Developmental Biology
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Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors
Chapters
Summary January 1st, 2017
This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.
Transcript
The overall goal of this procedure is to generate iPS cells from adult human peripheral blood mononuclear cells using non-integrating episomal vectors. This protocol can help scientists in the stem cell field who wish to generate iPS cells for disease monitoring, drug screening, and regular radio medicine. Our reprogramming protocol is very simple, yet highly efficient.
Following this procedure, one can easily master the technique of blood cell reprogramming. The quality of the plasma is as important for successful reprogramming. Contaminants may decrease reprogramming efficiency, therefore we recommend using Endofree kits to purify episomal plasmids.
iPS cells are more fragile than other cells. Preparing cells for more than one during passing gene may induce massive cell deaths, in particular at early passages. To begin this procedure, prepare all the required culture media according to the accompanying manuscript.
Then combine 10 mLs of fresh PB sample and ten mLs of PBS buffer into a 50 mL tube and mix well. Bring PBS to room temperature or 37 degrees Celsius prior to use. Next, add 10 mL of ficoll to the bottom of the tube.
This process should be carried out slowly to ensure that the ficoll layer does not mix with the PB.Centrifuge the mixture at 400 x g for 30 minutes with low acceleration and deceleration. After centrifugation, the PB MNCs are in the white layer located between the PB plasma and ficoll. Slowly aspirate 10 mL of PB plasma without disturbing the buff ficoll layer.
Then carefully harvest the white layer into a new 50 mL tube. The total volume of the collected cells from the white layer should be about three to six mL. After that, add PBS to bring the total volume to 30 mL and mix well.
Then centrifuge the sample at 400 x g for ten minutes. After ten minutes, remove the supernatant and resuspend the cell pellet in 20 mL of culture medium. Mix well and centrifuge the sample at 400 x g for five minutes to remove the majority of the platelets.
Subsequently, remove the supernatant and resuspend the cell pellet in one to two mL of IMDM. The cells may be used for immediate culture or frozen down for later use. For cryopreservation, add an equal amount of cryopreservation medium.
Aliquot the cells into cryovials and transfer them to a 80 degrees Celsius freezer immediately. In this procedure, prepare five mL of IMDM medium in a 15 mL tube. Quickly thaw the frozen PB MNCs in a 37 degrees Celsius water bath before transferring them to the tube with IMDM medium.
Centrifuge the sample at 400 x g for five to ten minutes. Afterward, remove the supernatant and resuspend the cell pellet in erythroid medium. Then count the cells under a microscope using a hemocytometer.
Subsequently, culture PB MNCs in a non-tissue culture treated six well plate with two mL of medium per well at 37 degrees Celsius in a 5%Carbon Dioxide, humidified incubator. One day three and day five, add one mL of fresh erythroid medium directly in each well without changing the medium. On day six, harvest PB MNCs for nucleofection.
One day before nucleofection, precoat the tissue culture treated six well plate with 0.1%gelatin at 37 degrees Celsius for 20 minutes. Thaw the inactivated MEF feeder cells in a 37 degrees Celsius water bath and immediately transfer them to a 15 mL tube containing five mL of MEF medium. Next, centrifuge them at 400 x g for five minutes and remove the supernatant.
Then remove the gelatin from the six well plate and resuspend the cell pellet in MEF medium. Afterward, seed the inactivated MEF cells in two mL of MEF medium in each well of a gelatin pre-treated six well plate and culture them at 37 degrees Celsius in a 5%carbon dioxide humidified incubator. On the day of nucleofection, aspirate the MEF medium and replace it with one mL of erythroid medium.
Then pre-equilibrate the culture plate for 10-30 minutes at 37 degrees Celsius in a 5%carbon dioxide humidified incubator. Add two micrograms PEV OCT4 to Sox2, one microgram PEV MYC, one microgram PEV KLF4, and 0.5 micrograms PEV Bcl-XL in a 1.5 mL sterile tube. Heat the tube at 50 degrees Celsius for five minutes to prevent contamination and then cool it down to room temperature.
Afterward, add 57 microliters of nucleofection buffer and 13 microliters of supplement buffer to the sample. Harvest two times ten to the sixth cultured PBMNCs to a five mL tube by centrifugation at 200 x g for seven minutes. After that, remove the supernatant and add the plasmid and nucleofection buffer mix to the cell pellet.
Then mix well by flicking the tube with a finger. Next, transfer the DNA in cell suspension to the cuvette provided by the kit and cap the cuvette. Select program U-008 on the nucleofection device.
Subsequently, insert the cuvette into the holder and press OK.Afterward, take the cuvette out of the holder. Add 0.5-1 mL of prewarmed erythroid medium to each cuvette and transfer the cells to the pre-equilibrated MEF plate immediately. For some samples, you may get thousands of colonies from one mil of blood.
If you wish to pick single colony for further culture you may need to seed one extra well with only 1 million cells. Right before placing the hypoxia chamber into the incubator, gently rock the chamber several times to create an even distribution of cells in the culture wells. Then transfer the plate to a hypoxia chamber.
Flush the chamber with a mixed gas for one to two minutes at a rate of 20 Liters per minute. Afterward, seal the chamber and culture the sample at 37 degrees Celsius. On day two post-nucleofection, add two mL of iPSC medium directly into each well.
On day four post-nucleofection, remove three mL of medium and add two mL of fresh iPSC medium. At this stage, most of the live cells have attached to the feeder layer. After day six post nucleofection, change the medium every two days by leaving 500 microliters of spent medium and adding two mL of fresh E8 medium supplemented with 0.25 millimolars sodium butyrate until days 14-18.
Shown here is a schematic of the protocol for reprogramming peripheral blood cells. This image shows the AP standing in bowl, and this one shows a typical ESC-like iPSC colony on day 14 after PB MNCs nucleofection. Here are representative FACS diagrams of iPSCs at passage five expressing TRA-1-60 or SSEA4.
Here are the representative confocal images of iPSC colonies expressing NANOG and OCT4. The images here show the H&E staining of terratoma, comprising all three germ layers. After watching this video, you should have a good understanding of how to generate integration free iPS cells from human peripheral blood mononuclear cells.
Once mastered, one can generate large quantities of iPS cells in three weeks, so hands on time will be less than three hours. We have developed a simple, episomal vector system for efficient blood cell reprogramming. We believe this affordable system can be easily adopted, in particular in small labs with limited resources.
Don't forget that working with human blood and plasmas can be hazardous, and precautions must be taken such as wearing gloves and lab coats while performing this procedure.
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