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 in ice and add it to the suspended cells. Add Sendai virus to the cells based on the manufacturer's recommendations.
   6. Seal the plate with a sealing film and centrifuge it at 1,150 x g for 30 min at 30 °C.
   7. After centrifugation, incubate the cells at 37 °C in 5% CO₂ overnight (O/N).
2. Cell transfer to feeder matrix (Day 1)
   1. The next day, coat a 24-well plate with vitronectin. Dilute the vitronectin solution in PBS for a final 5 µg/ml concentration. Add 1 ml of vitronectin to a well of a 24-well plate and incubate it at RT for at least 1 hr. Remove the coating solution before use. Coated plates can be stored in RT for 3 days.
   2. Transfer all the media containing the cells and the virus to the coated well.
   3. Collect the remaining cells with an additional 0.5 ml of fresh blood cell media and add it to the cell-containing well.
   4. Centrifuge the plate at 1,150 x g for 10 min at 35 °C.
   5. After centrifugation, remove the supernatant, add 1 ml of iPSC media, and maintain the cells at 37 °C in 5% CO₂ O/N.
3. Second cell transfer (Day 2)
   1. Coat the wells of a new 24-well plate with 5 µg/ml vitronectin, as described in step 1.2.1. Use one well of the plate for each transduction.
   2. Transfer the cell suspension from the first plate to the newly coated vitronectin plate.
      NOTE: If not needed, suspension cells can be discarded. The procedure mentioned in step 2.3 can be repeated 2-3 times with the suspension cells. If the steps are repeated, harvest the cells.
   3. Meanwhile, add 1 ml of iPSC (induced pluripotent stem cells) media to the well of the first plate, for maintenance, and incubate it at 37 °C in 5% CO₂ O/N.
   4. Maintain the attached cells at 37 °C and 5% CO₂ and perform a daily media change with fresh iPSC media. Colonies will appear on days 14-21 after transduction.
   5. Centrifuge the newly coated plate containing the suspension cells at 1,150 x g and 35 °C for 10 min.
   6. After centrifugation, incubate the cells at 37 °C in 5% CO₂ O/N.
   7. The next day, remove the supernatant and replace it with fresh iPSC media.
      NOTE: The procedure mentioned in step 1.3 can be repeated 2-3 times with the suspension cells. If the steps will be repeated, harvest the cells in the supernatant and repeat step 1.3.
   8. Maintain the attached cells with daily media changes until 80% confluency is reached.

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