Hi, my name is Brad Hamilton. I'm a scientist here in the research and Development department here at STEM Gen. Today we'll show you a procedure for how to reprogram human fibroblasts using lentiviral delivered reprogramming factors.
This procedure can be used to both generate IPS cells and study the reprogramming process. IPS cells are similar to ES cells in morphology proliferation and ability to differentiate and to all the tissue types of the body. Human IPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of destroying an embryo to obtain the cells.
The generation of patient-specific IPS cells circumvent an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non autologous transplanted cells. So let's get started. At several points in this protocol, you replace the medium of the developing IPSC or induced pluripotent stem cells with meth conditioned medium.
Preparing this medium takes six days. So begin at least a week before you want to start your experiment. To begin seed each well of a six.
Well plate with two times 10 to the fifth cf one meth feeder cells in two milliliters of meth growth. Medium incubate overnight at 37 degrees Celsius and 5%carbon dioxide. The next day, change medium to human E-S-I-P-S cell culture.
Medium incubate overnight at 37 degrees Celsius and 5%carbon dioxide. Collect the supernatant and replace with fresh medium every 24 hours for four days. Filter the supernatant through a 0.22 micrometer filter.
Supplement the collected supernatant with 15 nanograms per milliliter of BFGF basic fibroblast growth factor. You will end up with about 16 milliliters of meth conditioned medium store each day's collected supernatant separately at four degrees SIUs. Meth conditioned medium can be stored for one to two weeks at four degrees Celsius or for several months at minus 20 degrees Celsius.
Now that you have assured that your meth conditioned medium will be ready when you need it, let's talk about preparing the cells. The first task in generating IPSC is to use lentiviruses to transduce, human foreskin fibroblast cells, or BJ cells with four transcription characteristic of stem cells. To prepare for transduction seed BJ cells at a density of one times 10 to the fifth cells per well of a six well plate and culture.
The cells in two milliliters of growth, medium overnight at 37 degrees Celsius and 5%carbon dioxide on the same day. Begin preparing meth feeder plates by adding two milliliters of 0.1%gelatin diluted in water to a six well plate and incubating overnight at 37 degrees Celsius and 5%carbon dioxide. The gelatin coats the wells and serves as a matrix for thes.
Now the BJ cells are ready for lentiviral transduction to prepare the virus for transduction. Supplement two milliliters of fresh medium with six micrograms per milliliter of poly brain and four lentiviruses each expressing one of the human transcription factors. T four, SO two nanog and lin 28, such that each is at the same multiplicity of infection or MOI.
These lentiviruses are all included in the human reprogramming factor lentivirus set. To perform the transduction, simply replace the BJ cell growth medium with the virus containing medium. Gently rock the plate so that the medium will evenly coat the bottom.
Incubate overnight at 37 degrees Celsius and 5%carbon dioxide on the same day. Remove one vial of cf one meth feeder cells from liquid nitrogen and thaw. Remove the gelatin solution from the meth feeder plates, which are now gelatin coated to each.
Well add 0.2 times 10 to the fifth cells in two milliliters of meth growth.Medium. Incubate the MES overnight at 37 degrees Celsius and 5%carbon dioxide. The next day, detach the BJ cells with 0.05%trypsin EDTA and centrifuge at 200 Gs for five minutes.
Following centrifugation, aspirate the supernatant and resuspend the cells in growth medium. Remove the medium from the meth feeder plate and add two milliliters per well of the BJ cell suspension. The BJ cell concentration should be approximately five times 10 to the fourth cells per well.
Incubate overnight at 37 degrees Celsius and 5%carbon dioxide. 24 hours after receding the BJ cells on the meth feeder plate replace the medium with human E-S-I-P-S that is embryonic stem or induced pluripotent stem cell culture.Medium. Continue to change the medium every 24 hours for seven days.
On day seven, replace the ES cell culture medium with two milliliters of meth conditioned medium. The meth feeder cells have been providing the soluble factors needed to grow IPS cells. But now we need to add more of these factors to keep the culture healthy.
Meth conditioned medium contains all the soluble factors secreted by meth feeder cells incubate overnight at 37 degrees Celsius and 5%carbon dioxide. Looking through the scope, select a cell colony. Distinguished by a small compact dome like appearance in a fresh 24 well plate that you have preceded 24 hours previously with ccf one meth feeder cells recede the selected colony in human E-S-I-P-S culture.
Medium supplemented with 10 micromolar stem molecule Y 2 7 6 3 2. A dependent kinase inhibitor. Change the culture medium without supplementing with stem molecule Y 2 7 6 3 2 every 24 hours for the first seven days.
After seven days change medium to meth. Condition to medium continue to passage the cells until they show typical human ES cell morphology. Look for small, compact dome like colonies that have a sharp edge.
Colonies should be uniform and have a high ratio of nucleus to cytoplasm. At this point, you have developed colonies that have the morphology of ES cells, but do they also express genes characteristic of ES cells? To find out stain the colonies for markers of pluripotency such as TRA 180 1 TRA one 60 SSEA four s, SE, a three T four sox, two nanog and SSEA one To begin gently wash the cells three times with PBS.
Next, fix the cells with 500 microliters of 4%paraform, aldehyde or other fixative for 20 minutes at room temperature. After 20 minutes, wash the cells with PB S3 more times to remove excess fixative. Next block nonspecific binding by incubating the cells in 500 microliters of blocking buffer per well for one hour at room temperature After the blocking step, add 250 microliters of primary antibody diluted one to 100 in blocking buffer to each.
Well incubate the cells in primary antibody overnight at four degrees Celsius the next day. Wash the cells three times with PBS to remove unbound primary antibody. Incubate the cells with 250 microliters of secondary antibody diluted one to 300 and blocking buffer for one hour at room temperature, keeping away from light to avoid photobleaching after incubation with the secondary antibody.
Wash the cells three times with PBS to the third wash. Add DPI at one microgram per milliliter to stain the nuclei. Lastly, behold, analyze the gene expression of your IPS cells.
Under the microscope morphology results human four skin fiberblast cells where cot transduced with T four SOX two nanog and lin 28 morphological changes were observed as early as day four post transduction and the cluster of cells became more tightly packed at day 17 expression of plurry potency markers. To further characterize the isolated IPS cell colonies, we looked for the presence of common pluripotency markers expressed in ES cells. The colonies exhibited strong alkaline phosphatase activity.
Additionally, immunochemistry or ICC was performed on the IPS cell colonies with a panel of pluripotency marker specific antibodies including surface markers TA 180 1 TRA one 60 SSEA four and SS EEA three, as well as nuclear markers, T four, SOX two, and Nanog. The isolated IPS colonies were positive for all markers. The ICC results show that the IPS cells exhibited the appropriate pluripotency marker expression pattern, demonstrating that these IPS cells closely resemble undifferentiated human E es cells.
We've just shown you how to generate IPS cells by reprogramming human fibroblasts with stem gen's. Human reprogramming factor lentivirus set. When doing this procedure, it's important to take several factors into account.
First, the active virus to target ratio may need to be modified during the primary transduction step to achieve optimum transduction efficiency. Second, the growth condition of the target cells can impact. Reprogramming healthy and proliferative cells are more amenable to reprogramming.
Third, when modifying the protocol for different cell numbers, it is recommended that the target cell numbers be adjusted proportionally to the surface area of the culture dish. Lastly, applying rock inhibitors such as Y 2 7 6 3 2 should be considered to help ensure successful reprogramming as recent studies have demonstrated its utility in enhancing human yes colony survival. So that's it.
Thanks for watching and good luck with your experiments.
