CiRA | M&M

July 17, 2008
Ver. 2

Improved Method for Generation of Mouse iPS Cells
Center for iPS Cell Research and Application (CiRA)
Institute for Integrated Cell-Material Sciences (iCeMS)
Kyoto University

Since the first report of mouse induced pluripotent stem (iPS) cells in 2006, questions havebeen raised regarding the method of retroviral transduction and the necessity of antibiotic
selection, and various refinements have been made to the method of induction. The
pluripotency of Nanog-GFP+ mouse iPS cells has been demonstrated to be sufficient tocontribute to the germline in chimeric mice. Modification of plating density has also made it
possible to induce iPS cells from wildtype mouse cells in the absence of antibiotic selection
as well as by using only three factors, without t the oncogene c-Myc. In this protocol, we
describe a method for the induction of pluripotency by gene transfection into mouse
fibroblasts, as was previously detailed in ref. 1, with refinements to the previously publishedprotocol shown in red letters.

1. Materials
OPTI-MEMI (Invitrogen 31985-062)
See ref. 1 for other necessary materials, such as cells, vectors and reagents.

2. Cell culture
See ref. 1 for culture methods for MEF and TTF cells and PLAT-E packaging cells.

3. Establishment of mouse iPS cells
3.1.1 Construction of retroviral vectors: preparation of PLAT-E cells (Day 1)
As described in ref. 1. Follow the table below when plating PLAT-E cells.

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Dish size

Number of cells

100 mm dish3.6 X 106

60 mm dish1.5 X 106

35 mm (6-well plate)

6 X 105

Also note that it is necessary to prepare a sufficient number of PLAT-E cultures in order to beable to transfect each plate with a single plasmid in step 3.1.2 (below). For example,experiments using 4 genes (Oct4, Sox2, Klf4 and c-Myc) as well as Ds-Red require 5
separate dishes of PLAT-E cells.

3.1.2 Construction of retroviral vectors: Transfection into PLAT-E cells (Day 2)
The following procedures are for PLAT-E cells in 100mm dish. In the case of PLAT-E cells
in 60mm and 35mm dishes, use 1/3 and 1/6 volume of the transfection reagent in 100 mm
dish, respectively.

•Inject 0.3 ml of OPTI-MEMI into separate 1.5 ml tubes, one for each gene to betransfected.

•Add 27 μl of Fugene 6 transfection reagent by gently tapping to mix, and incubate atroom temperature for 5 minutes.

•

Add 9 μg of pMXs plasmid DNA (as a negative control for Oct4, Sox2, Klf4, c-Myc andDsRed) dropwise into separate solutions of Fugene 6/OPTI-MEMI, and mix bytapping with finger. Incubate at room temperature for 15 minutes.

IMPORTANT: Use appropriate controls. At CiRA, vectors encoding EGFP or DsRed are used tomonitor transfection efficiency, and we routinely achieve efficiencies of 80% or higher.
High-efficiency transfection is essential for the induction of iPS cells.

•On the next day (Day 3), aspirate the Fugene 6 transfection agent-containing
medium, add 10 ml of 10% fresh fetal bovine serum (FBS), and return to theincubator.

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3.2 Preparation of MEF, TTF cells (Day 3)•See ref. 1.

3.3 Retroviral transfection (Day 4)•See ref. 1. Make a mixture of equal parts of each of the retroviruses; however, whenattempting to establish iPS cells without antibiotic selection, use a ratio of 1:1:1:3
for Oct4, Sox2, Klf4 and DsRed. Aspirate the medium from the culture dish andreplace with the virus-containing medium and incubate overnight at 37°C, 5% CO2.

•

After 24 hours (Day 5), aspirate the medium and replace with fresh 10% FBS.

•After two more days (Day 7), aspirate the medium and replace with fresh 10% FBS.

3.4 Re-seed of MEFs to mitomycin C-treated SNL feeder cell culture•

Aspirate medium and wash with 10 ml PBS.

•Aspirate PBS, ad 1 ml 0.05% trypsin/0.53 mM EDTA, and incubate at 37°C for 10minutes.

•Add 9 ml medium, suspend the cells such all cells are separated (single cells), andcollect in a 50 ml tube.

•Calculate the number of cells, and re-seed the cells to a 100 ml culture dishcontaining SNL feeder cells following the table below, and culture overnight at 37°C,
5% CO2.

4 factors

3 factors (minus c-Myc)

Cell number

0.5 ~ 5 X 104

3.5 X 105Antibiotic selection

From Day 11

From Day 18

IMPORTANT: The frequency of iPS colony formation varies for number of experiments
performed. There is a risk that no colonies will form if the number of re-seeded cells on MEFs
is too low. Conversely, if too many cells are re-seeded, the growth of iPS cells will beaffected, and it will be difficult to isolate Nanog-GFP-positive colonies. For these reasons it
is recommended that re-plating be staged so as to allow the evaluation of optimal numberof cells. When establishing iPS cells using only three factors (minus c-Myc), the number of

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colonies will be approximately 10% that seen when using four factors, so it should not be aproblem to re-seed all cells which were retrovirus-infected.

•The next day (Day 9), replace the medium with 10 ml mouse ES cell culture medium.
Continue to replace with fresh mouse ES medium on alternating days thereafter.

3.5 Antibiotic selection•See ref. 1. However, note that antiobiotic selection should be performed at Day 11
when using 4 factors, but at Day 18 when using only 3 factors (minus c-Myc).

IMPORTANT: Delaying the antibiotic selection can yield a greater numbers of colonies. If you
experience difficulties obtaining cultures using three factors, it may be possible to obtain
more by selecting with puromycin at Day 25.

3.6 Isolation of mouse iPS colonies (from Day 25 on)•See ref. 1. However, when attempting to derive iPS cells without antibiotic selection,
pick cells that show silencing of DsRed.

IMPORTANT: Silencing of retroviruses serves as a good index of the degree (quality) ofpluripotency in iPS cells. It is advisable to transfect DsRed etc. for visualization of gene
expression at the time that the reprogramming factors are transfected. High-quality iPS
cells may be found in DsRed-negative colonies.

3.7 iPS cell culture, passaging, freezing and thawing•

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See ref. 1. Essentially similar to protocols used for mouse ES cells.

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4. References
1. Takahashi, K., et al. Induction of pluripotent stem cells from fibroblast cultures. Nat.
Protoc. 2, 3081-9 (2007)

2. Nakagawa, M., Koyanagi, M., et al. Generation of induced pluripotent stem cells without
Myc from mouse and human fibroblasts. Nat. Biotechnol. 10.1038/nbt1374 (2007)

Acknowledgments
We thank second-year Master’s student Ms. Mari Ohnuki for her great contributions to themanuscription of this protocol.

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