iPS cell induction from human T lymphocytes	  Keisuke Okita<day -1>
Prepare 6 well plate coated with MEF feeder cells. (3 X 105 cell/well)<day 0>1. Add 2 mL of T cell stimulation medium to each well 6 well plates seeded with MEF feeder cells andstore at 37˚C, 5% CO2.
2. Use Ficoll-paque Plus to purify mononuclear cells:a. To 10 mL of anti-coagulated blood (EDTA), add 10 ml of PBS.
b. In two 15 mL tubes, add 5 mL Ficoll-Paque and the gently add 10 mL of blood+PBS.
c. Spin tubes at 400 X g for 30 mins. at 18°C. Use slow acceleration and no brake.
d. Remove the plasma for the top fraction without disrupting the mononuclear cells at the interface.
e. Transfer the cells at interface to a 15 mL tube and add 12 mL of PBS.
f. Spin at 200 X g for 10 mins. at 18°C (no brake).
g. Resuspend cells in 3 mL of X-vivo 10 medium and count cells.
h. Prepare aliquots of 3 x 106 cells in 1.5 mL tube.
g. Spin at 200 X g for 10 mins. at 18°C (no brake). During spin, prepare nucleofection solution.
3. Nucleofection:
Amaxa Tcell Solution 81.8 µL
Supplement

18.2 µL

Plasmid mix (and GFP control) 3 µL
a. Aspirate supernatant completely by hand using a pipette.
b. Add nuclefection solution and suspend cells, be careful not to create any bubbles.
c. Perform nucleofection using pre-stored program V-024.
4. Plating, 106 to 104 cells per well:a. Immediately following nucleofection, add 800 µL of X-vivo 10 to the electroporation cuvette, andharvest the cells. Metal ions in the nucleofection solution are toxic to cells!
b. Plate the cells to incubated 6-well plate ranging from 90 µL (3 X 105 cells) to 9 µL (3 X 104 cells).

<day 2, 4, and 6>
Add additional 1.5 mL of primate ES cell medium per well.

<day 8>Replace medium with 1.5 mL of primate ES cell medium per well. Most T cells are floating. Confirm
that they are proliferating. If not, don't change medium for 2 more days.

* Medium replacement is performed every 2 days.

<day 20 to 30>
Pick colonies of about 2 mm diameter.

Reagents
Ficoll-paque Plus (GE Healthcare, 17-1440-02)

Dynabeads® Human T-Activator CD3/CD28 (Invitrogen, 111.31D)X-vivo 10 (Lonza, 04-380Q)
Human IL-2, recombinant (PeproTech, 200-02)
T cell stimulation medium in 1 well
Washed Dynabeads 5 µL
X-vivo 102 mL

IL-230 U/mL

* Preparation : <100 wells
Add Dynabeads (5 X number of wells) µL into 1 mL of X-vivo 10 in 1.5 mL tube.
Vortex 5 secs. then quick spin.
Place to magnetic rack for 1 min.
Remove supernatant and resuspend in (2 X number of wells) mL X-vivo 10 medium.
Add 30 U/mL IL-2.
Primate ES Cell Medium (ReproCELL, RCHEMD001)	  * Add bFGF (4ng/mL) before using.
Amaxa Human T Cell Nucleofector Kit (Lonza, VAPA-1002)
Nucleofector 2b (Lonza, AAB-1001)
Plasmid (Addgene, http://www.addgene.org/Shinya_Yamanaka)
Use following plasmid mixtures. Set 1 shows high efficiency. In set 2, we omitted WPRE
sequence and replaced shRNA against p53 with dominant negative form of mouse p53, which
exist in set 1.

Plasmid set 1

Plasmid set 2

pCXLE-hOCT3/4-shp53-F

0.83 µg

pCXLE-hSK

0.83 µg

pCXLE-hUL

0.83 µgpCXWB-EBNA1

0.5 µgpCE-hOCT3/4

0.63 µg

pCE-hSK

0.63 µg

pCE-hUL

0.63 µg

pCE-mp53DD

0.63 µgpCXB-EBNA10.5 µg

Optional
If you want to make iPS cells without genomic rearrangement, use following medium instead of T cellstimulation medium. But the induction efficiency is very low (1-10 colonies from 1 X 106 cells)
α-MEM containing 10% FCS and 10 ng/mL of cytokines (IL-3, IL-6, G-CSF, and GM-CSF)

Acknowledgement
I appreciate Dr. Eric Rulifson for his help of translation.

Reference1.

Okita, K., et al. An Efficient Non-viral Method to Generate Integration-Free Human
iPS Cells from Cord Blood and Peripheral Blood Cells. Stem Cells. 2012 Nov 29

2.

Okita, K., et al. A more efficient method to generate integration-free human iPS cells.

Nat Methods. 8, 409-12 (2011).