Protocol Reprogramming Human Fibroblasts into ips Cells using the Stemgent Reprogramming Lentivirus Set: Human OKSM

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1 STEMGENT Page 1 Reprogramming Lentivirus Set: Human OKSM OVERVIEW The following procedure describes the reprogramming of BJ Human Fibroblasts (BJ cells) to induced pluripotent stem (ips) cells using the Reprogramming Lentivirus Set: Human OKSM. Transduction efficiency can vary depending on factors such 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. Product Description Cat. No. Format Storage Reprogramming Lentivirus Set: Human OKSM ST Set -70 C Components Cat. No. Size Storage Reprogramming Lentivirus: Human Oct4* Reprogramming Lentivirus: Human Klf4* Reprogramming Lentivirus: Human Sox2* Reprogramming Lentivirus: Human c-myc* *Lentiviruses can be ordered separately. ST ml -70 C ST ml -70 C ST ml -70 C ST ml -70 C ADDITIONAL MATERIALS REQUIRED BJ Human Fibroblast (early passage, p6) ( Cat. No ) EMEM medium FBS HEPES (1M) Polybrene Stemfactor Fibroblast Growth Factor-basic ( Cat. No ) 10 mm Tris, ph 7.6 NutriStem XF/FF Culture Medium ( Cat. No ) Matrigel hesc-qualified Matrix (BD) 0.1% Gelatin in water DMEM Non-essential amino acids (100x) L-glutamine (200 mm) β-mercaptoethanol (55 mm) PBS 0.05% Trypsin/EDTA 6-well tissue culture plates 12-well tissue culture plates 15 ml conical tubes

2 STEMGENT Page 2 Reprogramming Lentivirus Set: Human OKSM MATERIAL PREPARATION BJ Medium 435 ml EMEM medium 50 ml FBS 15 ml HEPES (1M) Store BJ Medium at 4 C. Warm to room temperature before use. Transduction Medium Thaw one vial of each of the reprogramming lentiviruses Oct4, Klf4, Sox2, and c-myc. Once thawed, keep lentiviruses on ice. A multiplicity of infection (MOI) of 10 is recommended when using the Reprogramming Lentivirus Set: Human OKSM to reprogram BJ cells. Calculate the amount of transduction units (TU) needed by multiplying the number of cells plated by the MOI: (1 x 10 5 BJ cells) x (10 TU/cell) = 1 x 10 6 TU Determine the volume of each virus to add by dividing the TU s needed by the concentration of the virus (TU/ml). Concentrations are lot specific and can be found on the specification sheet. 1 x 10 6 TU 1 x 10 6 TU/ml = 1 ml 1 ml 1000 µl/ml = 1000 µl Add the calculated amount of each virus to a 15 ml conical tube and add polybrene to a final concentration of 6 µg/ml. Transduction Medium should be used immediately. Note: The MOI should be optimized for the target cell type to achieve the best transduction efficiency balanced with the lowest possible cytotoxicity. If transducing a different cell type than what is used in this protocol, the MOI may need to be adjusted. bfgf Solution 50 µg Stemfactor Fibroblast Growth Factor-basic 1 ml 10 mm Tris, ph 7.6 Briefly centrifuge the lyophilized vial of bfgf and reconstitute in 500 μl of 10 mm Tris, ph 7.6 to yield a 50 µg/ml solution. Aliquot and store at -20 C for up to 6 months. NutriStem Reprogramming Medium 500 ml NutriStem XF/FF Culture Medium 200 µl bfgf Solution Supplement NutriStem medium with 20 ng/ml of bfgf. Store NutriStem Reprogramming Medium at 4 C for up to 2 weeks.

3 STEMGENT Page 3 Reprogramming Lentivirus Set: Human OKSM 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. Gelatin Coated Plates Add 0.5 ml of 0.1% gelatin in water to each well of a 12-well plate. Incubate at 37 C for a minimum of 1 hour. Gelatin Coated Plates can be stored at 37 C for up to 1 week. MEF Medium 450 ml DMEM 50 ml FBS 5 ml Non-essential amino acids (100x) 5 ml L-glutamine (200 mm) 500 µl β-mercaptoethanol (55 mm) Filter-sterilize using a 0.22 µm pore size, low protein-binding filter. Store MEF Medium at 4 C. MEF Feeder Plates 1. Trypsinize and count gamma-irradiated feeder layer MEFs. 2. Dilute cells in MEF Medium to a density of 5 x 10 4 cells/ml. 3. Add 2 ml of cell solution to each well of a 12-well Gelatin Coated Plate for a final feeder layer cell count of 1 x 10 5 cells per well. 4. 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.

4 STEMGENT Page 4 Reprogramming Lentivirus Set: Human OKSM REPROGRAMMING TIMELINE DAY ACTIVITY MORPHOLOGY BJ Cell Preparation and Transduction Replating of Transduced BJ Cells ips Cell Colony Identification and Isolation 0 Plate BJ cells 1 Transduce BJ cells 2 Remove Transduction Medium and replace with BJ Medium 3 Incubate transduced cells 4 Change medium 5 Incubate transduced cells 6 Replate transduced cells onto Matrigel-coated plate with NutriStem Reprogramming Medium 7-14 Incubate transduced cells, changing medium every other day Incubate transduced cells, changing medium daily 28+ ips cell colonies are picked and plated in 12-well plates. True ips cell colonies can form earlier than day 28 and should be picked and transferred as they appear ips cells may begin to emerge as early as day 14. Monitor cultures accordingly 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 1) REPROGRAMMING PROCEDURE Prepare Cells for Reprogramming If using BJ cells, follow the plating protocol listed below. If using another cell type, further optimization may be required. 1. Plate BJ cells on a 6-well tissue culture plate at a final density of 1 x 10 5 cells per well. 2. Incubate overnight at 37 C and 5% CO 2. Transduce BJ Cells 3. The next day, prepare the Transduction Medium. 4. Aspirate the medium from one well of cells and replace with freshly prepared Transduction Medium.

5 STEMGENT Page 5 Reprogramming Lentivirus Set: Human OKSM 5. Incubate at 37 C and 5% CO 2 overnight. Culture Transduced Cells 6. After the overnight incubation, aspirate the medium and replace with 2 ml per well of BJ Medium. 7. Incubate 2 days at 37 C and 5% CO On day 4 post transduction, replace the medium with fresh BJ Medium. 9. Incubate 2 days at 37 C and 5% CO On day 6 post transduction, aspirate the medium from the well. 11. Wash the well twice with 2 ml of PBS. 12. Aspirate the PBS and add 1 ml per well of pre-warmed 0.05% Trypsin/EDTA. 13. Incubate at 37 C for 30 seconds. 14. Aspirate the Trypsin/EDTA and incubate at room temperature until the cells begin to detach from the plate. Note: Incubation times will vary between cell lines. Begin checking the culture after 1 minute. 15. Add 4 ml of NutriStem Reprogramming Medium and pipette across the well until all of the cells are detached. 16. Transfer the cell suspension to a 15 ml conical tube. 17. Count the cells and prepare a cell suspension of 5 x10 3 cells/ml in NutriStem Reprogramming Medium. 18. Aspirate the solution from a 6-well Matrigel-Coated Plate and add 2 ml per well of the cell suspension to each well for a final concentration of 1 x 10 4 cells per well. Note: Cells should be replated at an appropriate density specific for the cell type used. Typically, cells are replated at a density range of 3 x 10 5 cells per well to 5 x 10 5 cells per well. BJ cells are plated at a lower than normal density due to their growth rate. Plating at least 6 wells of transduced cells is recommended. If a low reprogramming efficiency is expected, increase the number of wells plated. 19. Incubate overnight at 37 C and 5% CO The next day, aspirate the medium and replace with 2 ml per well of NutriStem Reprogramming Medium. 21. Change the medium every other day for the first 7 days after replating. On the eighth day, begin changing the medium daily. 22. ips cell colonies should form within 3 weeks after replating. Single Colony Pickup, Passage and Expansion The following procedure uses feeder cells to culture ips cell colonies. If desired, ips cell colonies can be maintained in feeder-free conditions. 23. Manually pick colonies with ips cell morphology to a single well of a 12-well MEF Feeder Plate (see Figure 1 for morphology examples). a. Prepare a 12-well MEF Feeder Plate by aspirating the medium, washing

6 STEMGENT Page 6 Reprogramming Lentivirus Set: Human OKSM with 1 ml of PBS and adding 1 ml of NutriStem Reprogramming Medium. b. Locate an ips cell colony. Using a sterile glass picking tool, gently separate the identified colony from surrounding cells. c. Using the glass picking tool, gently divide the colony into clusters that contain 30 to 40 cells. d. Using the glass picking tool, gently detach each colony cluster from the tissue culture well. e. Using a P200 pipettor set at 100 μl, pipette the detached colony clusters out of the 6-well plate and into an individual well of the 12-well MEF Feeder Plate. 24. Incubate at 37 C and 5% CO Replace the medium daily with fresh NutriStem Reprogramming Medium. If there is no visible colony attachment or expansion by 8 days after picking, then those wells do not need to be maintained and can be discarded. DISCUSSION ips cell colonies can be replated onto 6-well plates for expansion of the line 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 Stemolecule Y27632 molecule ( Cat. No ) or the Stemolecule Thiazovivin ( Cat. No ), can be used when picking and passaging human ips cell colonies for expansion. Expanded ips cell colonies can be tested for pluripotency by using the Alkaline Phosphatase Staining Kit II ( Cat. No ) and staining for typical pluripotency markers such as Nanog ( Cat. No ), Oct4 ( Cat. No ), SSEA-3 ( Cat. No ) SSEA-4 ( Cat. No ), TRA-1-60 ( Cat. No ), TRA-1-81 ( Cat. No ) and Rex1 ( Cat. No ).

7 STEMGENT Page 7 Reprogramming Lentivirus Set: Human OKSM Day 15 Day 17 Day 19 Figure 1: Representative ips Cell Colonies BJ cells were transduced, re-plated and cultured following the Reprogramming Lentivirus Set: Human OKSM protocol. 5x phase contrast images of emerging (P0) ips cell colonies were taken on day 15, 17, and 19 post transduction. Matrigel is a trademark of BD. NutriStem is a trademark of Biological Industries.

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