirate the culture medium and wash with 10 ml of PBS.</li>
<li>Discard PBS, add 1 ml per dish of 0.05% trypsin/0.53 mM EDTA, and incubate at 37&deg;C for 10 min.</li>
<li>Add 9 ml of the culture medium, suspend the cells to a single cell, and transfer to a 15-ml tube.</li>
<li>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&deg;C, 5% CO2.</li>
<li>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&deg;C, 5% CO2.</li>
<li>After 24 aspirate the medium from a fibroblast dish, and add 10 ml of fresh ES cell medium. </li>
<li>Replace regular ES cell medium with medium containing Doxycycline to initiate the expression of the four genes; in other words, initiate reprogramming.</li>
<li>If using an Oct4-neo reporter, then initiate drug section anytime between 6 and 9 days. </li>
<li>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.</li>
</ol>
Step 3:
Picking up the iPS colonies
The Day before Picking:
<ol>
<li>Seed the required number of gelatin-coated 24-well plates with &gamma;-irradiated DR4 MEFs</li>
</ol>
Picking Clones on Day 1:
<ol>
<li>Feed the colonies on the plates 1-2 hours before picking.</li>
<li>Add 15 &mu;l of PBS to the wells of a V-bottomed 96-well dish.</li>
<li>Wash the dish containing the colonies to be picked once with PBS and add 10ml of PBS to the dish.</li>
<li>Pick the colonies with the small 5&mu;l pipet tips (set the P20 pipetman on 4 &mu;l). Transfer colonies to a well in the 96-well dish.</li>
<li>Add 20 &mu;l of trypsin to each well using the multi-channel (12) pipetor. Pipet up and down 3 times. Incubate at 37&deg;C for 4 minutes.</li>
<li>Add 100 &mu;l 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.</li>
<li>Grow the cells on the 24-well plate in a 37&deg;C, 5% CO2 incubator until the cells reach 80&ndash;90% confluency, feeding daily with ES cell medium. Cells will probably be ready to expand in 3-7 days. </li>
</ol>
Step 4
<ol>
<li>Expansion of iPS cells <ol>
<li>Aspirate the medium, and wash the cells with 1 ml of PBS.</li>
<li>Remove PBS completely, add 0.1 ml of 0.25% trypsin/1 mM EDTA and incubate at 37&deg;C for 10 min.</li>
<li>Add 0.4 ml of the ES medium and suspend the cells by pipetting up and down to single cell suspension.</li>
<li>Transfer the cell suspension to a well of 6-well plate, add 1.5 ml ES cell medium, and incubate in a 37&deg;C, 5% CO2 incubator until cells reach 80&ndash;90% confluency in 6-well plates. At this point, prepare frozen stock of the cells, as follows.</li>
</ol></li>
<li>Preparation of freeze stock <ol>
<li>Aspirate the medium, and wash the cells with 2 ml of PBS.</li>
<li>Remove PBS completely, add 0.5 ml of 0.25% trypsin/1 mM EDTA and incubate at 37&deg;C for 10 min.</li>
<li>Add 2 ml of the ES medium and suspend the cells by pipetting up and down to single cell suspension.</li>
<li>Transfer the cell suspension to a 15-ml tube, count the number of cells and spin down the cells.</li>
<li>Discard the supernatant, resuspend the cells with ES medium to the concentration at 2x106 cells per ml.</li>
<li>Prepare 2 ES cell freezing medium (20% DMSO in ES medium) and aliquot it at 0.5 ml per vial.</li>
<li>Transfer 0.5 ml of the cell suspension to freeze vials and mix gently.</li>
<li>Put the vials in a cell-freezing container and keep it at -80&deg;C overnight; transfer to liquid nitrogen the next day for long-term storage.</li>
</ol></li>
</ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Wernig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+.+In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">et al . In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epigenetic+remodeling+in+directly+reprogrammed+fibroblasts.">Global epigenetic remodeling in directly reprogrammed fibroblasts.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Brambrink, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+expression+of+pluripotency+markers+during+direct+reprogramming+of+somatic+cells.">Sequential expression of pluripotency markers during direct reprogramming of somatic cells.</a> Cell Stem Cell. 2, 151-159 (2008).</li></ol>

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.

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<td>Doxycycline hyclate</td>
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generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4

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.

Watch this Scientific Journal Video about Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 at JoVE.com

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

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

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

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Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
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The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...