Required Materials. Stemgent Reprogramming-Qualified NuFF Cells GSC-3002G 4 x 10 6 cells/vial Liquid Nitrogen

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1 Page 1 Overview This protocol for microrna-enhanced mrna reprogramming describes the combined use of the Stemgent mrna Reprogramming Kit, the Stemgent microrna Booster Kit, and the Stemgent Stemfect RNA Transfection Kit. Used in combination, the microrna-enhanced mrna Reprogramming System will support the reprogramming of 9 total wells of target cells in a 6-well plate format. This protocol outlines the procedure for reprogramming 3 wells of target cells in one experiment. mrna reprogramming experiments can be successfully performed under both atmospheric conditions (21% O 2 ) and decreased oxygen levels (5% O 2 ). However, the mrna reprogramming process has proven to be more efficient under lower oxygen levels, often yielding 2 to 5 times as many ips cell colonies. The ngfp control mrna should be included when preparing the mrna reprogramming cocktail, as it is useful to assess the transfection efficiency and to track morphological changes throughout the reprogramming process. Assessing morphological progression and ngfp expression over time can be an invaluable tool to evaluate the progress of a reprogramming experiment. Capture images for reference and monitor the cell cultures on a daily basis to compare the morphologies and mrna expression levels over the course of the experiment. Stemgent's StainAlive live-staining antibodies should be used to assess the pluripotency marker expression of the emergent ips cell colonies after the series of transfections is complete. Required Materials PRODUCT DESCRIPTION CAT. NO. FORMAT STORAGE Stemgent mrna Reprogramming Kit mrna Reprogramming Factors Set B18R Recombinant Protein (0.5 mg/ml) Pluriton Supplement (2500X) Pluriton Medium Stemgent microrna Booster Kit microrna Reprogramming Cocktail B18R Recombinant Protein (0.5 mg/ml) Stemgent Stemfect RNA Transfection Kit Stemfect RNA Transfection Reagent Stemfect Buffer x 200 μl 100 μl 0.2 ml 500 ml 2 x 35 μl 100 μl 750 μl 35 ml -70 C -70 C -70 C -20 C -70 C -70 C Stemgent Reprogramming-Qualified NuFF Cells GSC-3002G 4 x 10 6 cells/vial Liquid Nitrogen 4 C 4 C Stemgent bfgf, human recombinant μg -70 C Stemgent StainAlive Antibody StainAlive TRA-1-60 Antibody (DyLight 488) StainAlive TRA-1-81 Antibody (DyLight 488) Corning Matrigel Matrix, hesc-qualified Corning μl 100 μl 4 C 4 C Manufacturer s Instructions

2 Page 2 Reprogramming Timeline The System requires 2 microrna transfections and 11 mrna transfections in total. Emerging ips cell colonies are often able to be identified by morphology and live staining by Day 13, but cell colonies may be ready to be picked and replated earlier or later than shown on the timeline below, depending on the target cell type. Prepare Plate Transfect Identify Identify Pick Maintain DAY microrna microrna mrna Transfections STEP DESCRIPTION DAY 1 Material Preparation Prior to starting 2 Plate Cells -1 3 Transfect Cells 0 to 11 4 Identify Cells 12 to 13 5 Pick and Passage ips Cell Colonies 14 6 Maintain ips Cell Cultures 15 +

3 Page 3 Step 1 Material Preparation Material preparation must begin prior to starting the reprogramming experiment, including the generation of NuFF-Conditioned Pluriton Medium. All material preparation steps should be performed under sterile conditions in a biological safety cabinet. Appropriate storage and handling of the reagents is critical to the success of the reprogramming experiment. Aliquot all reagents into single-use volumes for consistency and to eliminate degradation caused by repeated freeze/thaw cycles. NOTE: Reagent and aliquot volumes in this protocol reflect the amount needed to reprogram 3 wells in one experiment. All aliquots must be prepared in single-use volumes. If reprogramming more or fewer than 3 wells in one experiment, scale all volumes accordingly. 1.1 Pluriton Medium Pluriton Medium must be conditioned on human newborn foreskin fibroblasts (NuFF cells) prior to beginning the reprogramming experiment. After thawing and aliquotting Pluriton Medium, proceed immediately to the Stemgent protocol: Generating NuFF-Conditioned Pluriton Medium, which can be found on our website in the protocols section. 1. Thaw a 500 ml bottle of Pluriton Medium at 4 C for 2 days. 2. Add 5 ml of penicillin/streptomycin (100X) to the bottle of Pluriton Medium (optional). 3. Transfer 250 ml of the Pluriton Medium to a separate sterile container and store at 4 C. This medium will be used to generate NuFF-Conditioned Pluriton Medium. 4. Aliquot remaining 250 ml of Pluriton Medium and store at -20 C.

4 Page Pluriton Supplement (2500X) Single-use aliquots are necessary to avoid repeated freezing and thawing cycles that can result in decreased activity of the Pluriton Supplement. One 2.5 μl aliquot of Pluriton Supplement will be used for each daily medium preparation for 3 wells of target cells. Each of the aliquots containing 5 μl of Pluriton Supplement will be used to support the daily medium exchange of one full 6-well plate. 1. Thaw the Pluriton Supplement on ice. Once thawed, keep the vial on ice at all times. 2. Briefly centrifuge the vial to collect the contents at the bottom of the tube. 3. Using RNase-free aerosol-barrier tips, pipet the contents of the vial to mix thoroughly. 4. Aliquot 2.5 μl of Pluriton Supplement into 54 individual sterile, low protein-binding microcentrifuge tubes. 5. Aliquot 5 μl of Pluriton Supplement into each of 13 sterile, low protein-binding microcentrifuge tubes. 6. Freeze and store the aliquots at -70 C. 1.3 B18R Recombinant Protein B18R protein is essential to modulate the cells innate immune response during repeated RNA transfections. The B18R protein must be supplemented in the cell culture medium to a final concentration of 300 ng/ml during each RNA transfection in order to maintain healthy cultures throughout the reprogramming experiment. Each vial of B18R Recombinant Protein contains 50 μg of protein (100 μl volume at 0.5 mg/ml stock concentration). 1. Thaw two 50 μg vials of B18R protein on ice. Once thawed, keep the vials on ice at all times. 2. Briefly centrifuge the vial to collect the contents at the bottom of the tube. 3. Pipet 4 μl of B18R protein directly to the bottom of 50 sterile, low protein-binding microcentrifuge tubes. 4. Freeze and store the B18R protein aliquots at -70 C until use.

5 Page mrna Reprogramming Cocktail Create an mrna reprogramming cocktail master mix and aliquot the cocktail into single-use volumes prior to beginning the reprogramming experiment. Combine all mrna factors according to the volumes in the table below. The mrna reprogramming cocktail, as prepared below, has a molar stoichiometry of 3:1:1:1:1:1 for the Oct4, Sox2, Klf4, c-myc, Lin28 and ngfp mrnas, respectively. Each mrna factor is supplied at a concentration of 100 ng/μl. 1. Thaw the individual vials containing each mrna reprogramming factor on ice. Keep mrna vials on ice at all times. 2. Briefly centrifuge the vials to collect the contents at the bottom of the tubes. 3. Using RNase-free aerosol-barrier tips, combine the mrna factors according to the table below in a sterile, 1.5 ml RNase-free microcentrifuge tube on ice. Oct4 mrna µl Sox2 mrna µl Klf4 mrna µl c-myc mrna µl Lin28 mrna 85.7 µl ngfp mrna µl mrna Cocktail µl 4. Pipet the contents of the tube to mix throughly. 5. Aliquot 30 µl of the mrna cocktail into 34 individual sterile RNase-free 1.5 ml microcentrifuge tubes. 6. Freeze and store the aliquots at -70 C. NOTE: Each aliquot is intended for one daily transfection. Once the single-use aliquots have been thawed they should not be refrozen.

6 Page microrna Cocktail Each vial of the provided microrna cocktail contains 35 µl of a 20 µm stock of mature micrornas in RNase-free water. Generate single-use microrna cocktail aliquots that will support a single transfection of 3 wells. Two microrna transfections are required per reprogramming experiment. 1. Thaw both vials of the microrna cocktail on ice. Keep the microrna cocktail on ice at all times. 2. Briefly centrifuge the vial to collect the contents at the bottom of the tube. 3. Using RNase-free aerosol-barrier tips, pipet the contents of the vial to mix thoroughly. 4. Aliquot 10.0 μl of the microrna cocktail into 7 sterile RNase-free 1.5 ml microcentrifuge tubes. 5. Store the microrna cocktail aliquots at -70 C until use. 1.6 Coat Reprogramming Wells with Matrix 1. The day prior to plating target cells for reprogramming, coat 3 wells of a 6-well tissue culture plate with Corning Matrigel Matrix, hesc-qualified, according to the manufacturer s instructions. 2. Seal the plate tightly in Parafilm M to prevent evaporation and store level at 4 C overnight.

7 Page 7 Step 2 Plate Target Cells Day Plate Target Cells This harvesting procedure is appropriate for human newborn foreskin fibroblasts in culture in a T75 flask, and may not be applicable to all target cell types. For target cells other than human newborn foreskin fibroblasts, harvest the cells according to an appropriate protocol and plate in the format described below. Reprogramming one well of human newborn foreskin fibroblasts is recommended as an experimental control. 1. Transfer the plate previously coated with Corning Matrigel Matrix, hesc-qualified, to the biological safety cabinet and allow it to equilibrate to room temperature for at least 1 hour. 2. Remove the culture medium from the T75 flask of cells to be harvested. 3. Add 10 ml of Phosphate Buffered Saline (PBS) to the culture surface of the flask to wash. Aspirate the PBS. 4. Add 3 ml of 0.05% Trypsin/EDTA to the culture surface of the flask and incubate for 3 to 5 minutes at 37 C and 5% CO Tap the flask to completely detach the cells from the culture surface. 6. Add 6 ml of human newborn foreskin fibroblasts cell culture medium (or appropriate target cell culture medium containing serum) to the flask to neutralize the Trypsin/EDTA. 7. With a 10 ml pipet, transfer the harvested cell suspension from the T75 flask to a 15 ml conical tube. 8. Centrifuge the cells for 5 minutes at 200 x g. 9. Remove the supernatant and resuspend the pellet in 5 ml of target cell medium. 10. Count the cells and calculate the live cell density. 11. Add a sufficient volume of target cell medium to dilute the cell suspension to a concentration of 2.5 x 10 4 cells/ml. 12. Aspirate the excess medium from the 3 wells previously coated with Corning Matrigel Matrix, hesc-qualified. 13. To each well to be reprogrammed, add 2.0 ml of the fibroblast suspension to a final cell density of 5.0 x 10 4 cells per well. 14. Incubate the cells overnight at 37 C and 5% CO 2. NOTE: Primary fibroblast cultures often times exhibit distinct differences in proliferation due to extended time in culture, response to cryopreservation, and disease-state. As a result, faster growing fibroblast cultures will receive less mrna cocktail dose per cell over the duration of the reprogramming experiment. Conversely slower growing fibroblast cultures will receive more mrna cocktail dose per cell. Therefore optimal reprogramming of adult normal and diseased patient fibroblast may require plating a couple of different cell densities. It is recommended to have one well at the control density of 5.0 x 10 4 cells. For the second well, if your fibroblasts are proliferating more rapidly than a control human foreskin fibroblast cell line, then we recommend a density of 2.5 x 10 4 cells. If your fibroblasts are proliferating less rapidly than a control human foreskin fibroblast cell line, then we recommend the second well density of 7.5 x 10 4 cells.

8 Page 8 Step 3 Transfect Target Cells Day 0 Transfect with microrna Cocktail The B18R protein must be present in the culture medium at a concentration of 300 ng/ml during every transfection. Therefore, a 2-hour B18R pretreatment step is required prior to the first microrna transfection on Day 0 to pre-suppress the cells innate interferon response. Subsequent culture and transfections from Day 1 to Day 11 will be carried out in NuFF-Conditioned Pluriton Reprogramming Medium that is already supplemented with B18R protein. 3.1 Pretreat cells with B18R Protein If reprogramming under low oxygen conditions (5% O 2 ), the medium should be equilibrated at low oxygen tensions prior to each medium exchange (steps 1 and 2, below). 1. Add 6 ml of NuFF-Conditioned Pluriton Medium (without Pluriton Supplement or B18R) to a sterile 15 ml conical tube with a vented cap. 2. Incubate the medium for 30 minutes at 37 C and 5% CO 2 to equilibrate the medium. 3. Thaw one aliquot of Pluriton Supplement and one aliquot of B18R protein on ice. 4. Add 2.4 μl of the Pluriton Supplement and 3.6 μl of B18R protein to 6 ml of the equilibrated medium to generate NuFF-Conditioned Pluriton Reprogramming Medium (with B18R). 5. Aspirate the target cell medium from each of the 3 wells to be transfected and add 2 ml of the NuFF-Conditioned Pluriton Reprogramming Medium (with B18R) to each well. 6. Incubate the cells for 2 hours at 37 C, at 5% CO 2 and appropriate oxygen tension.

9 Page microrna Transfection TUBE µl microrna Cocktail 64.5 µl Stemfect Buffer 75 µl Total TUBE 2 12 μl Stemfect RNA Transfection Reagent 63 μl Stemfect Buffer 75 μl Total microrna Transfection Complex 1. Allow the Stemfect Transfection Reagent and the Stemfect Buffer to equilibrate to room temperature for 30 minutes. 2. Thaw one 10.5 μl aliquot of microrna cocktail on ice (Tube 1). 3. Using RNase-free, aerosol-barrier pipette tips, add 64.5 μl of Stemfect Buffer to the tube containing the microrna cocktail and pipet gently to mix (Tube 1). 4. In a second sterile, RNase-free 1.5 ml microcentrifuge tube, add 63 μl of Stemfect Buffer (Tube 2). 5. Add 12 μl of Stemfect RNA Transfection Reagent to the Stemfect Buffer in Tube 2, and pipet gently to ensure that the transfection reagent is removed from the pipet tip. 6. Using a 100 μl RNase-free, aerosol-barrier tip, pipet gently but thoroughly to mix the Stemfect reagent with the buffer (Tube 2). 7. Using the same 100 μl pipet tip, transfer the contents of Tube 2 to the microrna cocktail solution in Tube 1 to generate the microrna transfection complex. Pipet gently 3 to 5 times to mix. 8. Incubate the microrna transfection complex at room temperature for 15 minutes to allow the microrna to properly complex with the transfection reagent. 9. Holding the plate at a 45 degree angle, add 50 μl of the microrna transfection complex in a dropwise fashion to the pool of medium in each of the 3 wells to be transfected. 10. After adding the transfection complex to the wells, gently rock the plate from side to side and front to back to distribute the transfection complex evenly. 11. Incubate the cells at 37 C, 5% CO 2, and the appropriate oxygen tension.

10 Page Equilibrate Medium If reprogramming in low-oxygen conditions (5% O 2 ), the NuFF-Conditioned Pluriton Medium must be equilibrated to the proper oxygen tension for at least 2 hours to overnight. 6 ml of medium will be used to exchange medium in 3 wells prior to transfection. If incubating the medium overnight, equilibrate 7 ml of medium to account for any evaporation. If reprogramming at normal oxygen conditions, this equilibration step is optional. 1. Add 7 ml of NuFF-Conditioned Pluriton Medium (without Pluriton Supplement or B18R) in a sterile 15 ml conical tube with a vented cap. 2. Incubate the medium overnight at 37 C and 5% CO 2 to equilibrate to the 5% oxygen tension. Day 1 to Day 3 Transfect with mrna Reprogramming Cocktail Cells undergoing reprogramming must be transfected with the mrna reprogramming cocktail every day. It is important to transfect the cells at the same time each day in order to maintain sufficient levels of the reprogramming transcripts to allow for continual expression of the mrna factors. A single-use aliquot of each reagent is used to prepare fresh transfection complex and medium each day. 3.4 Exchange Culture Medium 1. Thaw one aliquot of Pluriton Supplement and one aliquot of B18R protein on ice. 2. Add 2.4 μl of the Pluriton Supplement and 3.6 μl of B18R protein to 6 ml of the pre-equilibrated NuFF-Conditioned Pluriton Medium to generate NuFF-Conditioned Pluriton Reprogramming Medium (with B18R). 3. Aspirate the medium from each of the 3 wells and add 2 ml of the NuFF-Conditioned Pluriton Reprogramming Medium (with B18R) to each well. 4. Incubate the cells at 37 C, 5% CO 2, and the appropriate oxygen tension.

11 Page mrna Transfection TUBE 1 30 µl mrna Cocktail 45 µl Stemfect Buffer 75 µl Total TUBE 2 12 µl Stemfect RNA Transfection Reagent 63 μl Stemfect Buffer 75 µl Total mrna Transfection Complex 1. Allow the Stemfect Transfection Reagent and the Stemfect Buffer to equilibrate to room temperature for 30 minutes. 2. Thaw one 30 μl aliquot of mrna reprogramming cocktail on ice (Tube 1). 3. Using RNase-free, aerosol-barrier pipette tips, add 45 μl of Stemfect Buffer to the tube containing mrna reprogramming cocktail and pipet gently to mix (Tube 1). 4. In a second sterile RNase-free 1.5 ml microcentrifuge tube, add 63 μl of Stemfect Buffer (Tube 2). 5. Add 12 μl of Stemfect RNA Transfection Reagent to the Stemfect Buffer in Tube 2 and pipet gently to ensure that the transfection reagent is removed from the pipet tip. 6. Using a 100 μl RNase-free, aerosol-barrier tip, pipet gently but thoroughly to mix the Stemfect reagent with the buffer (Tube 2). 7. Using the same 100 μl pipet tip, transfer the contents of Tube 2 to the mrna reprogramming cocktail solution in Tube 1 to generate the mrna transfection complex. Pipet gently 3 to 5 times to mix. 8. Incubate the mrna transfection complex at room temperature for 15 minutes to allow the mrna to properly complex with the transfection reagent. 9. Holding the plate at a 45 angle, add 50 μl of the mrna transfection complex in a dropwise fashion to the pool of medium in each of the 3 wells to be transfected. 10. After adding the transfection complex to the wells, gently rock the plate from side to side and front to back to distribute the transfection complex evenly. 11. Incubate the cells at 37 C, 5% CO 2, and the appropriate oxygen tension.

12 Page Equilibrate Medium If reprogramming in low-oxygen conditions (5% O 2 ), the NuFF-Conditioned Pluriton Medium must be equilibrated to the proper oxygen tension for at least 2 hours to overnight. 6 ml of medium will be used to exchange medium in 3 wells prior to transfection. If incubating the medium overnight, equilibrate 7 ml of medium to account for any evaporation. If reprogramming at normal oxygen conditions, this equilibration step is optional. 1. Add 7 ml of NuFF-Conditioned Pluriton Medium (without Pluriton Supplement or B18R) in a sterile 15 ml conical tube with a vented cap. 2. Incubate the medium overnight at 37 C and 5% CO 2 to equilibrate to the 5% oxygen tension. Day 4 Co-Transfect with microrna Cocktail and mrna Reprogramming Cocktail On Day 4, the target cell culture must be transfected with both the mrna reprogramming cocktail and the microrna cocktail. Transfection of the microrna cocktail can be done immediately following transfection of the mrna reprogramming cocktail. 3.7 Co-Transfection 1. Exchange the culture medium on the 3 wells of target cells as described on Day 1, Section Prepare the mrna transfection complex and transfect the target cells exactly as described on Day 1, Section Prepare the microrna transfection complex and transfect the target cells exactly as described on Day 0, Section Equilibrate medium as described on Day 0, Section 3.3.

13 Page 13 Day 5 to Day 11 Transfect with mrna Reprogramming Cocktail The target cells must be transfected with the mrna reprogramming cocktail each day from Day 5 to Day 11, exactly as done from Day 1 to Day mrna Transfection 1. Exchange the culture medium on the 3 wells of target cells as described on Day 1, Section Prepare the mrna transfection complex and transfect the target cells exactly as described on Day 1, Section Equilibrate medium as described on Day 1, Section 3.6.

14 Page 14 Step 4 Day 12 Identify ips Cells 4.1 Culture and Monitor ips Cell Colonies Once the series of RNA transfections is completed, the primary reprogramming cultures should be maintained in NuFF-Conditioned Pluriton Reprogramming Medium for one to three additional days in order to allow the ips cell colonies to expand in size enough to be picked and passaged as primary colonies. After the series of RNA transfections is complete, NutriStem Culture Medium may be used in place of NuFF-Conditioned Pluriton Reprogramming Medium in this procedure. At this stage, the B18R protein is no longer needed in the culture medium. 1. Thaw one aliquot of Pluriton Supplement on ice. 2. Add 2.4 μl of the Pluriton Supplement to 6 ml of pre-equilibrated NuFF-Conditioned Pluriton Medium to generate NuFF-Conditioned Pluriton Reprogramming Medium. 3. Aspirate the medium from each of the 3 wells and add 2 ml of the NuFF-Conditioned Pluriton Reprogramming Medium to each well. 4. Incubate the cells at 37 C, 5% CO 2, and the appropriate oxygen tension. NOTE: Continue to culture the cells in this manner, exchanging the medium each day for 1 to 3 days, or until the ips cell colonies are large enough to be picked and passaged.

15 Page 15 Day Identify Colonies using a StainAlive Antibody Prior to manual isolation, the primary ips cell colonies can be identified using sterile, live-staining StainAlive DyLight 488 mouse anti-human TRA-1-60 or TRA-1-81 antibody to confirm the onset of these expression markers associated with pluripotency. NutriStem Culture Medium may be substituted in place of NuFF-Conditioned Pluriton Reprogramming Medium in this procedure. 1. Thaw one 2.5 μl aliquot of Pluriton Supplement on ice. 2. Add 3 ml of Pluriton Medium to a 15 ml conical centrifuge tube. 3. Add 30 μl of either StainAlive DyLight 488 TRA-1-60 or StainAlive DyLight 488 TRA-1-81 antibody to the centrifuge tube containing the medium. This will yield a 5 μg/ml solution of the StainAlive antibody. 4. Aspirate the medium from the 3 wells of cells to be stained and add 1.0 ml of the diluted antibody solution to each well. 5. Incubate the culture for 30 minutes at 37 C, 5% CO 2, and the appropriate oxygen tension. 6. Aspirate the diluted antibody medium from each well and add 1.5 ml of Pluriton Medium to wash. 7. Repeat the wash with an additional 1.5 ml of Pluriton Medium per well. 8. Add 2.4 μl of the Pluriton Supplement to 6 ml of pre-equilibrated NuFF-Conditioned Pluriton Medium to generate NuFF-Conditioned Pluriton Reprogramming Medium. 9. Aspirate the wash medium from each of the 3 wells and add 2 ml of the NuFF-Conditioned Pluriton Reprogramming Medium to each well. 10. Examine the cultures on a fluorescent microscope using a filter set for the appropriate wavelength. NOTE: For a DyLight 488-labeled antibody, the maximum excitation wavelength is 493 nm, and mean emission wavelength is 518 nm. 11. Identify and mark appropriate colonies for picking and expansion. Do not allow the cell cultures to be kept outside of the incubator for an extended period of time. 12. Return the cells to the incubator and continue to culture at 37 C and 5% CO Equilibrate 13 ml of NuFF-Conditioned Pluriton Medium, as described on Day 1, Section 3.6.

16 Page 16 Step 5 Pick and Passage ips Cells Day 14 All procedures in this picking protocol must be performed in a sterile environment. Picking can be performed with a stereo microscope in either a horizontal flow hood (positive pressure) or a static enclosure. Glass picking tools can be made from 9 Pasteur pipettes pulled to a closed, angled end over the controlled flame of an alcohol burner. 5.1 Pick and Replate Primary ips Cell Colonies Pick and replate no more than 6 colonies at one time to avoid keeping the cells out of the incubator for extended periods of time. To maintain clonal lines, transfer all of the pieces of each individual colony into a separate well of a 12-well plate (with appropriate matrix or feeder cells). Change pipet tips with each new colony to be transferred to avoid cross-contamination of clonal lines. NutriStem Culture Medium may be used in place of NuFF-Conditioned Pluriton Reprogramming Medium in this procedure. 1. Thaw one 5 μl aliquot of Pluriton Supplement on ice. 2. Add 4.8 μl of the Pluriton Supplement to 12 ml of pre-equilibrated NuFF-Conditioned Pluriton Medium to generate NuFF-Conditioned Pluriton Reprogramming Medium. 3. Aspirate the medium from 6 wells of a 12-well plate (previously prepared with new matrix as described in Section 1.6). 4. Add 1 ml of NuFF-Conditioned Pluriton Reprogramming Medium to each of the 6 wells. 5. Aspirate the medium from each well of primary ips cells, and replace with 2 ml of NuFF-Conditioned Pluriton Reprogramming Medium per well. 6. Using a phase-contrast or stereo microscope, locate ips cell colonies based on morphology and pluripotency marker expression. 7. Using a glass picking tool, gently separate the colony from the surrounding fibroblasts by circling the area to be picked. 8. Using the glass picking tool, gently divide the colony into approximately 4 to 8 pieces. It is important to break the colony into smaller cell aggregates, but not into single cells. 9. Using the glass picking tool, gently and completely detach the colony pieces from the tissue culture plate so that the cell aggregates are freely suspended in the medium. 10. Using a pipettor with a sterile large bore tip, transfer the detached colony pieces out of the reprogramming well and into an individual well of the prepared 12-well plate. NOTE: Transfer all of the pieces from one colony into a single well of the 12-well plate.

17 Page Repeat the picking and replating process for the next ips cell colonies. Pick one colony at a time and transfer the cell aggregates of each to a new well of the prepared 12-well plate. 12. After 6 ips cell colonies have been picked and replated, place both the 12-well plate and the primary reprogrammed colonies to the incubator at 37 C and 5% CO Repeat this process (steps 1 through 12) in increments of 6 ips cell colonies at a time until the desired number of colonies have been picked. Step 6 Maintain ips Cell Cultures Day 15+ Human ips cell cultures should be monitored and cared for every day, as the overall quality of the culture can change rapidly. Human ips cells are generally passaged every 4 to 7 days in culture, but the actual passaging schedule and split ratio for each passage will vary depending on the cell culture s quality and growth rate. Within the first few days of each passage, the proliferating cells grow easily in a monolayer colony. Once the colony becomes large, the proliferating cells begin to pile up, often causing unwanted spontaneous differentiation to occur. It is important to passage the cells before the cultures become overgrown. For maintenance and expansion, the ips cells should cultured in NutriStem Culture Medium on Corning Matrigel Matrix, hesc-qualified, or adapted to other proven human ips cell culture conditions. Between passages, the cell culture medium must be exchanged every day to provide necessary growth factors for the maintenance of human ips cells. If the cells were reprogrammed under low oxygen tension, the cultures can be transitioned to atmospheric oxygen conditions within the first passage. For the first few passages after picking colonies from the primary reprogrammed cultures, the cells should be passaged manually (without enzymes or centrifugation) at low split ratios to build dense cultures. The cells can be split using an enzymatic protocol for routine culture once there are a large number of human ips cell colonies in the well(s).

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