The overall goal of this study is to reprogram peripheral blood mononuclear cells into induced pluripotent stem cells, using serial plating by centrifugation. Originally, iPSCs, were generated by reprogramming fibroblasts. Today, various are used for reprogramming.
However, because of the accessibility of blood, PBMCs can be unaccepted cell source for further application of iPSCs, in drug screening, thesis modelling, and regenerative medicine development. Through the study, we will be sharing a protocol about the reprogramming of iPSCs using PBMCs by adding centrifugal force. This protocol will provide another option when reprogramming floating cells.
Demonstrating the procedure, we'll be using Nam, a grad student from my laboratory. To isolate the monocytic cells, first obtain at least ten milliliters of fresh blood from a blood draw in a cell preparation tube. Next, transfer the blood to a new 50-milliliter conical tube and dilute it with steril PBS at a ratio of one to four.
Afterward, add ten milliliters of density gradient media to a new 50-milliliter conical tube, and carefully layer the diluted blood on top of the density gradient media. Then, centrifuge the sample at 750 times g for 30 minutes at room temperature, without a centrifugation break. After 30 minutes, carefully transfer the buffy layer to a new 50-milliliter conical tube.
Add 30 milliliters of PBS, and wash the cells. Then, centrifuge the cells at 515 times g for five minutes at room temperature. Afterward, discard PBS, and resuspend the cells in 0.5 milliliters of blood cell media.
Following that, count the cells and plate them in a 24-well plate. Add PBS to the surrounding wells to prevent evaporation. Following that, stabilize the cells for five days at 37 degrees Celsius before transduction.
Add an additionally 0.5 milliliters of fresh blood cell media on day three to four, without disturbing the cells. In this procedure, transfer the blood cells to a 15-milliliter conical tube, and count them using a hemacytometer. Next, prepare three times ten to the fifth cells for transduction, and centrifuge the cells at 515 times g for five minutes at room temperature.
Afterward, discard the supernatant by suction, and resuspend the cells in 0.5 milliliters of blood cell media. Then, transfer the cells to a well of a non-coated 24-well plate. Thaw the Sendai virus mixture in ice, and add it to the suspended cells.
Seal the plate with a sealing film, and centrifuge it at 1, 150 times g for 30 minutes at 30 degrees Celsius. After centrifugation, incubate the cells at 37 degrees Celsius in 5%carbon dioxide overnight. To coat a 24-well plate with vitronectin, the next day, dilute the vitronectin solution in PBS to obtain a final concentration of five micrograms per milliliter.
Next, add one milliliter of vitronectin to a well, and incubate it at room temperature, for at least one hour. Afterward, remove the coating solution, and transfer all the media containing the cells and the virus to the coated well. Then, collect the remaining cells with an additional 0.5 milliliters of fresh blood cell media and add it to the cell-containing well.
Subsequently, centrifuge the plate at 1, 150 times g for ten minutes at 35 degrees Celsius, and then, maintain the cells at 37 degrees Celsius in 5%carbon dioxide overnight. For second cell transfer, coat the well with five micrograms per milliliter vitronectin as described previously. Use one well of the plate for each transduction, and transfer the cell suspension from the first plate, to the newly coated vitronectin plate.
Meanwhile, add one milliliter of iPSC media to a well of the first plate for maintenance, and incubate it at 37 degrees Celsius in 5%carbon dioxide, with a daily media change with fresh iPSC media. Colonies will appear on day 14 to 21, after transduction. Centrifuge the newly-coated plate, containing the suspended cells, at 1, 150 times g in 35 degrees Celsius for ten minutes.
After centrifugation, incubate the cells at 37 degrees Celsius in 5%carbon dioxide, overnight. The next day, remove the supernatant, and replace it with fresh induced pluripotent stem cell media. Maintain the attached cells with daily media change, until 80%confluency is reached.
A week before colony picking, seed one times ten to the fourth cells in a vitronectin-coated 100-millimeter dish. Next, prepare a vitronectin-coated 60-millimeter dish, by adding two milliliters of vitronectin solution, and incubate it at room temperature for at least one hour. By observing through a microscope, mark the colonies on the bottom of the plate with clear boundaries using a marker pen.
Remove the vitronectin solution from the new plate, and add six milliliters of induced pluripotent stem cell media, supplemented with ten millimolar RHO kinase. Subsequently, remove the culture medium from the cells, and wash them with three milliliters of PBS. Then, add one milliliter of iPSC colony-detaching solution, and incubate them for 30 seconds at room temperature.
After that, remove solution from the plate, and incubate it at room temperature for an additional 30 seconds. After 30 minutes, draw 200 microliters of media from the plate, and detach the targeted colonies by pipetting. Then, transfer the scattered colonies to a new 60-millimeter dish.
Incubate and maintain the cells at 37 degrees Celsius in 5%carbon dioxide until they reached passage ten. At the early stage of reprogramming, iPSCs were mixed with the attached non-reprogrammed differentiated cells. Before expansion, iPSCs and other cells were different to discriminate.
By seeding the cells in a low density, colonies that were large enough for picking were obtained. After isolating the colony, the cell clumps were dissociated into single cells, and culture. Pure iPSC colonies were seen afterwards.
After the pure induced pluripotent stem cells expanded, the quality of the cells was tested. Cells were stained with alkaline phosphatase to confirm the undifferentiated state of the induced pluripotent stem cells. Colonies derived from the isolated induced pluripotent stem cells all showed positive staining.
Pluripotent markers were confirmed by immunofluorescent staining. The reprogrammed cells highly-expressed pluripotent markers, such as SSEA4, Oct4, Sox2, TRA-1-81, Klf4, and most importantly, TRA-1-60, the expression of pluripotent markers was confirmed by RTPCR. Using serial plating by centrifugation, the attachment of transit use PBMC onto a metrics coated plate.
Attaches there are as pended, and iPSC colonies are repeat for a purification. Using this protocol, iPSC can be generated from PBMCs within a month. This protocol reduces the time required for the attachment of transduced cells and prevent the loss of reprogrammed cells that were not able to attach on its own.
However, the purification of fully reprogrammed colony-forming iPSCs can be critical, when attempting this procedure. Previously, we successfully reprogrammed PBMCs and mononuclear cells using this protocol. The generated blood cell derived iPCs can be broadly used in various fields of research.
This protocol can help the reprogramming process of other types of floating suspension cells, for further applications and studies.
