Name: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 (Video) | JoVE Journal
Uploaded: 2008-04-07T10:00:00.000+00:00
Duration: 13 min 2 s
Description: Learn to generate induced pluripotent stem cells from mouse embryonic fibroblasts using lentiviral delivery of Oct4, Sox2, c-Myc, and Klf4 transcription factors. This protocol enables cellular reprogramming for regenerative medicine research.

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5 minutes

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/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((et+al+.+In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Generation+of+germline-competent+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Global+epigenetic+remodeling+in+directly+reprogrammed+fibroblasts%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Sequential+expression+of+pluripotency+markers+during+direct+reprogramming+of+somatic+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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.

<table><tbody><tr><td>Doxycycline hyclate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>D9891-100G</td><td></td></tr></tbody></table>

generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4

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.

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., ...

Over 50 years ago, it was hypothesized that terminally differentiated cells such as skin fibroblasts could be forced to take on a pluripotent state similar to the embryonic stem cells. The basis for this concept was the observation that all cell types with a few minor exceptions, have the same genetic code and that the only difference between cell types is how the code is read. This ability to change the cellular identity of a differentiated cell to a pluripotent state is termed cellular reprogramming.After half a decade of research, it was recently shown that the forced expression of four transcription factors can reprogram skin fibroblasts to become pluripotent. The impact of this finding is huge. Beyond the importance for basic biology, it also overcomes the major ethical objections to the use of human embryonic stem cells for transplant therapy.In order for reprogramming to happen four genes that encode the transcription factors OC four sox two cmic and K four need to be packaged and put into a lentivirus tissue culture. Medium containing purified virus is then added to a cell culture dish containing a high density of fibroblasts, and within that dish, something amazing occurs. A small fraction of fibroblasts become infected with all four of the transcription factor carrying viruses and begin to de differentiate.They undergo drastic changes in their morphology and proliferation and begin to divide into large spiritual clusters of pluripotent stem cells formed. After two to three weeks of culture in embryonic stem cell conditions, colonies of reprogramed fibroblasts can be manually isolated and expanded in vitro. Once purified, these cells can be used to generate chimeric mice in which a certain percentage of cells derive from those de differentiated fibroblasts can contribute to different tissues of the mouse.In chimes in which the genetic material of the original fibroblasts has contributed to the germline, the genetic code be passed to the next generation and then the next and then the next. Although the use of a viral delivery system is currently an impediment for clinical applications, this will likely be overcome in short order. In this video, members of the yin lab will demonstrate how induced atory potent stem cells are derived from mouse embryonic fibroblasts.Hi, I'm Grant Welted from the laboratory of Rudolph Jish at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts. And I'm Tobias Bro brink also from ish lab. Today we will show you the generation of induced pluripotent stem cells by the introduction of four CDNAs OCT four SOX two CIC KF four using lentiviruses that express these four CDNAs from a Tet OCMV promoter.This allows for the precise control of the expression of these four transgenes after the infection of mouse Embryonic fibroblasts. The protocol we're showing you today involves the handling of lentivirus that can possibly infect human cells. It is critical that you always follow safe procedures and contact your safety office before you try to do this yourself.So let's generate some IPS cells in order to generate lentivirus that will convert mes to IPS cells. 2 9 3 cells must be transfected with three plasmids Paks two, which provides the required genetic elements for viral packaging. The viral backbone, which is an FUW TE OCMV lentivirus with one of the four CD aids T four SOX two, KLA four or CMIC PMDG, which expresses the envelope glycoprotein of vesicular stomatitis virus or VSV.This makes the virus that will generate atropic virus. So proper BL two plus procedures need to be followed. In this particular video, we are not actually handling infectious virus, so we are not wearing our typical gowns and are not in our BL two plus facility.Prior to transfection 2, 9 3 cells are thawed from liquid nitrogen and grown to 90%Co fluency cells are then transfected with FU gene and given 48 to 72 hours to express the virus, at which time the medium bathing the cells is filtered using a 0.45 micron filter and concentrated using ultracentrifugation after concentrating virus. We are now ready to infect MES in order to generate IPS clusters During the viral preparation steps, mouse embryonic fibroblasts passage less than three times are grown to 90%co fluency in 10 centimeter dishes, approximately two times 10 to the six cells per dish. In this particular experimental protocol, we are using MES that express the RTTA, which is critical for doxycycline expression and GFP from the endogenous OC four locus.MES are GFP negative, but true IPS cells become GFP positive. This marker allows for a method by which to identify reprogrammed mes aspirate the culture medium from the fibroblasts and wash with 10 mils of hippies, EDTA, discard the hippies EDTA and add five mils of one times trypsin and incubate at 37 degrees for 10 minutes. Trypsin will help lift the cells from the bottom of the dishes.Now add nine mils of the culture medium to resuspend the cells in a single cell suspension and transfer to a 15 mil tube. These cells are then spun down and pelleted. After resus suspending the pellet, the number of cells in the suspension are counted and the concentration is adjusted to eight times 10 to the four cells per mil.10 mils of the cell suspension is then transferred to a 10 centimeter dish coated with gelatin. Incubate the dish overnight at 37 degrees Celsius 5%CO2 following the overnight incubation medium from a fibroblast dish is aspirated and 10 mils of the virus containing medium is added to the meth. Incubate the cells from four hours to overnight at 37 degrees Celsius, 5%CO2.The next day aspirate the medium from a fiberblast dish and then add 10 mils of fresh ESL media. At this stage, cells can be frozen down, expanded, or prepared for the initiation of reprogramming. To initiate reprogramming, replace regular ESL medium with medium containing doxycycline to initiate the expression of the four genes.Place these cells in the incubator change the medium every day until the colonies become big enough to be picked. 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.Now that 20 days have passed, we can go ahead and pick IPS colonies before picking colonies. One should remember to see the required number of gelatin coated 24 well plates with gamma irradiated DR four myths one to two hours before picking. One should feed the colonies on the plates by adding fresh medium just prior to picking add 50 microliters of HEPA buffer to the wells of a V bottomed 96 Well dish immediately before picking, wash the dish containing the colonies to be picked once with PBS and add 10 mls of PBS to the dish IPS cell clusters can be picked under either a stereo or an inverted microscope.Pick the colonies with a small pipette tip and transfer them into a well in the 96 well dish containing PBS in order to remove the clusters. A circle is drawn with a pipette tip around the cluster to break up the maths. Now the reprogrammed cells can be extracted now using the multichannel pipetter, 20 microliters of tripsin are added to each.Well pipe it up and down three times, incubate at 37 C degrees for four minutes. After the four minute incubation, 100 microliters of ES medium are added to the tripps nice colonies. Pipe it up and down 10 times and transfer six 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 pipetter cells are then grown on the 24 well plate in a 37 cent degree, 5%CO2 incubator until the cells reach 80 to 90%Co fluency fitting deli with ES cell medium cells will probably be ready to expand in three to seven days A few days later. Now that the IPS cells are confluent, aspirate the medium and wash the cells with one mil of hippies completely Remove the hippies and add 100 microliters of one times trypsin and incubate at 37 degrees Celsius for 10 minutes. Following incubation with trypsin, add 400 microliters of ESL medium and suspend the cells by pipetting up and down and produce a single cell suspension.The 500 microliter single cell suspension from the 24 well is then transferred to a single well of a six well plate and incubated in a 37 degrees Celsius 5%CO2 incubator until the cells reach 80 to 90%co fluency in the six well plate. At this point, you can either proceed to amplify cells for southern analysis, blasts, injections, teratoma assays, or in vitro differentiation assays. And you should remember to freeze stock valves of these cells so that you do not lose this precious stock of IPS cells.We have just shown you how to induce pluripotency in mouse embryonic fibroblast using an inducible antiviral system. So thanks for watching and good luck with your experiments.

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Learn to generate induced pluripotent stem cells from mouse embryonic fibroblasts using lentiviral delivery of Oct4, Sox2, c-Myc, and Klf4 transcription factors. This protocol enables cellular reprogramming for regenerative medicine research.

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

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