Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

The overall goal of the following experiment is to generate integration free induced pluripotent stem cells from a small amount of peripheral blood. This is achieved by culturing isolated peripheral blood mononuclear cells in defined growth factors to yield an erythrocyte progenitor population that is very responsive to reprogramming. As a second step non integrating episomal plasmids carrying reprogramming genes are introduced into the erythroblasts by nucleo affection to initiate the reprogramming process.

Next, the nucle affected cells are plated onto a layer of irradiated mouse embryonic fibroblasts to allow for continued reprogramming ultimately successfully. Reprogrammed induced pluripotent stem cell colonies can be identified based on their morphological characteristics, alkaline phosphatase activity, positive immunochemistry for pluripotency markers and expression of endogenous pluripotency genes. The main advantage of this technique over existing methods like viral based reprogramming, is that this protocol ensures consistent generation of integration free IPSC Demonstrating the procedure today will be Jesse, myself, and Gretchen Johnson.

A technician in the lab Begin by diluting peripheral blood at a one-to-one ratio in DPBS. Then in a 15 milliliter round bottom polystyrene tube, carefully layer seven milliliters of the diluted blood onto three milliliters of room temperature fi call hi pack. After separating the sample for 30 minutes at 400 GS and room temperature, the pbmc will have settled in the cloudy white interface layer between the plasma and the fial high pack.

Using a sterile transfer pipette, transfer the P BMCs into a 15 milliliter conical tube and bring the volume up to 10 milliliters with fresh DPBS. Then after counting the cells, transfer two times 10 to the six P BMCs into a new 15 milliliter conical tube and spin down the remaining cells, freeze the pelleted cells at a two times 10 to the six pbmc per milliliter concentration in 90%fetal bovine serum with 10%dimethyl sulfoxide. Then after spinning down the P BMCs that were set aside, we suspend the pellet in two milliliters of freshly prepared expansion medium and plate them into one well of a 12 well tissue culture plate incubate the cells in a 37 degrees Celsius humidified incubator with an atmosphere of 5%oxygen, 5%carbon dioxide, and 90%nitrogen.

Then on day three and six, replace the media by collecting the cells from the well. Washing the well with two milliliters of QBSF 60 to collect any remaining cells, pelleting the cells by centrifugation, and then resus suspending the pellet in two milliliters of fresh expansion medium. Then return the cells to the same 12 well plate and continue the incubation On day nine, pipette the reprogramming plasmids into a sterile 1.5 milliliter einor tube as outlined in the table.

Next, mix the nucleo affection solution with the supplements supplied by the manufacturer and transfer the mixture into a sterile 1.5 milliliter upend DPH tube. Then dispense two milliliters of expansion medium into one well of a new 12 well plate and equilibrate the medium in the humidified 37 degree Celsius incubator until the cells are ready. Collect the cells, wash the cell culture well as just demonstrated and wash the cells in five milliliters of DPBS.

Then resus, suspend the cell pellet in 100 microliters of Nucle affection solution plus supplement taking care to avoid generating bubbles and transfer the cell suspension into the tube containing the reprogramming plasmid. Mix the cells by pipetting up and down once and transfer the entire solution to the bottom of a nucle affection vete. Then place the vete into the Nucle affection machine and run program T 0 1 9.

Now add 100 microliters of the EQUILIBRATED expansion medium to the nucle affection vete, and immediately collect the entire sample avoiding any white floating dead cell debris and deposit it into the well of the prewarm medium. Then place the cells back into the humidified incubator on day 10 or 11. Coat each well of a six well tissue culture plate with gelatin with one milliliter of 0.1%gelatin in HBSS, and then place the plate in the humidified incubator or at least five minutes during the incubation transfer one vial of thaw, two times 10 to the six 3000 centigrade irradiated mouse, embryonic, fibroblasts, or mes into 10 milliliters of prewarm meth media, and then centrifuge the cells while the cells are spinning down.

Aspirate the gelatin solution from the six well plate, and then resuspend the mes in 12 milliliters of fresh meth media. Immediately dispense two milliliters OFMs into each well of the gelatin coated.Six. Well plate and place the plate back into the humidified incubator until day 12.

On day 12, collect the nucle affected cells from the well into one 15 milliliter conical tube, washing the well as demonstrated to collect any remaining cells. After spinning down the cells, we suspend the pellet in 12 milliliters of freshly prepared reprogramming medium, and dispense two milliliters of cells into each well of feeder cells. In the six well plate next centrifuge the plate for 30 minutes at 75 GS and room temperature, and then place the plate back into the humidified incubator, changing the medium every other day for one to two weeks.

Once the inducible pluripotent cell colonies appear, begin feeding the cells with freshly prepared human embryonic stem cell medium containing histone deacetylase inhibitors three days after nucle affection and before plating, the nucle affected cells onto the inducible meth. The efficiency of a successful NU nuclear affection should be estimated by fluorescence microscopy For EGFP, for example, shown here is a typical nuclei affection experiment with approximately five to 10%of the total cell population expressing EGFP reprogrammed inducible pluripotent stem cell colonies will begin to appear approximately two weeks after NU nuclear affection. The colonies are generally circular with well-defined borders and may be identified by their characteristic human embryonic stem cell morphology, including small tightly packed cells with a high nuclear to cytoplasmic ratio and visible nucleoli of the representative lines that have been completely characterized.

All test positive for alkaline phosphatase activity express the standard pluripotency markers and reactivate endogenous pluripotency genes Once mastered. This technique can consistently generate integration free IPSC in four weeks if it is performed properly. Following this procedure, differentiation studies can be performed to answer questions about any tissue type.