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.
