scientificprotocols authored about 7 years ago
Authors: Yasong Wu, Yuan Li, Baoming Qin & Duanqing Pei
This protocol describes 4 methods to reprogram MEFs into iPSCs: the pMXs-based retroviral 4-factor, the polycistronic lentiviral 4-factor, the doxycycline inducible 4-factor secondary, and the iCD1 medium based Sox2+Oct4 2-factor systems.
pMXs-based retroviral 4-factor system
Day 1
Plate Plat-E cells in Plat-E medium at 8×10e6 cells per 10-cm dish. Distribute the cells evenly over the dish.
Day 2
In the evening, transfect Plat-E cells from day 1 (cells should be 70-80% confluent and evenly distributed) using the modified calcium phosphate transfection method1 as follows:
Replace the medium of the 10-cm dish with 5 ml fresh Plat-E medium. For each factor use one 15 ml tube, add:
Day 3
In the early morning, 8-12 hours after transfection, replace the medium of the transfected Plat-E cells with 10 ml Plat-E medium/10 cm dish.
Day 4
In the morning, split OG2 MEFs at 16,000-20,000 cells/well of P12 plate. Distribute the cells evenly over the well.
In the evening, start the first infection as follows:
Day 5
Repeat the infection a second time as in day 4 using the second round of viral supernatant.
Discard the Plat-E cell dishes.
Day 6
Change the medium of the infected OG2 MEFs to FBS-based ESC medium. Change new ESC medium every other day in the first 3-5 days before cells get confluent and every day afterwards.
Day 22
18 days post infection count iPSC colonies that are GFP positive.
Polycistronic lentiviral 4-factor system
The following protocol is based on the publication (2), with minor modifications.
Day 1
Plate HEK293T cells in HEK293T medium (the same with Plat-E medium) at 8×10e6 cells/10-cm dish. Distribute the cells evenly over the dish.
Day 2
In the evening, transfect HEK293T cells from day 1 (cells should be 80-90% confluent and evenly distributed) as follows:
Replace the medium of the 10-cm dish with 5 ml fresh HEK293T medium. In a 15 ml tube, add:
Day 3
In the morning, 8-12 hours after transfection, change the medium of the transfected HEK293T cells with 10 ml fresh HEK293T medium.
Day 4
In the evening, split MEFs at 16,000-20,000 cells/well of P12 plate. Distribute the MEFs evenly over the well.
Harvest the supernatant of the transfected HEK293T plates using a syringe and filter through a 0.45 μm filter. The virus supernatant can be stored at 4℃ for 3-5 days or in -80℃ for longer time.
Discard the HEK293T dishes.
Day 5
In the morning, start the first infection as follows:
Day 17
12 days post infection, wash the cells twice using DPBS, fix the cells using 4% paraformaldehyde in room temperature for 15 minutes, stain the colonies using SSEA1 primary antibody (ab#16825, Abcam, 1/100 dilution) and green fluorescence secondary antibody Alexa Fluor® 488 Goat Anti-Mouse IgG (#A-11029, Invitrogen, 1/200 dilution), then count the SSEA1+/dTomato- colonies using a fluorescence microscope.
Doxycycline inducible secondary 4-factor system
Day 1
Revive the OG2 secondary MEFs (with the doxycycline (DOX)-inducible 4F transgenes inserted into Col1a locus3) in MEF medium.
Day 2
In the evening, split OG2 secondary MEFs at 16,000-20,000 cells/well of P12 plate.
Day 3
In the early morning, the medium was changed into mouse ESC medium containing 2 μg/ml doxycycline and 50 μg/ml vitamin C. Change new ESC medium every other day in the first 3-5 days before cells get confluent and every day afterwards.
Day 15
12 days post infection count GFP positive iPSC colonies.
iCD1 medium based Sox2+Oct4 2-factor system
Day 1 to 5, the same with pMXs-based 4-factor reprogramming system above. Transfect Sox2 and Oct4 only.
Day 6
Change the medium of the infected OG2 MEFs to iCD1+BMP4 medium4. Change new iCD1+BMP4 medium every other day in the first 3-5 days before cells get confluent and every day afterwards.
Day 16
12 days post infection count GFP positive iPSC colonies.
For all the 4 methods, at 3-4 days after infection there will be obvious mesenchymal to epithelial transition observed. Cells should proliferate very fast in the early 4F reprogramming, with certain degree of apoptosis because of c-Myc expression. For the pMXs-based retroviral 4F system, the doxycycline inducible secondary 4F system, and the iCD1 medium based Sox2+Oct4 2F system, there should be GFP+ colonies appearing at the indicated time. For the polycistronic lentiviral 4F system, after staining SSEA1 with green fluorescence secondary antibody, the SSEA1+ and dTomato- colonies represent the iPSCs.
We thank all members of the Pei and Esteban labs for their support.
Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming
Yasong Wu, Yuan Li, Baoming Qin & Duanqing Pei, Pei's Lab, Guangzhou Institutes of Biomedicine and Health
Correspondence to: Baoming Qin (qin[email protected]), Duanqing Pei (pei[email protected])
Source: Protocol Exchange (2015) doi:10.1038/protex.2015.001. Originally published online 18 February 2015.