Derivation of Induced Pluripotent Stem Cells from Human Fibroblasts Using a Non-integrative System in Feeder-free Conditions

[Abstract] Induced pluripotent stem cells (iPSCs) are genetically reprogrammed somatic cells that exhibit features identical to those of embryonic stem cells (ESCs). Multiple approaches are available to derive iPSCs, among which the Sendai virus is the most effective at reprogramming different cell types. Here we describe a rapid, efficient, safe, and reliable approach to reprogram human fibroblasts into iPSCs that are compatible with future iPSCs uses such as genome editing and differentiation to a transplantable cell type.

modeling, but for the development of therapeutic strategies for pathologies that involve tissue degeneration. Furthermore, the iPSCs promise relies on a safe replenishable cell source derived in chemically defined media and free of random DNA integration.
Reprogramming somatic cells into iPSCs requires the forced expression of transcription factors that support the pluripotent state, including OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN-28 (Takahashi and Yamanaka, 2006;Takahashi et al., 2007;Yu et al., 2007). Multiple approaches are available to deliver the transcription factors into the cells, including those that require integration into the host chromosomes (Takahashi and Yamanaka, 2006;Kane et al., 2010). Exogenous DNA integration can lead to unpredictable effects on the quality of the cells and safety after transplantation. Other approaches include DNA based vectors that exist episomally (Yu et al., 2011;Weltner et al., 2012) and thus, decrease the possibility of integration, and finally those that do not integrate into the host genome and are known as transgene-free. The transgene-free methods include mRNA (Warren and   2. Inspect the fibroblast culture for the desired confluency (more than 70%), aspirate the culture medium and rinse twice with 2 ml DPBS.
3. Add 500 µl TrypLE TM Express enzyme and incubate at 37 °C and 5% CO2 for 2 to 5 min or until fibroblasts have detached. TrypLE TM is used because it has lower cell toxicity than standard Trypsin and it is an animal-free product.   8. Incubate the plate overnight in a 37 °C incubator with a humidified atmosphere of 5% CO2.

Day 1: Remove virus
After 24 h, aspirate medium with viruses and add 2 ml fresh fibroblast culture medium. Expect to see cytotoxicity 24-48 h post-transduction, this is an indication of high uptake of the virus. See Figure   2.

Days 2 to 3
Remove 2 ml of used medium and add 2 ml of fibroblast culture medium. Expect to see changes in cell morphology.

Day 4: Transition to defined medium
There are many different chemically defined culture medium commercially available for deriving and maintaining iPSCs. We have used mTeSR1 medium (Stem Cell Technologies), STEMFLEX (Life technologies) and mTeSR plus (Stem Cell Technologies) agnostically.

Day 5
1. Remove 1 ml of culture medium and add 1 ml of fresh mTeSR1.

Day 6
1. Remove 1.5 ml of culture medium and add 1.5 ml of fresh mTeSR1.

Day 7
Remove 2 ml of medium culture medium and add the same volume of fresh mTeSR1.

Days 8-20
Feed cells daily with 2 ml until colonies are ready to be passaged. Remove partially reprogrammed and differentiated colonies by scraping them before medium change. Fully reprogrammed colonies have round shape with well-defined borders, cells display identical morphology with a high ratio of nucleus to cytoplasm and prominent nucleoli. In contrast, partially reprogrammed colonies have undefined borders, an amorphous shape, and are composed of different types of cells.  4. Use a 100 µl pipettor to scrape fragments and collect them. Immediately transfer the fragments to the Matrigel coated plates with mTeSR1 + 10 µM ROCK inhibitor.
5. Rock plate back-and-forth and side-to-side to evenly distribute the cell fragments and incubate overnight at 37 °C with a humidified atmosphere of 5% CO2.
6. Feed cells daily until ready to passage (usually every 6 to 7 days).
7. Repeat Steps 1 to 10 for 7 passages. We found that > 95% of the iPSCs clones at passage 7, are transgene free.

IPSCs cloning and expansion
After passage 7, IPSCs are passage with EDTA buffer, as follow: 1. Coat 6 wells of a 6-well tissue culture plate with Matrigel and place in incubate at 37 °C for 30 min.
2. Prior to use, allow the Matrigel-coated plate to equilibrate to room temperature for at least 1 h.
3. Just before dissociating cells for passaging, aspirate the liquid Matrigel solution from the wells and replace with 1 ml of mTeSR1 + 10mM Y27632 cell culture media per well. Set aside.
5. Add 2 ml of room temperature 0.5 mM EDTA solution to the cells. 6. Incubate the culture at 37 °C for 3 to 5 min, or until cells begin to separate uniformly throughout the entire colony. Do not allow the cultures detach in the EDTA solution.
7. As soon as the cells appear rounded and uniform separation is seen throughout the colonies, carefully aspirate the EDTA solution from the well. Do not rinse.
8. Immediately add 1 ml of mTeSR1 + 10mM Y27632. With a 5 ml pipet, take up the 1 ml of media from the well, and very gently dispense it against the culture surface to dissociate the cells from the dish. Repeat 1 to 2 more times, if needed. 9. Be careful not to over-pipet the cell suspension. 10. Dispense the cells gently into the 15 ml conical tube containing an additional 3 ml of pre-warmed media.
11. Pipet the solution very gently 1 time to mix, and dispense 1 ml of the cell suspension drop-wise into each of the 6 new Matrigel-coated wells, and immediately rock plate back-and-forth and side-to-side to evenly distribute the colony pieces across the well. 12. Incubate undisturbed at 37 °C and 5% CO2 overnight. 13. Replace cell culture media every day with 2.5 ml of fresh mTeSR1, warmed to room temperature.
14. Monitor cells daily and passage as needed.

Characterization and cryopreservation
Cells are expanded for cryopreservation at passage 10. Then, iPSCs are characterized ( Figure 4) and prepare for downstream usage in genome engineering experiments or differentiation into specific cell types. Characterization includes: