Induced Pluripotent Stem Cell Biology Protocols

The information contained below is a compilation of various accredited sources. For your convenience, Sigma^® product numbers have been added where applicable, however, some of these products have not been tested in iPS cell applications.

• Generating iPSc from MEFS through forced expression of Sox-2, Oct-4, c-Myc, and Klf4
• In Vitro Differentiation of iPS

Generating iPSc from MEFS Through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Thaw out cells and plate for lentivirus production.

• Prepare 9 ml of DMEM+ medium (DMEM; D5671, FBS; F2442, Penicillin/Streptomycin; P0781) in a 15 ml tube.
• Remove a vial of frozen stocks from the liquid nitrogen tank and put the vial in a 37 °C water bath until most (but not all) cells are thawed.
• Remove cells from freezing vial and place into the 15 ml tube from step 1.
• Centrifuge at 180 g for 5 min., and then discard the supernatant.
• Resuspend the cells with 10 ml of DMEM+ medium, and transfer to a gelatin-coated 100 mm dish. Incubate the cells in a 37 °C, 5% CO₂ incubator until they are 80–90% confluent.
• Passage of the cells: wash cells with PBS (P7059), aspirate the PBS, and add 4 ml per 10 cm dish of 0.05% trypsin/0.53 mM EDTA (T4049), and incubate for 1 min at 37 °C.
• Detach cells from dishes by tapping, resuspend with 10 ml DMEM+ medium, and transfer to a 15 ml tube. Centrifuge at 180 g for 5 min., and aspirate the supernatant.
• Add appropriate volume of DMEM+ medium, and break up the cells into a single cell suspension by pipetting up and down several times.
• Count the number of cells and adjust the concentration to 8x10⁵ cells per ml with FP medium.
• Seed cells at 8x10⁶ cells (10 ml) per 100 mm culture dish, and incubate overnight at 37 °C, 5% CO₂.

Transfect cells with lentivirus plasmids and harvest virus

• For each 10 cm plate, use 10 µg of FUW-tetO-cDNA (Oct4, Sox2, Klf4 or c-Myc) lentivirus backbone with 2.5 µg of pMD-G and 7.5 µg of pPax2.
• While aliquoting the three plasmids into a tube, deliver 30 l of Fugene 6 transfection reagent into a second tube containing 500 µl of DMEM without serum and mix gently by finger tapping and incubate for 5 min. at room temperature.
• Add the DNA mix drop-by-drop into the Fugene 6/DMEM-containing tube, mix gently by finger tapping and incubate for 20 min.
• Add the DNA/Fugene 6 complex dropwise into the dish of cells, and incubate overnight at 37 °C, 5% CO₂.
• Next morning: Aspirate the transfection reagent–containing medium, add 5 ml of fresh ES cell medium, and return the cells to the incubator.
• At 48 hours and 72 hours after transfection, collect the medium from the cells by using a 10 ml sterile disposable syringe, filtering it through a 0.45 mm pore size cellulose acetate filter, and transferring into a 15 ml tube.
• Concentrate the virus supernatant by ultracentrifugation. Resuspend the virus pellet in desired volume and make amixture of equal parts of the medium containing Oct-3/4-, Sox2-, Klf4- and c-Myc-lentiviruses.
• While making the virus, prepare Oct4-neo/rtTA or Oct4-GFP/rtTA MEFs (passage <3) to 90% confluency in 10 cm dishes (2x10⁶ cells per dish).
• Aspirate the culture medium and wash with 10 ml of PBS (P7059).
• Discard PBS, add 1 ml per dish of 0.05% trypsin/0.53 mM EDTA (T4049), and incubate at 37 °C for 10 min.
• Add 9 ml of the culture medium, suspend the cells to a single cell, and transfer to a 15 ml tube.
• Count the number of cells, and adjust the concentration to 8 10⁴ cells per ml. Transfer 10 ml of cell suspension (8x10⁵ cells) to a 10 cm dish coated with gelatin (G1393). Incubate the dish overnight at 37 °C, 5% CO₂.
• Aspirate the medium from a fibroblast dish, and add 10 ml of the virus-containing medium. Incubate the cells from 4h to overnight at 37 °C, 5% CO₂.
• After 24 aspirate the medium from a fibroblast dish, and add 10 ml of fresh ES cell medium.
• Replace regular ES cell medium with medium containing Doxycycline (D9891) to initiate the expression of the four genes; in other words, initiate reprogramming.
• If using an Oct4-neo reporter, then initiate drug section anytime between 6 and 9 days.
• Change the medium every day until the colonies become big enough to be picked up. Colonies should first become visible approximately a week after the initiation of reprogramming. They should become large enough to be picked up around day 20.

Picking up the iPS colonies

The Day before Picking:

Seed the required number of gelatin-coated 24-well plates with γ-irradiated DR4 MEFs

Picking Clones on Day 1:

• Feed the colonies on the plates 1–2 hours before picking.
• Add 15 µl of PBS (P7059) to the wells of a V-bottomed 96-well dish.
• Wash the dish containing the colonies to be picked once with PBS and add 10 ml of PBS to the dish.
• Pick the colonies with the small 5 µl pipet tips (set the P20 pipetman on 4 µl). Transfer colonies to a well in the 96-well dish.
• Add 20 µl of trypsin (T4424, T4549, T4674) to each well using the multi-channel (12) pipetor. Pipet up and down 3 times. Incubate at 37 °C for 4 minutes.
• Add 100 µl of ES medium to the trypsinized colonies. Pipet up and down 10 times. Transfer 6 clones at a time from the 96-well dish to a 24-well dish using a tip at every other position on the 12 channel pipetor.
• Grow the cells on the 24-well plate in a 37 °C, 5% CO₂ incubator until the cells reach 80–90% confluency, feeding daily with ES cell medium. Cells will probably be ready to expand in 3–7 days.

Expansion of iPS cells

• Aspirate the medium, and wash the cells with 1 ml of PBS.
• Remove PBS completely, add 0.1 ml of 0.25% trypsin/1 mM EDTA and incubate at 37 °C for 10 min.
• Add 0.4 ml of the ES medium and suspend the cells by pipetting up and down to single cell suspension.
• Transfer the cell suspension to a well of 6-well plate, add 1.5 ml ES cell medium, and incubate in a 37 °C, 5% CO₂ incubator until cells reach 80–90% confluency in 6-well plates. At this point, prepare frozen stock of the cells, as follows.

Preparation of freeze stock

• Aspirate the medium, and wash the cells with 2 ml of PBS (P7059).
• Remove PBS completely, add 0.5 ml of 0.25% trypsin/1 mM EDTA (T4049) and incubate at 37 °C for 10min.
• Add 2 ml of the ES medium and suspend the cells by pipetting up and down to single cell suspension.
• Transfer the cell suspension to a 15 ml tube, count the number of cells and spin down the cells.
• Discard the supernatant, resuspend the cells with ES medium to the concentration at 2x10⁶ cells per ml.
• Prepare 2 ES cell freezing medium (20% DMSO in ES medium) and aliquot it at 0.5 ml per vial.
• Transfer 0.5 ml of the cell suspension to freeze vials and mix gently.
• Put the vials in a cell-freezing container and keep it at –80 °C overnight; transfer to liquid nitrogen the next day for long-term storage.

Protocol taken in part from Journal of Visualized Experiments, G.Grant Welstead, Tobias Brambrink, Rudolf Jaenisch; Whitehead Institute for Biomedical Research, MIT; April, 2008.

In Vitro Differentiation of iPS

• Transfer the clusters of iPS cells, which were collected via collagenase IV (C5138, C1889), to poly (2-hydroxyrthyl methacrylate)-coated (P3932) dish in DMEM/F12 (D6421, D6434) containing 20% serum replacement (S0638, S9388, S2640), 2 mM L-glutamine (G8540), 1x10^-4 M nonessential amino acids, 1x10^-4 M 2-mercaptoethanol (M7522), and 0.5% penicillin and streptomycin (P0781). Change the medium every day.
• After 8 days as floating cultures, transfer EBs to gelatin-coated (G9391, G9136, G1890, G1393) plate and culture in the same medium for another 8 days.
• For cardiomyocyte differentiation, keep iPS cells on Matrigel-coated (E1270) plate in MEF-CM supplemented with 4 ng/ml bFGF (F0291) for 6 days. Replace than the medium with RPMI1640 (M3817, R8758, R5886) plus B27 supplement medium supplemented with 100 ng/ml human recombinant activin A (A4941) for 24 hours, followed by 10 ng/ml human recombinant bone morphologenic protein 4 (B2680) for 4 days.
• Keep the cells in RPMI/B27 without any cytokines. Change the medium every other day.

Protocol compiled from Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors; Takahashi K., Tanabe K., Ohnuki M., Narita M., Ichisaka T., Tomoda K., Yamanaka S.; Cell Vol. 131, 861–872, November 2007.

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