Home › Cell Biology › Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions
Cell Biology JoVE (Open Access) Citable · DOI

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

DOI: 10.3791/53225-v
What you'll learn
  • ✓ Prepare and activate human peripheral T cells for reprogramming
  • ✓ Generate iPSCs from T cells using Sendai virus vectors in feeder-free culture
  • ✓ Expand and maintain T cell-derived iPSCs in matrigel conditions
Protocol

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Difficulty
advanced
Total time
~3-4 weeks (T cell activation ~3-5 days, reprogramming ~2-3 weeks, expansion ongoing)
Model organism
Human peripheral T cells (primary)
Biosafety
BSL-2

Steps

1
Prepare and activate human peripheral T cells

Isolate and culture human peripheral T cells with activation stimuli to prepare them as starting material for reprogramming. This step conditions the T cells to a metabolic state amenable to pluripotency induction.

▶ 01:19
2
Transduce activated T cells with Sendai virus vectors

Introduce Sendai virus vectors encoding reprogramming factors (Oct4, Sox2, Klf4, c-Myc) into activated T cells on matrigel-coated plates in feeder-free conditions. Monitor transduction efficiency and initial cell responses.

▶ 04:05
3
Expand derived iPSC colonies in feeder-free culture

Culture emerging iPSC colonies on matrigel substrate, passage reprogrammed cells, and maintain pluripotency in defined medium conditions without feeder cell support.

▶ 07:11
4
Characterize generated T cell-derived iPSCs

Validate iPSC identity and pluripotency through morphology assessment, marker expression, and functional assays to confirm successful reprogramming from peripheral T cells.

▶ 08:28
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