STEMGENT Page 1 OVERVIEW The following protocol describes the reprogramming of one well of mouse embryonic fibroblast (MEF) cells into induced pluripotent stem (ips) cells in a 6-well format. Transduction efficiency can vary depending on such factors as the amount of virus used, the type of cell transduced, the use of cationic polymers, and the type of media used. If using cell types or conditions different from what is listed below, the amounts of virus used may need to be optimized for your experiment. Product Description Cat. No. Format Storage Reprogramming Ecotropic Retrovirus Set: OSKM 00-0042 1 set -70 C Product Components Cat. No. Size Storage Reprogramming Ecotropic Retrovirus: Mouse Oct4* Reprogramming Ecotropic Retrovirus: Mouse Sox2* Reprogramming Ecotropic Retrovirus: Mouse Klf4* Reprogramming Ecotropic Retrovirus: Mouse c-myc* 07-0042 250 µl -70 C 07-0044 250 µl -70 C 07-0043 250 µl -70 C 07-0045 250 µl -70 C Reprogramming Ecotropic Retrovirus: GFP 250 µl -70 C *Ecotropic Retroviruses can be ordered separately ADDITIONAL MATERIALS REQUIRED Oct4-GFP MEF (P2) ( Cat. No. 08-0028) 0.1% Gelatin in water DMEM ES cell-qualified FBS Non-essential amino acids (100X) β-mercaptoethanol (55 mm) Matrigel hesc-qualified Matrix (BD) Polybrene Knockout DMEM (Invitrogen) Stemfactor Recombinant Mouse LIF (1 x 10 7 U/ml) ( Cat. No. 03-0011) PBS 0.05% Trypsin/EDTA 6-well tissue culture plates 4-well tissue culture plates 24-well tissue culture plates 96-well tissue culture plates 15 ml conical tubes, logo, and all other trademarks are property of Inc. 2010 Inc.
STEMGENT Page 2 MATERIAL PREPARATION Gelatin Coated Plates 1.5 ml 0.1% gelatin in water to each well of a 6-well plate 0.5 ml 0.1% gelatin in water to each well of a 4-well or 24-well plate Incubate overnight at 37 C. Gelatin Coated Plates can be stored at 37 C for up to 3 days. MEF Medium 450 ml DMEM 50 ml ES cell-qualified FBS 5 ml Non-essential amino acids (100X) 0.5 ml β-mercaptoethanol (55 mm) Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. Store MEF Medium at 4 C. Matrigel Coated Plates Coat a 6-well tissue culture plate with Matrigel hesc-qualified Matrix according to the manufacturer s instructions. If plates have been stored at 4 C, move to room temperature 1 hour prior to use. Transduction Medium 250 µl Reprogramming Ecotropic Retrovirus: Mouse Oct4 250 µl Reprogramming Ecotropic Retrovirus: Mouse Sox2 250 µl Reprogramming Ecotropic Retrovirus: Mouse Klf4 250 µl Reprogramming Ecotropic Retrovirus: Mouse c-myc 6 µg/ml polybrene Thaw one vial of each virus and add the vial contents to a 15 ml conical tube. Add 1 ml of MEF Medium to the tube. Add polybrene to a final concentration of 6 µg/ml and pipet to mix. Transduction Medium should be used immediately. Mouse ES Cell Medium 425 ml Knockout DMEM 75 ml ES cell-qualified FBS 5 ml Non-essential amino acids (100X) 0.9 ml β-mercaptoethanol (55 mm) 50 µl Stemfactor Recombinant Mouse LIF (1 x 10 7 U/ml) Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. Store Mouse ES Cell Medium at 4 C for up to 1 month., logo, and all other trademarks are property of Inc. 2010 Inc.
STEMGENT Page 3 MEF Feeder Plates The day before MEF Feeder Plates are needed, thaw gamma irradiated feeder layer MEFs and dilute to a concentration of 1 x 10 5 cells/ml in MEF Medium. 4-well Gelatin Coated Plate 400 µl of cell suspension 24-well Gelatin Coated Plate 400 µl of cell suspension Incubate overnight at 37 C and 5% CO 2. REPROGRAMMING TIMELINE DAY ACTIVITY MORPHOLOGY MEF Cell Preparation and Transduction ips Cell Colony Identification and Isolation -1 Plate MEF cells 0 Transduce MEF cells 1 Change medium to Mouse ES Cell Medium 2-8 Incubate transduced cells, changing medium on day 5 and day 8 9-11 Incubate transduced cells, changing medium on day 9 and day 11 13+ Incubate transduced cells, changing medium daily. ips cell colonies are picked and plated in 12-well plates. True ips cell colonies can form earlier than day 13 and should be picked and transferred as they appear. ips cell colonies typically begin to emerge around day 10 but GFP expression is not usually evident until day 12 to 14. Pick ips cell colonies within a few days of proper morphology identification. As the overall cell confluence increases, they will become more difficult to isolate (see Figure 2). REPROGRAMMING OCT4-NEO MEFs Prepare Cells for Reprogramming If using Oct4-GFP MEFs (P2), follow the plating protocol listed below. If using another cell type, further optimization may be required. 1. Plate Oct4-GFP MEFs on a 6-well Matrigel Coated Plate at a final density of 1 x 10 5 cells per well. 2. Incubate overnight at 37 C and 5% CO 2., logo, and all other trademarks are property of Inc. 2010 Inc.
STEMGENT Page 4 Note: Seeding Oct4-GFP MEFs at this density should result in a confluency that is ideal for reprogramming. See Figure 1 for an example of correct cell density. Transduce MEFs 3. After the overnight incubation, carefully aspirate the medium from one well of the 6-well plate of Oct4-GFP MEFs by tilting the plate so as not to disturb the cells. Replace with Transduction Medium.Incubate the cells overnight at 37 C and 5% CO 2. 4. After the overnight incubation, aspirate the medium as described in step 3 and add 2 ml of Mouse ES Cell Medium. 5. Incubate at 37 C and 5% CO 2. 6. Using Mouse ES Cell Medium, change the medium every 3 days for the first 2 medium changes. 7. Change the medium every other day for the next 2 medium changes. 8. Finally, change the medium every day until all colonies have been picked for expansion and testing. Select ips Cell Colonies The Oct4-GFP MEFs used in this protocol contain a GFP transgene driven by the endogenous Oct4 (pou5f1) promoter. The expression of GFP from the endogenous Oct4 locus allows for the identification of mouse ips cell colonies that are able to maintain pluripotency independently of exogenous transcription factor expression. We recommend manually picking and passaging mouse ips cell colonies that have both good morphology and homogenous GFP expression. Single Colony Pick and Passage If picking cells encoding a GFP reporter gene, analyze the culture and mark mips cell colonies expressing GFP prior to picking. If the transduced cells do not contain a GFP or similar reporter, they must be picked according to proper morphology alone. In this case, more colonies should be picked to ensure enough of the picked cells continue to expand in culture after passaging. Note: Multiple colonies may be picked at one time, however, cells should be returned to the incubator within 30 minutes. Cells also should not remain in PBS for longer than 30 minutes. 1. Manually pick colonies with ips cell morphology and reporter expression (if applicable): a. Using a sterile glass picking tool, gently separate the identified colony from surrounding cells. b. Using the glass picking tool, gently detach each colony from the tissue culture well., logo, and all other trademarks are property of Inc. 2010 Inc.
STEMGENT Page 5 c. Using a 10 μl pipettor set at 5 μl, pipette the detached colony out of the 6-well plate and into an individual well of a 96-well plate containing 15 μl of PBS. d. Trypsinize each individually isolated ips cell colony by adding 20 μl of 0.05% trypsin/edta per well and incubating for 2 minutes at 37 C. Pipet the cell solution in the well to dissociate the colonies to single cells. 2. Replate the contents of each well of the 96-well plate into individual wells of a 24-well MEF Feeder Plate containing 0.5 ml of Mouse ES Cell Medium. 3. Incubate the 24-well plates for up to 7 days at 37 C and 5% CO 2. Replace with fresh Mouse ES Cell Medium every 48 hours and monitor the cultures daily for ips cell colony growth and proliferation. 4. Trypsinize and passage individual wells from the 24-well plate that exhibit colony expansion and ips cell morphology at a 1:8 split ratio into 4-well MEF Feeder Plates for pluripotency analysis. Also passage into an appropriate size format for ips cell expansion and banking. 5. Monitor cultures for equivalent ips cell colony growth and morphology. 6. Continue to passage and culture ips cell colonies for use in experimentation and banking. DISCUSSION ips cell colonies can be replated onto 24-well plates and may need several passages before becoming fully established. Any reagent that can enhance the survival and cloning efficiency of human embryonic stem (ES) cells, such as the Y27632 molecule ( Cat. No. 04-0012) or Thiazovivin ( Cat. No. 04-0017), can be used when picking and passaging human ips cell colonies for expansion. Expanded ips cell colonies can be tested for pluripotency by Alkaline Phosphatase staining ( Cat. No. 00-0009) and staining for typical pluripotency markers such as SSEA-1 ( Cat. No. 09-0005), Nanog ( Cat. No. 09-0020), Oct4 ( Cat. No. 09-0023), TRA-1-60 ( Cat. No. 09-0010), Rex1 ( Cat. No. 09-0019), and TRA-1-81 ( Cat. No. 09-0011)., logo, and all other trademarks are property of Inc. 2010 Inc.
STEMGENT Page 6 Figure 1. Oct4 GFP MEF Density Before Reprogramming Oct4 GFP MEFs were seeded at 1 x 10 5 cells per well and incubated overnight. This image shows cells that are at the correct confluency for reprogramming. Figure 2. Representative ips Cell Colonies Oct4 GFP cells were transduced following the Reprogramming Ecotropic Retrovirus Set: Mouse OSKM protocol. 20x phase contrast image of emerging (P0) ips cell colonies was taken on day 15 post transduction. Knockout DMEM is a trademark of Life Technologies Matrigel is a trademark of BD, logo, and all other trademarks are property of Inc. 2010 Inc.