Sendai Virus–Mediated Reprogramming of Human Blood Cells into Induced Pluripotent Stem Cells

Take a culture of human peripheral blood mononuclear cells (PBMCs).

Add Sendai virus encoding reprogramming transcription factors. Cover and seal the plate.

Centrifuge to enhance virus–cell contact and incubate.

Following virus fusion, the viral RNA expresses transcription factors that activate cellular pluripotency genes and repress lineage-specific genes to initiate reprogramming.

During this, a subset of transduced PBMCs converts into induced pluripotent stem cells (iPSCs) and expresses adhesion proteins.

Transfer them to a matrix-coated well and spin down the cells. Replace the supernatant with a growth medium.

Upon incubation, iPSCs adhere to the matrix, while transduced non-adherent PBMCs remain suspended.

Transfer the suspension to a coated well and centrifuge. Add the medium to the original well and incubate both.

The remaining transduced PBMCs initiate reprogramming, convert into iPSCs, and adhere. This step maximizes the retention of reprogrammed cells.

Replace the medium daily to sustain iPSC growth.

In this procedure, transfer the blood cells to a 15-milliliter conical tube, and count them using a hemocytometer. 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.