1. Seed HDF cells in 10cm dishes (20-30% confluent) one day before viral infection, including one dish used for GFP control.

2. The following day, dilute 50μl of each Excellgen KOSM-adenovirus in 4ml complete culture medium.

3. Aspirate medium from 10cm dishes and add diluted virus (4ml) to cells. Return dishes to a CO2 incubator for one hour.

4. Aspirate virus-containing medium, and replenish with 10ml iPSC medium.

5. Change medium every 48 hours.

6. Repeat adenoviral gene transduction every other 5 days as transgene expression by adenovirus will only last 5~7 days, depending on the rate of cell division. Longer expression can be expected for cells with slower cell dividing rates.

7. Wait for 2-4 weeks for iPSC colonies to form.

8. Once iPSC colonies form, prepare Mytomycin C treated MEF (mouse embryonic fibroblasts) feeder cells in a 24-well plate (80% confluent) at a concentration of 10μg/ml for 3 hours in an incubator at 37°C followed by 2X PBS wash.

9. Pick colonies manually into 96-well plate and trypsinize in 96-well plate.

10. Transfer the trypsinized cells from each of the 96 wells into 24-well MEF coated plate.

11. Wait for another 1-2 weeks for iPSC colonies to develop (change medium every 48 hours)

12. When MEF cells become too old (about 2 weeks) or a lot of iPSC colonies have developed in the 24-well plate, prepare MEF feeder layer as described above in 6-well plate to expand.

13. Trypsinize the cells (both MEF and iPSC cells) and spin down at 1000Xg.

14. Seed iPSCs into 6-well MEF coated plate for expansion.

15. Using the same procedures above, iPSCs can be further expanded to bigger culture vessels to meet your lab needs for iPSC analysis or down-stream applition