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Cell Culture

Induced Pluripotent Stem Cell Reprogramming 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

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.

Time course of human iPS colonies generated using Human SimpliconTM RNA Reprogramming Kit

Time course of human iPS colonies generated using Human SimpliconTM RNA Reprogramming Kit (Cat. No. SCR550). The transfected HFFs were replated onto inactive MEFs at Day 10, Colonies start to emerge from Day 15-16 and are more obvious around day 17-20 (A, B). Colonies are ready to be picked at Day 27. Alkaline phosphatase staining of hiPS colonies.


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.

Human foreskin fibroblasts

Human foreskin fibroblasts (Cat. No. SCC058) infected with STEMCCA Constitutive Polycistronic (OKSM) Lentivirus undergo morphological changes and may form colonies that are compact and have ES-like morphology (A, C), but are not yet fully reprogrammed iPS cells. Passage 6 human foreskin fibroblasts were infected following the reprogramming protocol outlined in the STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Kit (Cat. No. SCR530). Cells were stained live with SSEA-4 PE and TRA-1-60 FITC antibodies at Day 15 post-infection. At this timepoint, human iPS-like colonies are positive for SSEA-4 (B, D) and negative for TRA-1-60 (B, D), indicating that the cells are not yet fully reprogrammed.
 

Fully reprogrammed human iPS cells express human pluripotent markers

Fully reprogrammed human iPS cells express human pluripotent markers, TRA-1-60 FITC (D, E, G, H, green) and SSEA-4 PE (C, F, G, H, red) while downregulating the fibroblast marker, CD13 PE (data not shown). Cells were stained with cell permeable Hoechst nuclear dye (B, C, D, H, blue). Fully reprogrammed human iPS cells exhibit Hoechst dim phenotype (see colony center in B, C, D, H) while non-iPS and differentiated cells exhibit a Hoechst bright phenotype (see the periphery of the colony in B, C, D, H, which is surrounded by fibroblast cells and are Hoechst bright). Human iPS colonies at passage 5 were used for live staining. Human foreskin fibroblasts (Cat. No. SCC058) were reprogrammed using the STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit (Cat. No. SCR530).


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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