Method Article
Somatic cell reprogramming using NF-κB inhibitory strategies
https://doi.org/10.1038/protex.2015.057
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This protocol describes somatic cell reprogramming methodologies using NF-κB inhibitors step-by-step. Thus, we have used NF-κB pharmacological and genetic inhibitory strategies for the reprogramming of human fibroblasts from normal and accelerated ageing. These strategies can be used for implementing reprogramming methodologies in ageing conditions.
• HEK-293T (ATCC).
• Human primary fibroblasts (Coriell cell repository).
• IKK-VII inhibitor (Selleckchem).
• Sodium salicylate (Sigma).
• pMX-OCT4, SOX2, KLF4 and c-MYC plasmids (Addgene).
• pBABE-IκBα-SR and IKK2-KI plasmids (Addgene).
• pCL-Ampho retroviral packaging plasmid (Addgene).
• HEK-293T medium: DMEM medium supplemented with 10% of fetal bovine serum (FBS).
• Human fibroblasts medium: DMEM medium supplemented with 15% FBS, nonessential amino acids, 2-mercaptoethanol and sodium pyruvate.
• Human iPSC medium: DMEM-F12 medium supplemented with 20% knockout serum replacement (KSR), nonessential amino acids, 2-mercaptoethanol, penicillin/streptomycin, GlutaMax, and bFGF (4 ng/mL).
Cell reprogramming with NF-κB pharmacological inhibitors
Retroviral vectors expressing human OCT4, SOX2, KLF4 and c-MYC were transfected in HEK-293T cells (5x106 cells per 10 cm plate each) along with packaging plasmids to produce retrovirus.
Human primary fibroblasts were seeded at a density of 1x105 cells per well in 6-well plates.
Twenty four h after plating, cells were infected with retroviral supernatants and the same procedure was repeated every 12 h for 2 days (a total of four additions) (Figure 1).
The day after last infection (day 3), medium was replaced with fibroblasts medium, and kept for three more days. NF-κB inhibitors were added at this point and maintained along the reprogramming process (IKK-VII, 0.5 µM and sodium salicylate, 0.2 mM) (Note 1).
At day 7, cells were trypsinized and reseeded on feeder plates (Note 2).
On day 8, medium was changed to human iPSC medium.
Cultures were maintained with daily medium changes for 4 weeks. At that point, colonies were isolated and expanded on a feeder layer or Tra-1-60 stained for evaluating reprogramming efficiency.
Cell reprogramming with NF-κB genetic inhibitors
Retroviral vectors expressing human OCT4, SOX2, KLF4, c-MYC, IκBα-SR or IKK2-KI were transfected in HEK-293T cells (5x106 cells per 10 cm plate each) along with packaging plasmids to produce retrovirus.
Human primary fibroblasts were seeded at a density of 1x105 cells per well in 6-well plates.
Twenty four h after plating, cells were infected with retroviral supernatants and the same procedure was repeated every 12 h for 2 days (a total of four additions).
Cells were trypsinized at day 7 and reseeded on feeder plates. On day 8, medium was changed to human iPSC medium and cultures were maintained with daily medium changes for 4 weeks.
Notes
We have observed that NF-κB-inhibition during the first two weeks is sufficient to achieve the complete effect on reprogramming efficiency. Thus, NF-κB inhibitors could be removed from iPSC medium with no effect on reprogramming efficiency after that time point.
Human iPSCs were cultured on mitomycin-inactivated mouse embryonic fibroblasts (MEFs).
No conflict of interest
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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