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, a