iPSC Reprogramming Protocol Using Sendai Virus-Mediated Gene Transfer

This protocol allows the efficient generation of integration-free iPS cells from a small amount of peripheral blood (<1 ml). Peripheral blood mononuclear cells (PBMCs) are cultured to expand the erythroblast (EB) population. They are then used to derive iPS cells using four recombinant Sendai viral vectors, expressing the four reprogramming factors Oct4, Sox2, Kfl4, and c-Myc.

• Thaw 1 vial of PBMCs into 10 mL of QBSF-60 and centrifuge at 300 RCF for 10 min. Resuspend the pellet in 2 mL of EM + primocin and transfer to 1 well of a 12-well plate, incubate at 37°C.
• Transfer cells to a sterile 15 mL conical tube and wash 1×with 1 mL of QBSF-60 to collect non- and loosely adherent cells. Scrape the well with a cell scraper to collect all cells if necessary.
• Centrifuge cells at 300 RCF for 10 min and resuspend in 2 mL of fresh EM + P/S. Continue to culture in 1 well of a 12-well plate.
• EM media expands the erythroblast population from PBMCs. A 2-fold expansion should occur in about 9-12 days with an initial decrease in cell number. When cells are noticeably dividing and have reached the appropriate density, perform FACS to monitor erythroblast expansion using antibodies to erythroblast cell surface markers. When more than 90% of the cells express CD36 and CD71, you can proceed to transduction.
• Transfer cells to a sterile 15 mL conical tube and wash 1×with 1 mL of QBSF to collect non-adherent and loosely adherent cells.
• Count cells. Centrifuge 2.5×105 cells in a 15 mL conical tube add 1 mL of fresh EM+P/S plus viruses and transfer to one well of a 12-well plate.
• Spinoculation. Centrifuge plate at 2250 rpm at 25°C × 90 min.
• While centrifuging, divide the remaining cells into two tubes, centrifuge, and save one tube for RNA and one for DNA.  Move the centrifuged plate to the incubator and maintain it at 37°C, 5% CO₂, 5% O₂, and 90% N₂.
• Following 6-8 hours, add 1 mL of fresh EM + P/S to cells (for a total of 2 ml of EM + P/S).
• Collect and centrifuge cells at 300 RCF in a conical tube for 10 min and resuspend in 2 mL of fresh EM+P/S.  Plate MEFs onto 0.1% gelatin-coated 6-well TC plates.
• Collect cells into a 15 mL conical tube and centrifuge at 300 RCF for 10 min. Resuspend cells in 6 mL of iPSC media plus growth factors as in EM medium.
• Plate 1 mL/well into a 6-well MEF plate. Add 1.5 mL/well of iPSC media plus growth factors for a total of 2.5 mL/well. Centrifuge plate at 500 rpm at 25°C×30 min.
• Feed cells on day 5 with 2.5 mL of iPSC media w/o growth factors. Feed cells on day 7 with 2.5 mL of iPSC: hESC (1:1) media. Aspirate and discard floating cells with each feed.
• Once small colonies appear, feed cells daily with 2 mL of hESC media. Add additional MEFs as needed (1×/wk).
• Each colony is picked into one well of a 12-well or 24-well plate pre-coated with MEFs on gelatin in 1 mL/well of hESC media containing 10 μM ROCK inhibitor.
• Feed cells in two days, then daily thereafter with 1 mL of hESC media, and continue to expand clones for characterization.

Creative Bioarray Relevant Recommendations

• Creative Bioarray offers a broad range of iPSC reprogramming kits that are useful to stem cell scientists interested in performing reprogramming trials, including the Episomal iPSC Reprogramming Kit, RNA iPSC Reprogramming Kit, Retrovirus iPSC Reprogramming Kit, Lentivirus iPSC Reprogramming Kit, and iPSC Protein Reprogramming Kit.

4 Sendai viral vectors each expressing Oct3/4, Sox2, Klf4, and c-Myc are used for transduction. We typically transduce 2.5×10⁵ cells with 10 MOI of each of the four viruses (0.01%-1% efficiency).

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