Name: Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System (Video) | JoVE Journal
Uploaded: 2009-11-13T20:00:00.000+00:00
Duration: 11 min 48 s
Description: Learn to generate induced pluripotent stem cells from mouse embryonic fibroblasts using a Dox-inducible lentiviral system with four transcription factors. This protocol enables controlled reprogramming and pluripotency marker analysis.

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1. Viral Transduction of MEFs
 <ol>
<li>The day before beginning your experiment, coat a 15 cm cell culture dish with 0.2% gelatin sterile filtered in ddH2O. Incubate the plate overnight at 37°C and 5% CO2.</li>
<li>Aspirate the liquid from the gelatin coated dish, and seed MEF cells (we used Nanog-GFP/rtTA MEF cells) at a density of 4x105 cells per dish. Incubate the cells in 30 ml of MEF growth medium (450ml DMEM supplemented with 50 ml FBS, 5 ml 100x non-essential amino acids, 5 ml penicillin/streptomycin, 5 ml 200 mM L-glutamine and 0.5 ml 55mM β-mercaptoethanol) for two days at 37°C and 5% CO2 until approximately 80% confluent.</li>
<li>Aspirate the medium and add 30 ml of MEF growth medium supplemented with concentrated lentivirus (4 x 100 μl virus stock solution + 29.6 ml growth medium). Rock the dish gently to ensure even distribution of the medium. Incubate the cells overnight at 37°C and 5% CO2.</li>
</ol>
 2. Doxycycline Induced Reprogramming
 <ol>
<li>20-24 hours post-transduction, trypsinize the transduced cells, centrifuge at 200 x g for 5 minutes and resuspend in ES/iPS growth medium (450 ml Knockout DMEM supplemented with 50ml ES cell-qualified calf serum, 5 ml penicillin/streptomycin, 5 ml 200 mM L-glutamine, 0.5 ml β-mercaptoethanol and 25 μl LIF) . Seed transduced MEF's at an appropriate concentration for the cell culture dish size (we used 2.5 x 105 cells per 10 cm dish for reprogramming efficiency experiments and colony isolation, and 2 x 104 cells per well in 4 well plates for ICC experiments). Incubate overnight at 37°C and 5% CO2. Note: any remaining transduced cells can be frozen in liquid nitrogen for future analysis.</li>
<li>Aspirate the medium and replace with ES/iPS medium prepared fresh and supplemented with doxycycline to a final concentration of 2 μg/ml (we also added medium without DOX as a negative control). Incubate at 37°C and 5% CO2.</li>
</ol>
 3. Immunocytochemical Analysis of Transduction Efficiency
48 hours post-doxycycline induction, determine transduction efficiency by immunohistochemistry(ICC). Carry out ICC testing on cells replated in 4 well plates. All volumes listed in the following protocol should be adjusted according to the cell culture plate size.
<ol>
<li>Wash the cells gently once with PBS (without Mg2++ or Ca2+).</li>
<li>Fix the cells with 500 μl of 0.5 ml 4% paraformaldehyde in PBS for 15 minutes at room temperature.</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Permeabilize cells by adding 500 μl of ice-cold 0.2% Tween®-20 in PBS. Incubate for 10 minutes at room temperature.</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Block non-specific binding with 200 μl blocking buffer for one hour at room temperature.</li>
<li>Incubate the cells with 200 μl of the specific primary antibody overnight at 4°C (we used Oct4, Klf4, Sox2 and c-Myc).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Incubate the cells with 200 μl of secondary antibody for 1 hour at room temperature, protecting the plates from light (we used Donkey anti-Rabbit Rhodamine conjugate, Donkey anti-Goat AlexaFluor® 594 conjugate, Donkey anti-Mouse Rhodamine conjugate and Donkey anti-Rabbit Rhodamine conjugate).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Add DAPI (final concentration 2 μg/ml in PBS) and incubate 10 minutes to visualize nuclei.</li>
<li>Wash the cells gently with PBS.</li>
<li>Add anti-fade Aqua-Mount before imaging using an inverted fluorescent microscope.</li>
</ol>
4. Isolating and expanding iPS Cell Colonies
<ol>
<li>After initiating the reprogramming process (as described in section 2), monitor the cultures and replace medium every 48 hours. We used doxycycline containing medium to culture the cells for the first twelve days and then subsequently removed doxycycline from the medium to ensure that the iPS colonies manually picked for expansion would be DOX independent.</li>
<li>Cells should be monitored daily for morphological changes indicative of the reprogramming process; or when using cells like Nanog-GFP/rtTA MEF, monitor morphology and GFP fluorescence to identify reprogrammed colonies. Each experiment will be different, but colonies are generally large enough for isolation between 16 and 22 days. Colonies identified during this time frame can then be manually isolated and trypsinized for expansion and analysis.</li>
<li>The day before isolating and trypsinizing the reprogrammed colonies, prepare a 24-well plate by seeding with gamma-irradiated feeder layer MEFs at a density of 5 x 104 cells per well. Incubate overnight at 37°C and 5% CO2.</li>
<li>Manually pick each iPS colony and trypsinize to dissociate the cell aggregates. Re-plate the iPS cells in ES/iPS medium in individual wells of the 24-well plate pre-seeded with gamma-irradiated feeder layer MEFs. Wells should be seeded at a density of 2 x 105 cells/well. Incubate at 37°C and 5% CO2. Change media every 24 hours.</li>
<li>Monitor iPS colonies daily for growth and GFP fluorescence. We incubate our cultures for 6 days before passaging.</li>
<li>Determine which wells of the 24-well plate are uniformly expressing GFP. Wells that have good GFP expression can be trypsinized and passaged 1:8 into 4-well plates that have been pre-seeded with gamma-irradiated feeder layer MEFs. These plates can be used for pluripotent marker analysis by ICC and AP staining.</li>
</ol>
5. Immunocytochemical Analysis for Pluripotency
Our ICC testing was carried out on cells expanded in 4 well plates. All volumes listed in the following protocol should be adjusted according to the cell culture plate size.
<ol>
 <li>Wash the cells gently twice with PBS (without Mg2++ or Ca2+).</li>
<li>Incubate in 0.5 ml of ice-cold 0.2 % Tween 20 in PBS per well for 10 minutes.</li>
<li>Wash the cells gently three times with PBS.</li>
<li>Block non-specific binding with 200 μl blocking buffer for one hour at room temperature.</li>
<li>Incubate the cells with 200 μl of the specific primary antibody overnight at 4°C (we used SSEA-1, Nanog and Oct4).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Incubate the cells with 200 μl of secondary antibody for 1 hour at room temperature, keeping away from light (we used Donkey anti-Mouse Rhodamine conjugate and Donkey anti-Rabbit Rhodamine conjugate).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Add DAPI (final concentration 2 μg/ml in PBS) and incubate 10 minutes to visualize nuclei.</li>
<li>Wash the cells gently with PBS</li>
<li>Add anti-fade Aqua-Mount before imaging using an inverted fluorescent microscope.</li>
</ol>
6. Alkaline Phosphatase (AP) Staining of iPS Cell Colonies
<ol>
<li>iPS colonies from section 4 can also be analyzed for AP activity utilizing commercially available kits and following the manufacturer’s protocol.</li>
</ol>
Part 7. Representative Results
The Stemgent DOX Inducible Mouse TF Lentivirus Set can be used to reprogram MEFs to iPS cells. After transduction of the MEFs, expression of transcription factors Oct4, Sox2, Klf4 and c-Myc can be detected in cells treated with doxycycline (DOX+), but little or no expression can be detected in untreated (DOX-) cells (figure 1). Morphological changes will progress over time (12 days of DOX treatment in this example) to generate larger, more ES cell-like colonies with defined colony edges and three dimensional growth (figure 2a). When DOX is removed, there is a noticeable reversion of cellular morphology for some ES cell-like colonies, however, many of the colonies maintained their iPS morphology (figure 2a). These iPS colonies, when picked and passaged, display typical pluripotency marker expression of Alkaline Phosphatase (AP), Nanog, Oct4 and SSEA-1 (figure 3). The type of MEF cells used in this experiment (Nanog-GFP/rtTA MEF cells) express GFP from the endogenous Nanog locus when reprogrammed to the pluripotency state. GFP expression can therefore be used as a preliminary indicator for successful reprogramming (figure 3).
<img src="/files/ftp_upload/1447/1447fig1.jpg" alt="Cell fluorescence microscopy chart; gene expression with/without Dox; Oct4, c-Myc, Klf4, Sox2." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1: Immunocytochemistry (ICC) analysis 48 hours post-DOX induction. The far left panel (-DOX) is a representative negative control for expression of the four transduction factors without DOX induction. Correctly expressed transcription factors were confirmed by corresponding antibodies (shown in red), stained with DAPI to visualize the nucleus.
<img src="/files/ftp_upload/1447/1447fig2.jpg" alt="Cell culture comparison in various stages, microscopy images with GFP fluorescence analysis, diagram." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2: Morphological conversion of Nanog-GFP/rtTA MEFs to the iPS cell state. A) 100x phase contrast imaging of cells demonstrating the compaction and conversion of MEFs into iPS colonies over time from day 3 (D3+Dox) to day 22 (D22). DOX was withdrawn on day 12. Upper left panel: 20x negative control image from –DOX plate. B) 200x phase-contrast and GFP fluorescence images of highlighted day 18 post-DOX induction iPS colony. C) 200x phase-contrast and GFP fluorescence images of additional day 18 post-DOX induction iPS colony.
<img src="/files/ftp_upload/1447/1447fig3.jpg" alt="Cell differentiation analysis; immunofluorescence microscopy; GFP, Rhodamine, DAPI staining results." data-altupdatedat="1787630408000" data-alt="Figure 3"> Figure 3: Analysis of iPS colonies. (A) Phase contrast microscopy and AP staining of an iPS colony (200x). (B) Pluripotency marker analysis: Left panel shows phase contrast overlay with GFP reprogramming reporter expression. GFP expression reflects the endogenous Nanog expression level. Middle panel shows ICC staining for pluripotency markers. Right panel shows DAPI staining to visualize the nuclei (100x).

<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>Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+pluripotent+stem+cells+from+adult+mouse+liver+and+stomach+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Generation of pluripotent stem cells from adult mouse liver and stomach cells.</a> Science. 321, 699-702 (2008).</li>
<li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li>
<li>Murry, C. E., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+embryonic+stem+cells+to+clinically+relevant+populations%3A+lessons+from+embryonic+development%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.</a> Cell. 132, 661-680 (2008).</li>
<li>Lerou, P. H., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+potential+of+embryonic+stem+cells%5BTitle%5D)+AND+%22Blood+Rev%22%5BJournal%5D)">Therapeutic potential of embryonic stem cells.</a> Blood Rev. 19, 321-331 (2005).</li>
<li>Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li>
</ol>

The author, Brad Hamilton, is employed by Stemgent that produces reagents and instruments used in this article.

These results demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set can be used to efficiently generate iPS colonies by inducing the ectopic expression of transduced transcription factors in mouse embryonic fibroblasts. When designing reprogramming experiments, several variables should be considered to optimize the efficiency of reprogramming. First, it is possible to modify the active virus-to-target cell ratio (i.e. M.O.I.) during the primary infection step to increase or decrease the transduction efficiency, thereby affecting the number of integrated viruses in the target cell population. Second, adjusting the length of time the cells are exposed to DOX can affect the efficiency of iPS cell colony generation. Third, the proliferative capacity of the target cells can impact reprogramming, as cells which are actively growing and dividing are more amenable to reprogramming. Lastly, when modifying the protocol for different cell numbers or different size tissue culture dishes, it is recommended that target cell numbers be adjusted proportionally to the surface area of the culture dish.

<table><tbody><tr><td>DOX Inducible Mouse TF Concentrated Lentifirus Set</td><td>Stemgent</td><td>00-0003</td><td></td></tr></tbody></table>

generation of induced pluripotent stem cells by reprogramming mouse embryonic fibroblasts with a four transcription factor, doxycycline inducible lentiviral transduction system

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Hi, my name is Brad Hamilton. I'm in the research and development department here at STEM Gen. Today we will show you a procedure for how to generate induced pluripotent stem cells from mouse embryonic fibroblasts.This procedure can be used to both generate induced pluripotent stem cells and to study the reprogramming process. So let's get started. Begin by seeding mouse embryonic fibroblasts, or meth cells onto a 15 centimeter gelatin coated dish at a density of four times 10 of the fifth cells per dish.Now add 30 milliliters of meth growth medium and incubate the cells for two days at 37 degrees Celsius and 5%carbon dioxide until they are approximately 80%confluent. When finished, incubating aspirate the medium and add 30 milliliters of growth. Medium supplemented with concentrated lentivirus.Rock the dish gently to ensure even distribution of the medium. Incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide with viral transduction complete. Let's move on to doxycycline induced reprogramming.To begin reprogramming 20 to 24 hours Post transduction trypsin is the transduced cells and centrifuge them at 200 Gs for five minutes. Next, carefully aspirate the media off of the cell pellet and resuspend the cells in growth medium. Once suspended seed, the transduced mes at an appropriate concentration for the cell culture dish size you are using.Here we are using 2.5 times 10 to the fifth cells per 10 centimeter dish for reprogramming efficiency experiments and IPS colony isolation and two times 10 to the fourth cells per well in four well plates For immunochemistry experiments. To monitor transduction efficiency, incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide. At this stage, the cells can be frozen in liquid nitrogen for future analysis if need be.The next day, aspirate the medium and replace it with fresh growth medium that has been supplemented with doxycycline to a final concentration of two micrograms per milliliter. We include a negative control containing medium without doxycycline. Finally incubate the cells at 37 degrees Celsius and 5%carbon dioxide.We have initiated the reprogramming process. In general. Pluripotent stem cell colonies will be large enough for isolation after 16 to 22 days.Before we demonstrate that procedure, we need to first verify transduction efficiency using chemistry to determine the transduction efficiency. Immunochemistry testing is carried out on the cells replated in four well plates 48 hours after doxycycline induction. All of the volumes listed in this protocol should be adjusted according to the cell culture plate size begin by washing the cells gently.Once with PBS not containing magnesium or calcium ions, fix the cells with 500 microliters of 4%paraform aldehyde in PBS for 15 minutes. At room temperature, wash the cells again gently two times with PBS. Next, perme the cells by adding 500 microliters of ice cold 0.2%Tween 20 in PBS incubate for 10 minutes at room temperature.After washing the cells two more times. Add 200 microliters of blocking buffer for one hour at room temperature to block non-specific antibody binding. Now add 200 microliters of the desired primary antibody.Here we are using the pluripotency markers T 4K LF four, SOX two, and cmic. Incubate the cells overnight at four degrees Celsius the next day. After washing the cells gently two times with PBS, incubate them with 200 microliters of secondary antibody for one hour at room temperature, protecting the plates from light.Here we are using the appropriate secondary antibodies conjugated with fluoro fours for visualization, using an inverted fluorescent microscope following incubation. Wash the cells two more times and then add DPI and incubate again for 10 minutes to visualize nuclei. Finally, after a last wash, add antifa aqua mount before imaging the cells with the inverted fluorescent microscope to determine the transduction efficiency.Begin isolation and expansion of pluripotent stem cells. After initiating the reprogramming process, monitor the cultures every day and replace appropriate medium every 48 hours. We use doxycycline supplemented medium to culture the cells for the first 12 days and then subsequently remove the doxycycline from the medium to that.The induced pluripotent stem cell or IPS colonies manually picked for expansion are doxycycline. Independent cells should be monitored daily for morphological changes indicative of the reprogramming process. Each experiment will be different, but colonies are generally large enough for isolation between 16 to 22 days after DS induction the day before you begin the process of isolating and trypsin reprogrammed colonies.Prepare a 24 well plate by seeding with a gamma irradiated feeder layer of mets at a density of five times 10 to the fourth cells per well. Incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide. The next day manually pick each IPS colony and tryps anize it to dissociate the cell aggregates repl the IPS cells in mouse embryonic stem cell medium in the individual wells of the preceded 24 well plate incubate the cells at 37 degrees Celsius and 5%carbon dioxide.Changing the media every 24 hours. Monitor the IPS colonies daily for growth and GFP fluorescence. We incubated our cultures for six days before packaging to four well plates for pluripotency analysis.Determine which wells of the 24 wheel plate are uniformly expressing GFP wells that have good GFP expression can be tripps, inized, and passaged one to eight into four well plates that have been preceded with gamma irradiated feeder layers of mes. These plates can then be used for ICC analysis for pluripotency to begin pluripotency analysis. Gently wash the cells twice with PBS not containing magnesium or calcium ions.Add 0.5 milliliters per well of ice cold 0.2%between 20 in PBS and incubate the cells for 10 minutes. After three more washes in PBS block, non-specific binding with 200 microliters of blocking buffer for one hour at room temperature. Now add 200 microliters of the desired primary antibody.Here we used SSEA one nanog and OCT four incubate the cells overnight at four degrees Celsius. After washing the cells gently twice, incubate them with 200 microliters of secondary antibody for one hour at room temperature, keeping them away from light. Here we are using the appropriate secondary antibodies conjugated with fluoro force for visualization, using an inverted fluorescent microscope after another two washes, add DPI and incubate the cells for 10 minutes.Now after a final wash, add antifa aqua mount before imaging the cells with the inverted fluorescent microscope. IPS colonies can also be analyzed for alkaline phosphatase activity by using commercially available kits. The stem gent docs inducible mouse TF lentivirus set can be used to reprogram mes to IPS cells after transduction of the MES expression of transcription factors.T four, SOX two, KLF four and seic can be detected in cells treated with doxycycline, but little or no expression can be detected in untreated cells. Morphological changes will progress over time to generate larger more ES cell-like colonies with defined colony edges and three dimensional growth. When docs is removed, there is a noticeable reversion of cellular morphology for some ES cell-like colonies.However, many of the colonies maintained their IPS morphology. These IPS colonies when picked and passaged display. Typical pluripotency marker expression of alkaline phosphatase, nano T four and SSEA one.The type of meth cells used in this experiment expressed GFP from the endogenous nag locus. When reprogrammed to the pluripotency state GFP expression can therefore be used as a preliminary indicator for successful reprogramming. We've just shown you how to induce reprogramming of mouse embryonic fibroblasts to induce pluripotent stem cells using a four transcription factor doxycycline inducible antiviral system.When doing this procedure, it's important to remember that when designing reprogramming experiments, several variables should be considered to optimize the efficiency of reprogramming. First, it is possible to modify the active virus to target cell ratio during the primary infection step to increase or decrease the transduction efficiency, thereby affecting the number of integrated viruses in the target cell population. Second, adjusting the length of time the cells are exposed to docs can affect the number of IPS colonies generated.Third, the proliferative capacity of the target cells can impact reprogramming as cells, which are actively growing and dividing are more amenable to reprogramming. Lastly, when modifying the protocol for different cell numbers or different size tissue culture dishes, it is recommended that target cell numbers be adjusted proportionally to the surface area of the culture dish. So that's it.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 a Dox-inducible lentiviral system with four transcription factors. This protocol enables controlled reprogramming and pluripotency marker analysis.

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System (Video) | JoVE Journal

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

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