This protocol describes the testing of gene products for enhancement of reprogramming to iPS cells from human embryonic fibroblasts. OSKM factors together with the gene of interest are expressed uisng lentiviruses.
Method Article
Reprogramming efficiency of human embryonic fibroblasts using polycistronic lentiviral expression of reprogramming factors OSKM. Testing of factors that enhance efficiency
https://doi.org/10.1038/protex.2013.032
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This protocol describes the testing of gene products for enhancement of reprogramming to iPS cells from human embryonic fibroblasts. OSKM factors together with the gene of interest are expressed uisng lentiviruses.
see the associated publication
see the associated publication
see the associated publication
This protocol is associated with the following reference:
W.W.M. Pim Pijnappel, Daniel Esch, Marijke P.A. Baltissen, Guangming Wu, Nikolai Mischerikow, Atze J. Bergsma, Erik van der Wal, Dong Wook Han, Hermann vom Bruch, Sören Moritz, Phillip Lijnzaad, A.F. Maarten Altelaar, Katrin Sameith, Holm Zaehres, Albert J. R. Heck, Frank C.P. Holstege, Hans R. Schöler and H.Th. Marc Timmers (2013). A central role for TFIID in the pluripotent transcription circuitry. Nature, in press.
Day 1
• Plate 293T cells into 10 cm dishes (4 dishes per virus) in a final volume of 9 ml MEF medium per dish. Plate enough cells to obtain 70% confluence on the next day
Day 2
• Transfect the 293T cells from day 1 (cells should look 70% confluent, nicely spread, evenly distributed, without clumps) with Fugene 6 (Promega) as follows:
Perform for each virus a separate transfection.
Use for 4 dishes (this will yield 1000 μl virus after concentration)
Tube 1: 328 μl Optimem with 72 μl Fugene 6
Tube 2: In a total volume of 400 μl Optimem:
12 μg LV-OSKM or LV-gene of interest
8 μg psPAX2
4 μg pCMV-VSVG
Incubate both tubes for 5 minutes at room temperature. Add tube 2 to tube 1, mix gently by flicking and incubate 15 minutes at room temperature.
Add 200 μl of the mixture per 10cm dish, shake and incubate for 72 hours.
Day 4
• Plate F134 human embryonic fibroblasts at 100,000 cells per well in a 6 wells plate in MEF medium (the dish does not need to be gelatinzed).
Day 5
• Collect supernatant of dishes with transfected 293T cells and filter through 0.45 μm PVDF filters. Spin filtered supernatant by ultra-centrifugation at 20 krpm in an SW32Ti rotor (Beckman) for 2 hr at 4 °C. Remove supernatant by decanting and dissolve the pellet into 1000 μl DMEM low glucose. Use the virus directly to infect cells or store virus for less than 1 week at 4 °C or for longer periods in aliquots at -80 °C.
Note: When virus production was efficient a white/brown pellet will appear after ultra centrifugation
• Infect the plated F134 cells from Day 4 with 50 μl of concentrated OSKM virus and 50 μl of the concentrated virus expressing the gene of interest. Add to each well 6 µg/ml protamine sulfate directly into the medium and mix by swirling.
Note: use titration of the amount of OSKM virus to determine the optimal amount of virus that gives a moderate amount of iPS colonies to allow for testing enhancers of reprogramming efficiency.
Day 6
• Replace medium of the infected F134 cells by washing three times with PBS followed by MEF medium
Note: is is also possible to perform this step one day later.
Optimal: Check after washing fluorescent signal of tomato red. 48 hours after infection, a light tomato red signal should appear in at least 40 % of the cells. After a couple of days this signal will increase.
Day 10
• Plate CF-1 MEF feeder cells with 1,7 x 10e4 cells per cm2 into a gelatinized 6 wells plate.
Note: these feeders will be used for 13 days. It is therefore important not to plate the feeders earlier than day 10.
Day 11
• Trypsinize infected F134 cells and plate 10,000 cells per well onto the feeders plated at day 11. Change medium to iPS medium.
Day 12 and onwards
• Daily refresh the medium with iPS medium.
Day 29: count the amount of colonies with iPS-like morphology, and/or process for Tra1-60 colony staining or single cell FACS analysis.
Medium composition:
MEF medium
-445ml DMEM low glucose (PAA, E15-005)
-50ml Fetal Bovine Serum (FBS, Invitrogen # 16000-044)
-5ml Pen/Strep/Glu 100x (PAA, P11-013)
iPS medium
-390ml DMEM/F12 (Invitrogen, # 21331046)
-100ml KO serum replacement (Invitrogen # 10828)
-5mL Non-essential amino acids (PAA, M11-003)
-5mL Pen/Strep/Glu 100x (PAA, P11-013)
-1ml β-Mercaptoethanol (Invitrogen # 31350010)
-1ml basic fibroblast growth factor (bFGF, 5 μg/ml stock solution in 0.1% BSA/PBS, Peprotech 100-18b)
Sterile filter through 0.2μm filter
Store at 4°C for up to 2 weeks
29 days
see details in the protocol
When usign low lentiviral titers that result in a few colonies after OSKM infection, co-infection with TAF4 virus shoudl lead to a strong increase in the amount of colonies and Tra1-60 positive cells after FACS analysis.
This protocol is associated with the following reference:
W.W.M. Pim Pijnappel, Daniel Esch, Marijke P.A. Baltissen, Guangming Wu, Nikolai Mischerikow, Atze J. Bergsma, Erik van der Wal, Dong Wook Han, Hermann vom Bruch, Sören Moritz, Phillip Lijnzaad, A.F. Maarten Altelaar, Katrin Sameith, Holm Zaehres, Albert J. R. Heck, Frank C.P. Holstege, Hans R. Schöler and H.Th. Marc Timmers (2013). A central role for TFIID in the pluripotent transcription circuitry. Nature, in press.
We thank Martina Radstaak and Boris Burr for technical assistance
None declared.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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