Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

The overall goal of this procedure is to generate induced pluripotent stem cells from human peripheral blood mononuclear cells obtained from frozen buffy coats using a cost-effective and virus free protocol. This is accomplished first by the isolation of PB MNCs from Buffy coats after whole blood centrifugation without a density gradient followed by expansion in a specific blood culture medium. The second step is to transfect the Buffy coat PB MNCs with episomal plasmids by electroporation.

The transfected PB MNCs are then plated onto fresh mouse embryonic fibroblast feeder cells. The final step is to manually pick induced pluripotent stem cells from the culture and transfer them onto new feeder cell plates for expansion. Ultimately, the efficient reprogramming of PB MNCs into induced pluripotent stem cells with a typical human morphology can be shown by immunofluorescence and measuring gene expression.

The main advantage of this technique over existing methods is that the degeneration of virus free IPCs from frozen OTs reduces cost working time and manual steps for operators during sample processing. Another advantage to this technique is that it allows for the use of frozen OTs stored in large scale Biobank gathered from population study as a source material for IPS production. Begin with collecting eight milliliters of venous peripheral blood into a sodium citrate buffered plastic tube.

Store the blood at 25 degrees Celsius and process it within 12 hours. The first processing step is to centrifuge the blood at 2000 Gs for 15 minutes at four degrees Celsius. Use a swinging bucket rotor and do not apply the centrifugal brakes from the separation.

Collect the cloudy buffy coat. This is the layer between the upper plasma phase and the lower phase. The buffy coat is the enriched P-B-M-N-C fraction transfer about 500 microliters from each eight milliliter sample to a cryo vial tube.

Now Resus suspend the buffy coat by adding an equal volume of two x freezing medium, and then freeze the vial in a controlled rate freezing container at minus 80 degrees Celsius for up to one week. For longer storage, the cells can be stored in liquid nitrogen to begin thaw A frozen one milliliter suspension of PB MNCs obtained from frozen buffy coats in a 37 degree Celsius water bath. Then dilute the cells to two milliliters with PBS.

Next, move the dilution to a 50 milliliter tube and add 10 milliliters of red cell lysis buffer. Let this mixture incubate at room temperature for about 10 minutes. After 10 minutes, increase the total volume to 50 milliliters with sterile PBS.

Then spin down the cells at 300 Gs for 10 minutes at room temperature. After the spin, discard the supernatant and wash the pellet with 50 milliliters of PBS. Now spin the cells again and re suspend them in one milliliter of PBM and C medium.

Take an aliquot of resuspended cells and measure the cell density using a counting chamber. Then adjust it to 2 million cells per milliliter. Now establish a suspension culture.

Load the cells into a well of a standard 12 Well plate and incubate them for 48 hours. Change the media every other day, aspirate the cells using a pipette and transfer them to a 15 milliliter conical tube. Centrifuge the loaded tube at 300 GS for 10 minutes at room temperature.

Then discard the supernatant and resuspend the cells in one milliliter of fresh P-B-M-N-C medium. A red blood cell contamination is usual during the first days of PB MNC culture. Before returning the cells to a new, well check them under an inverted microscope when they are at about 80%Co fluency split the culture during the transfer.

Two weeks after starting the PB MNC culture, the cells should be ready to transfect. Prepare four plasmids one carrying T three four and S-H-R-N-A against P 53 1 carrying SOX two and KLF four one carrying lmic and lin 28 and one plasmid carrying EGFP. To begin collect the PB MNCs in a 15 milliliter conical tube and count the number of viable cells using triam blue.

Then prepare 2 million viable cells, centrifuge them and resuspend the cells in a transfection mix containing 100 microliters of resuspension buffer T and one microgram of each of the four plasmids. Now fill the transfection container with three milliliters of electrolytic buffer. E two, aspirate the transfection mix into a 100 microliter tip and eject the sample vertically into the prepared electroporation tube.

Then ate the cells using three pulses After the electroporation, re suspend the cells in two milliliters of pre-war PBM and C medium with 0.25 millimolar sodium butyrate. Then count the number of viable cells as before and transfer the suspension into one well of an uncoated six well plate. Set the plate to incubate with gentle agitation for 24 hours.

Maintain the transfected cells without splitting them for three days from the day of transfection on opposite days. Replace the medium with two milliliters of freshly prepared P-B-M-N-C medium with 0.25 millimolar sodium butyrate one day in advance of establishing the co-culture, prepare the MES first coat, the wells of a six well plate with one milliliter of basement membrane matrix and let the plates incubate at 37 degrees Celsius for 15 minutes. Then aspirate the basement membrane matrix and plate 200, 000 MEFs into each well with two milliliters of DMEM containing 10%FBS the next day, collect the PB MNCs in a one milliliter tip.

Transfer them to a 15 milliliter conical tube and spin them down. Discard the supernatant and resuspend the pellet in two milliliters of fresh medium. Then plate the cells onto the meth coated wells and incubate the plate After two days.

Replace the medium with two milliliters of IPSC medium with 0.25 millimolar sodium butyrate, and continue replacing the IPSC media every other day. After about 22 to 25 days, the first colonies of I PSCs should be visible by 30 to 35 days. The colonies should be ready to pick and replay the day before packaging the IPSC colonies plate meth feeder cells onto a 12.

Well plate at 125, 000 cells per well and incubate them overnight. The next day, replace the medium on the meth cells with one milliliter of supplemented IPSC medium. Then under a dissecting microscope, use a needle to pick one colony at a time for larger colonies that form small clumps.

Transfer them individually by pipette, transfer one colony into each well of the meth coated plate and incubate the plate to passage. The IPSC colonies initially use the same picking technique, but after two or three passages, use the enzyme based procedure isolated PB MNCs showing a typical rounded shape were expanded in specific blood culture medium for 14 days, and then transfected with episomal plasmids 10 to 15 days post transfection. Small rounded bright colonies with defined margins and embryonic stem cell-like morphology began to appear about 20 to 30 days after the transfection.

Highly compact IPSC colonies with sharp edges appeared and increased in size until they were large enough to pick for the first few packaging steps. The IPSC colonies maintained their undifferentiated state and showed a typical human embryonic stem cell morphology. Increased magnification shows individual cells within the colonies and the high nucleus to cytoplasm ratio.

Selected IPSE clones were passed about 10 to 15 more times. About 20 meta phases from several independent cultures were analyzed for genetic stability. All the samples showed a normal gram after five to 10 in vitro expansions.

These clones were characterized for their stemness and pluripotency. The clones were positive for alkaline phosphatase staining. Quantitative R-T-P-C-R verified that the clones expressed endogenous pluripotent genes at much higher levels than the starting PB MNCs.

While levels of endogenous cmic and KLA four remained more or less the same expression of exogenous episomal transgene could not be detected in IPCs, thus indicating the successful activation of endogenous pluripotent genes. Finally, immunofluorescence analysis revealed that the IPCs expressed pluripotency markers such as OC three four SSEA four TRA 180 1, SOX two, and TRA one 60. After watching this video, you should have a good understanding of how to generate IPCs from ko, isolated from human peripheral blood in a cost effective manner, and using non integrating episomal plasmids.