Protocol Using a Dox-Inducible Polycistronic m4f2a Lentivirus to Reprogram MEFs into ips Cells

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1 STEMGENT Page 1 OVERVIEW The following protocol describes the transduction and reprogramming of one well of Oct4-GFP mouse embryonic fibroblasts (MEF) using the Dox Inducible Reprogramming Polycistronic Lentivirus Set: Mouse 4F2A. Target cells are plated in a conventional 6-well format for transduction and are subsequently replated at a lower cell density in a 6-well plate for doxycycline (dox) induction to initiate the reprogramming process. If desired, two wells can be reprogrammed at the same time to ensure the production of a sufficient number of induced pluripotent stem (ips) cells. Product Description Cat. No. Format Storage Dox Inducible Reprogramming Polycistronic Lentivirus Set: Mouse 4F2A ST set -70 C Components Cat. No. Size Storage Dox Lentivirus m4f2a ST x 25 μl -70 C Lentivirus rtta 2 x 50 μl -70 C Dox Lentivirus GFP 50 μl -70 C ADDITIONAL MATERIALS REQUIRED Oct4-GFP MEF (P2) ( Cat. No ) MEF Medium Gelatin Coated Plates mipsc Culture Medium Dox Solution (2 mg/ml) ( Cat. No ) mipsc Dox-Induction Medium MEF Feeder Plates 0.05% trypsin/edta Trypan blue 15 ml conical tubes 6-well tissue culture plates 4-well tissue culture plates 24-well tissue culture plates 96-well tissue culture plates MATERIAL PREPARATION Gelatin Coated Plates 1.5 ml 0.2% gelatin in water to each well of a 6-well plate 0.5 ml 0.2% gelatin in water to each well of a 4-well plate Incubate overnight at 37 C. Gelatin Coated Plates can be stored at 37 C for up to 3 days., logo, and all other trademarks are property of Inc Inc.

2 STEMGENT Page 2 MEF Medium 450 ml DMEM 50 ml Defined FBS (Atlas) 5 ml non-essential amino acids (100X) 5 ml L-glutamine (200 mm) 5 ml penicillin (5,000 U/ml)-streptomycin (5,000 g/ml) 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. Lentiviral Transduction Medium Add 2 ml of MEF Medium to a 15 ml conical tube Add 1 x 10 6 TUs (MOI 10) of Dox Lentivirus 4F2A Add 2 x 10 6 TUs (MOI 20) of Lentivirus rtta Determine the volume of each virus to add by dividing the TU s needed by the concentration of the virus (TU/ml) found on the specifications sheet. Viral stock solutions have been validated to maintain their titer after one additional freeze/thaw cycle. Store any remaining viral stock solution at -80 C. Prepared Lentiviral Transduction Medium should be used immediately. Note: When using the Dox Inducible Reprogramming Polycistronic Lentivirus Set: Mouse 4F2A to transduce Oct4-GFP MEFs, we recommend a multiplicity of infection (MOI) of 10. The MOI should be optimized for your target cell type of interest to achieve the best transduction efficiency balanced with the lowest possible cytotoxicity. When transducing cells using an MOI other than 10, calculate the amount of lentivirus to use (in TUs) by multiplying the desired MOI by the number of cells plated in the well to be transduced. mipsc Culture Medium 425 ml Knockout DMEM (Invitrogen) 75 ml ES Cell-Qualified, Defined FBS (Hyclone) 5 ml non-essential amino acids (100X) 5 ml L-glutamine (200 mm) 5 ml penicillin (5,000 U/ml)-streptomycin (5,000 g/ml) 0.5 ml β-mercaptoethanol (55 mm) 25 μl Stemfactor Recombinant Mouse LIF (1 x 10 7 U/ml) ( Cat. No ) Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. Store mipsc Culture Medium at 4 C for up to 2 weeks., logo, and all other trademarks are property of Inc Inc.

3 STEMGENT Page 3 Dox Solution (2 mg/ml) 10 mg Stemolecule Doxycycline hyclate ( Cat. No ) 5 ml ddh 2 O Add 0.5 ml of ddh 2 O to the vial of doxycycline hyclate. Mix the suspension by inversion until all of the doxycycline has dissolved and the solution is clear. Transfer the suspension to a 15 ml conical tube. Add another 0.5 ml of ddh 2 O to the vial and mix to dissolve any additional doxycycline. Transfer the suspension to the 15 ml conical tube. Bring the stock solution volume up to 5 ml with ddh 2 O. Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. Aliquot in 1.5 ml microcentrifuge tubes and store at -20 C. Freeze/thaw cycles of Dox Solution (2 mg/ml) should be minimized. mipsc Dox-Induction Medium 10 ml mipsc Culture Medium 10 μl Dox Solution (2 mg/ml) Prepare mipsc Dox-Induction Medium fresh daily. Thaw Dox Solution on ice and add to prewarmed mipsc Culture Medium. MEF Feeder Plates 1. Trypsinize and count gamma-irradiated feeder layer MEFs. 2. Dilute cells in MEF Medium to a density of 1 x 10 5 cells/ml. 3. Add 0.5 ml of cell solution to each well of a 4-well or 24-well Gelatin Coated Plate for a final feeder layer cell count of 5 x 10 4 cells per well. 4. Add 2.5 ml of cell solution to each well of a 6-well Gelatin Coated Plate for a final feeder layer cell count of 2.5 x 10 5 cells per well. 5. Incubate overnight at 37 C and 5% CO 2. MEF Feeder Plates are used for the expansion of ips cell colonies. Prepare these plates one day before they will be needed to allow attachment of the MEFs overnight. REPROGRAMMING OCT4-GFP MEFs Prepare Cells for Reprogramming If using Oct4-GFP MEFs (P2), follow the thawing and plating protocol outlined below. If using a different cell type, follow in-house or manufacturer s protocols for thawing and plating for reprogramming. 1. Thaw one vial of Oct4-GFP MEFs (P2) cells in a water bath until only a small ice crystal remains. 2. Submerge briefly in ethanol to sterilize and transfer to a biological safety cabinet. 3. Transfer the contents of the vial into a 15 ml conical tube and add 5 ml of fresh MEF Medium. 4. Centrifuge for 5 min at 200 x g. 5. Aspirate the supernatant and resuspend the cell pellet in 2 ml of MEF Medium., logo, and all other trademarks are property of Inc Inc.

4 STEMGENT Page 4 6. Count live cells using trypan blue and a hemacytometer. 7. Add enough MEF Medium to bring the cell suspension to a concentration of 5 x 10 4 live cells/ml. 8. Add 2 ml of the cell solution to each well of a 6-well Gelatin Coated Plate for a final concentration of 1 x 10 5 cells per well. 9. Incubate overnight at 37 C and 5% CO 2. Note: Plating at this density should allow the cells to be approximately 30 to 50% confluent for transduction the following day. Over-confluent cultures can hinder the transduction efficiency of the experiment. Refer to Figure 1 for an example of an appropriate cell confluency for transduction of Oct4-GFP MEFs. Transduce MEFs 10. Approximately 24 hours after plating the MEFs, prepare the Lentiviral Transduction Medium. 11. Aspirate the MEF Medium and add the Lentiviral Transduction Medium to the well of cells to be reprogrammed. 12. Ensure that the medium is evenly distributed by gentle rocking of the cell culture dish. Incubate overnight at 37 C and 5% CO 2. Replate Transduced MEFs 13. Twenty to 24 hours post-transduction, aspirate the Lentiviral Transduction Medium from the well. 14. Wash the well with 2 ml of PBS. 15. Aspirate the PBS and add 1 ml of 0.05% trypsin/edta. 16. Incubate the plate for 2 minutes at 37 C and 5% CO Add 2 ml MEF Culture Medium to neutralize the trypsin/edta. 18. Pipet the medium across the surface of the well until the cells appear completely detached. 19. Transfer the cell solution to a 15 ml conical tube. 20. Centrifuge the cells for 5 minutes at 200 x g. 21. Remove the supernatant. 22. Resuspend the cell pellet in 1 ml of MEF Medium. 23. Mix the cell solution gently in order to create a uniform suspension of single cells. 24. Count the total number of cells in solution using a hemacytometer. 25. Add the appropriate amount of MEF Medium to bring the cell suspension to 2.5 x 10 4 cells/ml. 26. Aspirate the gelatin from a 6-well Gelatin Coated Plate and add 1 ml per well of MEF Medium to 4 of the wells. 27. Add 1 ml of the cell suspension to each of the 4 wells for a final plating density of 2.5 x 10 4 cells per well., logo, and all other trademarks are property of Inc Inc.

5 STEMGENT Page Aspirate the gelatin from a 4-well Gelatin Coated Plate. 29. Add 0.5 ml of the cell suspension to each of the 4 wells for a final plating density of 1.25 x 10 4 cells per well. Note: Each 4-well plate will be sufficient to assess the transduction efficiency for 1 of the 4 reprogramming transcription factors by immunocytochemistry (ICC). To assess the transduction of multiple transcription factors within the polycistronic virus, plate an additional 3 to 4 wells for each ICC experiment. 30. Incubate both plates overnight at 37 C and 5% CO 2. Note: Any remaining transduced cells that are not replated at this point can be frozen in liquid nitrogen using standard cryopreservation techniques for future reprogramming experiments. Induce Reprogramming using Dox hours post-transduction, aspirate the MEF Medium from the wells in the 6-well plate. 32. Add 2 ml of mipsc Dox-Induction Medium to 3 of the wells. 33. Add 2 ml of mipsc Culture Medium to 1 well, which will serve as a negative control. Note: See Figure 2 for reference. 34. Aspirate the MEF Medium from each well of the 4-well plate. 35. Add 0.5 ml of mipsc Dox-Induction Medium to 2 of the wells. 36. Add 0.5 ml of mipsc Culture Medium to 2 of the wells. Note: See Figure 3 for reference. 37. Incubate plates at 37 C and 5% CO 2. Determine Transduction Efficiency 48 hours post induction, assess the transduction efficiency using the cells cultured in the 4-well plate. Perform ICC staining for 1 transcription factor per 4-well plate. For each transcription factor to be tested, incubate a dox-induced and non-induced well with both the primary and secondary antibodies. The remaining dox-induced well should be used as a negative control by just incubating with the secondary antibody. See the diagram in Figure 3 for experimental configuration of each 4-well plate. Follow the ICC staining procedure as described in s General for Immunocytochemistry. Change Medium and Remove Dox Replace the medium in the 6-well plate with freshly prepared mipsc Dox-Induction Medium every 48 hours. Dox can be removed from the cell culture medium beginning as early as day 12 post induction; replace the medium daily with mipsc Culture Medium at this point. recommends removing dox 16 days post-induction, which allows for the identification of dox-independent ips cell colonies that do not require exogenous, logo, and all other trademarks are property of Inc Inc.

6 STEMGENT Page 6 expression of the reprogramming factors to maintain characteristic ips cell morphology. Prolonged culturing of cells undergoing reprogramming in mipsc Dox-Induction Medium will result in the development of more colonies with the characteristic ips cell morphology. However, in our experience, a larger proportion of these colonies do not develop the ability to demonstrate dox-independence when returned to mipsc Cell Medium. If dox-induction is carried out longer than 16 days it may be necessary to pick more colonies to ensure enough true ips cell clones are selected for expansion. Select ips Cell Colonies The Oct4-GFP MEFs used in this protocol contain a GFP transgene targeted to the endogenous Oct4 (pou5f1) locus. 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 retain good morphology and homogenous GFP expression after culturing in the absence of dox for a minimum of 3 days. Single Colony Pickup, Passage, and Expansion If picking cells encoding a GFP reporter gene, analyze the culture and mark mips cell colonies expressing GFP prior to picking. Colonies showing proper morphology and expressing the Oct4-GFP reporter 3 days after removing dox are highly likely (>90%) to expand and proliferate in culture as mouse ips cells. 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 than 12 colonies should be picked to ensure enough of the picked cells continue to expand in culture after passaging. Note: Do not pick more than 12 colonies at a time in order to ensure that the cells do not remain out of the incubator or 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. 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 mipsc Culture Medium., logo, and all other trademarks are property of Inc Inc.

7 STEMGENT Page 7 3. Incubate the 24-well plates for up to 7 days at 37 C and 5% CO 2. Replace with fresh mipsc Culture 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 independent of dox 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. After 3 to 5 days, cultures can be tested for pluripotency by alkaline phosphatase staining ( Cat. No ) and by staining for typical pluripotency markers such as SSEA-1 ( Cat. No ), Nanog ( Cat. No ), and Oct4 ( Cat. No ). For more information, refer to the protocols on pluripotency analysis. 7. Continue to passage and culture ips cell colonies for use in experimentation and banking., logo, and all other trademarks are property of Inc Inc.

8 STEMGENT Page 8 Figure 1. Oct4-GFP MEFs (P2) 10X Oct4-GFP MEFs 24 hours after thawing and plating for reprogramming. Cell density is approximately 30%. Figure 2. 6-Well Plate Layout Typical set-up of a 6-well plate used for induction and culture of ips cells., logo, and all other trademarks are property of Inc Inc.

9 STEMGENT Page 9 Figure 3. 4-Well Plate Layout used to Assess Transduction Efficiency Well 1 is used for transduction efficiency assessment. These dox(+) cells are stained with both primary and secondary antibodies as well as DAPI for nuclear identification. Well 2 serves as a negative control. These dox(+) cells are stained with DAPI and the secondary antibody only to evaluate background signal from the antibody. Well 3 is a negative control for dox-induction. These dox(-) cells are stained with primary and secondary antibodies as well as DAPI to assess both the specificity of the primary antibody and to monitor successful induction with dox. This well of dox(-) cells should have little to no expression of the transcription factors, but dox-inducible systems have demonstrated levels of basal expression in the absence of dox and should therefore be monitored. Well 4 is a double negative control, and can be omitted from the plate if there is a shortage of transduced cells for replating. These dox(-) cells are stained with secondary antibody and DAPI. Knockout DMEM is a Trademark of Life Technologies., logo, and all other trademarks are property of Inc Inc.