Developmental Biology
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Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions
Chapters
Summary November 27th, 2017
This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.
Transcript
The overall goal of this procedure is to efficiently derive induced pluripotent stem cells by means of episomal reprogramming from two different kinds of fetal stem cells. This method can help provide a source of pluripotent stem cells for tissue engineering, disease modeling, basic research, or longitudinal studies. The main advantage of this technique is that the reprogramming and the maturation of iPSCs transpires in chemically defined conditions while being available early for potential pediatric cell-based therapies.
Demonstrating this procedure will be Steven McClellan, the principal investigator, and myself. Obtain placentas as soon as possible following birth. Cut nine-squared centimeter segment of the amnion, and wash it in a 50-millimeter centrifuge tube with 30 milliliters of PBS supplemented with antibiotic, antimycotic solution.
Remove blood clots from the membranes, and then use a pair of fine scalpels to mince the membranes into fine pieces in a sterile 10-centimeter tissue culture dish. Use the scalpel blades to transfer the minced membrane tissue mass into one tissue dissociation tube containing 4.7 milliliters of RPMI-1640 medium. Mix in the dissociation enzymes.
Mount the tube onto the tissue dissociator, and run program h_tumor_01. Then incubate the tube at 37 degrees Celsius on a rocking platform for 30 minutes. Further dilute the suspension with 35 milliliters of RPMI-1640, and apply to a 70-micron strainer placed over a 50-milliliter centrifuge tube.
Centrifuge for five minutes at 200 times g at room temperature. After discarding the supernatant, resuspend the pellet in five milliliters of RPMI-1640, and count the cells using a hemocytometer. Seed the cells in cell clusters at a density of roughly 10, 000 cells per square centimeter into tissue culture flasks with freshly prepared AFMC medium supplemented with antibiotic, antimycotic solution.
Harvest low passage number amniotic fluid and membrane stem cells for reprogramming from one T75 flask by adding two milliliters of cell detachment enzyme to the cell sheet. Incubate at 37 degrees Celsius for five to eight minutes. After centrifugation at 200 times g for 45 minutes, resuspend the pellet in one milliliter of PBS, and mix well to wash away serum components.
Count the cells using a hemocytometer. Then adjust the cell density to 100, 000 cells per milliliter of PBS, and now quad into 1.5-milliliter microcentrifuge tubes. Place the microcentrifuge tubes in five-milliliter polystyrene tubes to allow centrifugation in a regular swing rudder, and centrifuge at 200 times g for four minutes at room temperature.
After the spin, invert the tubes, and discard the supernatant into a waste container, and then perform an additional centrifugation step at 200 times g for three minutes. After the spin, use a 200-microliter pipette to carefully aspirate the remaining liquid from the tubes. Place transfection tips, tube, resuspension buffer, and electrolytic buffer into the tissue culture cabinet.
Move the transfection device close so that its tube station can be placed directly into the cabinet. Fill one transfection tube with three milliliters of electrolytic buffer, and mount the tube into the station by pushing it all the way inside the slot. Take previously prepared aliquots of reprogramming plasmid solution out of the minus 80 degrees Celsius storage, and allow them to thaw at room temperature in the culture cabinet.
Set the transfection parameters on the device to deliver one pulse of 40 milliseconds at 950 volts. Resuspend the pellet containing 100, 000 cells in 10 microliters of resuspension buffer. Working quickly, add 1/10 of the reprogramming plasmid solution to the resuspended cells.
Mount the transfection tip onto the transfection pipette. Then aspirate the cell suspension into the transfection tip carefully avoiding formation of air bubbles. Insert the transfection pipette into the transfection tube.
And press the start button on the screen at the transfection device. Wait for the screen message regarding the success of the transfection. Then remove the pipette from the tube immediately.
Expel the suspension into one well of the target six-well plates. Mix in the medium from a neighboring well, and distribute the suspension equally into both wells. Repeat the transfection for each microcentrifuge tube with cells.
Begin by adding 30 microliters of 0.5 millimolar EDTA, and PBS to five PCR tubes. Then load a tip onto a 10-microliter pipette set to two microliters. Hold the pipette tip at an angle at the colony edge, and carefully and gradually scrape the whole colony off the surface.
Immediately aspirate the whole colony into the pipette tip, and transfer it into one of the prepared PCR tubes with EDTA. After repeating the process with four other colonies, incubate at room temperature for four to six minutes. Use a larger pipette tip to pipette each cell suspension up and down gently to break the colony down into smaller clumps.
Avoid creating a single-cell suspension. Then plate the suspension directly into the target well of a recombinant vitronectin-coated 24-well plate. After repeating the process for the remaining colonies, incubate for three to six days.
After four to six days, when the colonies have grown and become compact, wash the cell sheets with 300 microliters of 0.5 millimolar EDTA, and aspirate immediately. Then add another 300 microliters, and incubate at room temperature for five minutes. After aspirating the EDTA, set a one-milliliter pipette to capacity, mount to one milliliter wide bore tip, and aspirate E8 medium from the target well of a vitronectin-coated six-well plate.
Squirt a stream of the medium at one well of the source plate to wash off the iPSC culture. Then transfer the suspension into the target well, and pipette up and down several times to break up the colonies into clumps of 20 to 40 cells. After plating all cells, ensure that the clumps are distributed evenly by gently rocking and shaking the plate.
Then return the plate into the incubator for clump adhesion and colony growth. The amniotic fluid and membrane stem cells, which represent source cells for reprogramming display a typical mesenchymal morphology elongated and phase-bright. The cells acquire epithelial properties after they undergo the mesenchymal-to-epithelial transition, which leads to the formation of colonies with cobblestone-like cells.
These colonies proliferate and create irregular cellular masses of MET cells. At the later stages of reprogramming, colonies of fully reprogrammed cells emerge, individually discernible cells with well-defined borders. These fully reprogrammed cells are present alongside MET colonies that are more numerous.
A fully reprogrammed isolated mature clone is shown here. Following iPSC derivation and maturation, they can be subjected to detail characterization, and direct the differentiation into a variety of cell types. Don't forget that working with human tissues that have not been tested for the presence of pathogens can be extremely hazardous, and precautions such as personal protective equipment, and working in the biosafety cabinet should always be taken while performing this procedure.
After watching this video, you should have a good understanding of how to induce pluripotency in fetal stem cells, what reagents and equipment to use, and how to routinely culture pluripotent stem cells.
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