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

Abstract

Source: Rim, Y. A., et al. Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation. J. Vis. Exp. (2016).

This video demonstrates the generation of induced pluripotent stem cells (iPSCs) from human peripheral blood mononuclear cells using Sendai virus–mediated reprogramming. The viral transcription factors activate pluripotency networks and suppress lineage genes, enabling a subset of cells to acquire adhesion properties and establish iPSC colonies. The protocol enriches and maintains these reprogrammed cells through selective matrix binding and daily media renewal.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Transduction by Sendai Virus

1. Transduction (Day 0)
   1. Collect and transfer the blood cells to a 15-ml conical tube and count them using a hemocytometer.
   2. Prepare 3 x 10⁵ cells per transduction and centrifuge the cells at 515 x g for 5 min at RT (room temperature).
   3. Discard the supernatant by suction and resuspend the cells in 0.5 ml of blood cell media.
   4. Transfer the cells to a well of a non-coated 24-well plate.
   5. Thaw the Sendai virus mixture i....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plasticware
50 ml Cornical Tube	SPL	50050
15 ml Cornical Tube	SPL	50015
10 ml Disposable Pipette	Falcon	7551
5 ml Disposable Pipette	Falcon	7543
12-well Plate	TPP	92012
24-well Plate	TPP	92024
iPSC Generation and Culture Materials
CytoTune-iPSC Sendai Reprogramming Kit	Life Technologies	A16518
TeSR-E8 Media	STEMCELL Technologies	5940	iPSC media
Vitronectin	Life Technologies	A14700
TrypLE express (TrypLE)	Life Technologies	12604-039
ReleSR	STEMCELL Technologies	12604-039	Colony detaching solution