The overall goal of this procedure is to generate human induced pluripotent stem cells with Sendai virus. This is accomplished by first preparing and plating fibroblast cells to be transduced. The next step of the procedure is to infect the cells with Sendai virus.
Then the infected fibroblast cells are transferred onto fresh mouse embryonic fibroblast feeder cells. The final step is to manually pick the reprogrammed. Now pluripotent stem cells, ultimately the reprogramming of fibroblast cells into I PSCs, without the use of trans genes, can be shown through immuno labeling and R-T-P-C-R.
The main advantage of this technique over existing method is that it enhance the reprogramming efficiency without remaining transing and in a cost effective manner For this protocol, have ready human fibroblasts cultured in DMEM. With FBS transfer these cells to 24 well plates for the experiment plate the cells in six serial dilution to determine the best density for cell attachment. Each row of the 24 well plate can accommodate one dilution series of cells.
Thus four cell types can be tested on one plate, incubate the dilution for 24 hours. The next day select wells at three levels of confluence for transduction. One at a high level of 80 to 90%confluence, one at about 60%confluence, and the third at about 30%Confluence an hour or more before the transduction.
Replace the media with 300 microliters of fresh fibroblast media for the transduction thaw. One set of Sendai virus tubes at 37 degrees Celsius for a few seconds, and then finish. Thawing them at room temperature.
Centrifuge the thawed viruses at 6, 000 G for 10 seconds and store them on ice in one micro centrifuge tube. Make a mixture of the viruses calculated according to the number of cells to be transduced. Calculation details can be found in the text protocol.
Mix the viruses well with gentle pipetting. Now to the most dense cells, add two volumes of virus. Add one volume of virus to the middle density cells and add a half volume of virus to the least dense selection.
Swirl the plate and let it incubate overnight for the reaction to take place the next day. Replace the media with 500 microliters of fresh fibroblast media. Do this again two days later, three days after the second media change made on the transduced cells.
Prepare meth cells in 60 millimeter dishes with DMEM containing 10%FBS plate 500, 000 cells per dish and incubate them overnight. The next day, replace the media on the meth cells with fresh media and then proceed with setting up the co-culture. First, remove the media from the transduced fibroblasts and wash them once with DPBS.
Next, add 200 microliters of 0.25%trypsin EDTA to each transduced well of cells within five minutes. Pool all the detaching cells into a single 15 milliliter conical tube. Spin down the cells at 1, 350 G for four minutes.
Then remove the supernatant and wash the pelleted cells with about five milliliters of fresh medium to neutralize the trippin. Now collect the cells with another four minute spin at 1, 350 G.Next plate. Most of the cells as six serial dilutions in meth.
The first and last dilutions may not be needed depending on the death rate of the cells. Save some of the cells for an RNA extraction and incubate the plates overnight. The next day, replace the medium with human ES media supplemented with 10 micromolar of RO kinase inhibitor Y 2 7 6 3 2.
To improve cell survival during the following days, change the media to normal unsu supplemented human ES media. After a week of culturing, start checking the dishes every few days for the formation of ES colony like cell clumps. Once they appear, monitor growth.
Three weeks after the transduction, the colonies of ES cell clumps should be ready for expansion. Have prepared meth feeder cells in 24 well plates just as they were prepared for 60 millimeter plates. Before picking any colonies, change the media on the meth cells to ES media with 10 microliters of Y 2 7, 6, 3, 2.
Pick one colony at a time from the dishes. Transfer the colony to a 15 milliliter tube with solution there. Break up the colony using the pipetter.
Then cover one me. Well with the broken up colony. Ultimately load each well with one broken up colony and load as many wells as possible.
Maintain the cells with daily media changes and expand them to six well plates and then 60 millimeter dishes. Typically, fibroblasts infected by Sendai virus do not show any morphological changes until after five days. Then the cells take on a round shape with a larger nucleus and smaller cytoplasm.
This small scale protocol includes several parameters that can be optimized, such as fibroblast density, virus titer, and plating density to the mes. Transduction of cells at different co fluency shown in different colors can also be optimized. After a week of co-culture with meth feeder cells, partially reprogrammed fibroblasts have a loose slike shape and are easily picked as opposed to being collected using trypsin.
Fully reprogrammed, I PSCs are clearly identifiable from partially reprogrammed cells. Fully reprogrammed cells are tightly packed and colonies can have clear boundaries. Partially reprogrammed cells make loose clusters that are easily detached.
After expanding the IPSC clones for several weeks, staining for stem cell markers is warranted compared to H nine cells. The IPCs are positive to several expected antibodies, including SSEA four, OCT four, TRA 81, and nano G supporting their pluripotent quality. Using R-T-P-C-R transgene expression was not noted in human IPSC clones.
After 10 primers for exogenous OCT four, SOX two, klf four and Cmic were used with positive control samples that showed strong transgene expression levels. After watching this video, you should have a good understanding of how to reprogram somatic cells to induce the poly potent stem cells and how to select those, completely reprogram the cells from the other cells.
