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

Materials and Reagents

1. Falcon^® 6-Well Flat-Bottom Plate, Tissue Culture-Treated (Falcon, catalog number: 38016 )
2. Serological pipettes multiple sizes (Corning)
3. Falcon 15 ml conical centrifuge tubes (Falcon, catalog number: 352097 )
4. Falcon 50 ml conical centrifuge tubes (Falcon, catalog number: 352070 )
5. Cells: Human fibroblast expanded from skin biopsies preferentially at passage 1 to passage 5. Early passage fibroblast can be obtained from a commercial source (i.e., ATCC), a repository (i.e., Corriell Institute) or expanded in the lab from skin biopsies (Vangipuram et al., 2013 describes the procedure in detail)
6. Corning^® Matrigel^® hESC-Qualified Matrix (Corning, catalog number: 354277 )
7. CytoTune^TM-iPS 2.0 Sendai Reprogramming Kit (Thermo Scientific, catalog number: A16518 )
8. DMEM/F-12 (Gibco, catalog number: 11320033 )
9. Fetal Bovine Serum (Gibco, catalog number: 16000044 )
10. MEM NEAA (Gibco, catalog number: 11140050 )
11. Pen Strep (Gibco, catalog number: 15-140-122 )
12. mTeSR^TM 1 (STEMCELL Technologies, catalog number: 85850 )
13. TrypLE^TM Express Enzyme (1x) (Gibco, catalog number: 12605036 )
14. Anti-Human TRA-1-60, Mouse monoclonal (Thermo Fisher Scientific, catalog number: 41-1000 )
15. Anti-Human OCT4, Rabbit polyclonal, IgG1 (Abcam, catalog number: ab19857 )
16. Anti-Human SOX2 Antibody, Rabbit polyclonal IgG1 (Abcam, catalog number: ab97959 )
17. STEMdiff Trilineage Differentiation kit, STEMCELL Technologies, catalog number: 0 5230 )
18. Universal Mycoplasma Detection Kit (ATCC, catalog number: 30-1012K )
19. Dulbecco’s PBS (DPBS) without Calcium and Magnesium (Gibco, catalog number: 14190136 )
20. Polybrene Hexadimethrine Bromide (Sigma-Aldrich, catalog number: H9268 )
21. 0.5 M EDTA (Gibco, catalog number: 15575020 )
22. Fibroblast culture medium (see Recipes)
23. 5 mM EDTA solution (see Recipes)
24. 10 mM Y27632 stock solution (see Recipes)
    

Equipment

1. Tissue culture incubator Heracell^TM 150i (Thermo Fisher Scientific, catalog number: 51026283 )
2. Beckman Avanti J-30I Refrigerated with plate adapters (Beckman Coulter, catalog number: 17039 )
3. Tissue culture laminar flow hood (NuAire Class II Type A2 NU-540)
4. Evos XL core microscope (Thermo Scientific, catalog number: AMEX1100 )
5. Countess II FL Automated Cell Counter (Thermo Fisher Scientific, catalog number: AMQAF1000 )
6. Water bath Isotemp (Fisher Scientific, catalog number: S35936 )
7. Portable Pipet-aid (Drummond, model: DP-101 , catalog number: 4-000-101)
8. Micro pipettes (Eppendorf)
   

Procedure

Day -1: Plate dermal fibroblast for reprogramming
Use low-passage (passage 1 to 5) human fibroblasts for reprogramming experiments. The reprogramming efficiency decreases with each passage. Hence, we don’t recommend reprogramming fibroblast after passage 6. A schematic timeline and representative pictures can be seen in Figure 1.

1. Coat two wells of a 6 well plate with 2 ml of 5 mg/ml Matrigel and make sure it covers the entire well. Incubate at 37 °C for 30 min.
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.
4. Add 1 ml fibroblast culture medium and transfer cell suspension to a 15-ml conical tube.
5. Centrifuge at 200 x g for 5 min. Remove and discard supernatant.
6. Resuspend cells in 1 ml of fresh fibroblast culture medium. Recipe available in the recipe section.
7. Count fibroblasts using the desired method (e.g., Countess Automated Cell Counter), and plate 1 x 10⁵ cells/well (10,000 cells/cm²) in the Matrigel-coated 6-well dish in 2 ml of fibroblast culture medium.
8. Incubate 24 h at 37 °C with a humidified atmosphere and 5% CO₂.
   
   
   Figure 1. Derivation of induced pluripotent stem cells from fibroblast using Sendai virus and chemically defined medium. A. Schematic view of general reprogramming procedure including culture medium used. B. Images of fibroblast morphology changes during the early stages of reprogramming. Scale bars: 650 μm.
   
   

Figure 2. Images of morphological changes seventy-two hours after transduction with Sendai virus. Scale bars: 650 μm.

1. Remove 500 μl of fibroblast culture medium and add 500 μl of fresh mTeSR1.
   

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

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

1. When colonies are ready to pick, prepare one Matrigel coated dish per colony to be passage, and incubate at 37 °C for at least 30 min.
2. Aspirate Matrigel from plates and add 2 ml mTeSR1 + 10 µM ROCK inhibitor.
3. Use a 22 gauge needle or a pulled glass pipette to cut colonies in a grid-like pattern into small fragments. See Figure 3.
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.
   
   
   Figure 3. Images of colonies cut in a grid-like pattern into small fragments. Scale bars: 650 μm.
   
   

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.
4. Wash the cells with 2 ml 0.5 mM EDTA, aspirate.
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.
    
    

1. Immunofluorescence staining of cell surface markers such Anti-Human TRA-1-60, Mouse monoclonal and transcription factors such as anti-Human OCT4, Rabbit polyclonal, IgG1 and anti-Human SOX2 Antibody, Rabbit polyclonal IgG1.
2. Pluripotency and trilineage differentiation capabilities (using the STEMdiff Trilineage Differentiation kit, and the Taqman hPSC Score card Panel.
3. Test for mycoplasma contamination using commercially available kits such as The Universal Mycoplasma Detection Kit.
4. G-banding Karyotype.
   
   
   Figure 4. Characterization of iPSC derived from fibroblast. A. Bright field images of iPSCs, B. Immunofluorescence staining for pluripotency markers Tra-1-60 and OCT4. C. Taqman ScoreCard assay for pluripotent iPSCs (left panel) and iPSCs differentiated into endoderm, mesoderm and ectoderm (right panel). D. G-banding karyotype.
   

Recipes

1. Fibroblast culture medium
   The following recipe is to prepare 500 ml of fibroblast culture medium. Aseptically mix the following:
   445 ml high glucose DMEM
   50 ml heat-inactivated fetal bovine serum (FBS)
   5 ml non-essential amino acids
2. 5 mM EDTA solution
   Dilute 0.5 ml of 0.5 M EDTA in 500 ml DPBS without Calcium and Magnesium. Store at room temperature
3. 10 mM Y27632 stock solution
   Aseptically add 3.122 ml of sterile water to 10 mg of Y27632
   Mix thoroughly by pipetting, aliquot in Eppendorf’s and store at -80 °C
   Avoid repeated freezing and thawing
   

Acknowledgments

This protocol was developed as part of standard operations procedures of the Human Pluripotent Stem Cell core facility at UNC-CH. This work is supported by the UNC Office of Research.

Competing interests

The authors do not have any financial or non-financial interest in the subject matter or materials discussed in this manuscript.

Ethics

This research was carried out in according with the UNC-CH institutional guidelines.

References

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