Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

1.Solution and Medium Preparation

1. Basement Membrane Matrix (Purified from Engelbreth-Holm-Swarm Tumor)
   1. Slowly thaw the basement membrane matrix (5 ml) on ice at 4 °C overnight.
   2. Dilute the stock solution 1:1 with 5 ml of ice cold, sterile DMEM/F-12 medium in a sterile, pre-chilled 15 ml conical tube. Dispense aliquots into pre-chilled, 1.5 ml sterile micro centrifuge tubes and immediately store at -20 °C.
   3. Prior to usage, thaw the frozen 1:1 basement membrane matrix aliquot overnight at 4 °C. At the time of use, further dilute the 1:1 aliquot another 50 fold with ice cold, sterile DMEM/F-12 medium to a final dilution of 1:100.
   4. Add the appropriate amount of the 1:100 diluted basement membrane matrix solution to the appropriate tissue culture (TC) vessels and incubate at 37 °C for 1 hr. Typically, use 3 ml of the diluted basement membrane matrix solution to coat a TC dish of 20 cm² surface area, coat all other types of TC dishes/plates at a ratio of 0.15 ml of diluted matrix per cm² of surface area. Aspirate the remaining solution prior to addition of any culture medium and cells.
2. Fibroblast Medium
   1. Thaw a 100 ml bottle of ES Qualified Fetal Bovine Serum (ES-FBS) at 4 °C overnight.
   2. Add 50 ml of the thawed ES-FBS and 5 ml of MEM Non-Essential Amino Acid Solution (1x) to 445 ml of DMEM medium. Sterilize the combined components through a 0.22 µm filter and store the medium at 4 °C for up to 4 weeks.
3. iMEF Medium
   1. Thaw a 100 ml bottle of ES-FBS at 4 °C overnight.
   2. Add 50 ml of the thawed FBS, 5 ml of MEM Non-Essential Amino Acid Solution (1x), and 500 µl of 2-mercaptoethanol to 445 ml of DMEM medium.
   3. Sterilize the combined components through a 0.22 µm filter and store the medium at 4 °C for up to 4 weeks.
4. Basic Fibroblast Growth Factor solution (b-FGF)
   1. Add 10 µl of Serum Replacement to 990 µl of D-PBS and thoroughly mix by pipetting up and down.
   2. Add the 1 ml of the 1% (v/v) Serum Replacement solution to one vial of 10 µg lyophilized b-FGF. Thoroughly mix by gently pipetting up and down.
   3. Aliquot the 10 µg/ ml b-FGF solution into 200 µl aliquots in micro-centrifuge tubes and store at -20 °C until needed for up to 4 weeks.
5. Human Pluripotent Stem Cell (hPSC) Medium
   1. Thaw a 100 ml bottle of Serum Replacement at 4 °C overnight.
   2. Add 100 ml of the thawed Serum Replacement, 5 ml of MEM Non-Essential Amino Acid Solution (1x), and 500 µl of 2-mercaptoethanol to 395 ml of DMEM/F-12 medium.
   3. Sterilize the combined components through a 0.22 µm filter and store the medium at 4 °C for up to 4 weeks.
   4. At the time of usage, pre-warm the hPSC medium to 37 °C and supplement with 4 ng/ml of bFGF.
6. iMEF Conditioned Medium (CM)
   1. Add 18 ml of 0.1% gelatin to a T-175 Culture flask and incubate at 37 °C for 1 hr.
   2. Remove the 0.1% gelatin solution after one hour of incubation. Add 9.1 x 10⁶ mitotically-inactivated mouse embryonic fibroblasts (iMEF) in 45 ml of iMEF medium and incubate in the 37 °C incubator overnight.
   3. Following overnight incubation, remove the old iMEF medium and wash once with 25 ml of D-PBS for 5 min at room temperature.
   4. Aspirate off the D-PBS wash and add 90 ml of pre-warmed hPSC medium, supplemented with 4 ng/ml of bFGF. Incubate in the 37 °C incubator for exactly 24 hr.
   5. After 24 hr of incubation, aseptically remove the hPSC medium from the iMEF flask and sterilize through a 0.22 µm filter. Aliquot into 50 ml conical tubes and freeze at -20 °C until needed. iMEF CM can be stored for up to 6 months at -20 °C.
   6. Add another 90 ml of pre-warmed hPSC medium supplemented with 4 ng/ml of bFGF to the iMEF flask. Incubate in the 37 °C incubator for exactly 24 hr. Repeat the harvesting of CM for up to 7 days.
   7. At the time of usage, pre-warm the iMEF-CM to 37 °C and supplement with 4 ng/ml of bFGF.
7. E8 Medium
   1. Thaw a 10 ml E8 supplement at 4 °C overnight.
   2. Remove 10 ml of the E8 basal medium and aseptically add the contents of the thawed E8 supplement to the remaining basal medium. Mix thoroughly and sterilize through a 0.22 µm filter. Store the complete medium at 4 °C for up to 2 weeks.
   3. At the time of usage, pre-warm the aliquot of the medium to be used to room temperature. It is not recommended to pre-warm E8 Medium to 37 °C.
8. Cell Sorting Buffer
   1. Prepare fresh sorting buffer right before cell sorting by adding EDTA solution (1 mM final concentration), HEPES buffer (25 mM final concentration) Penicillin/Streptomycin solution (100 units/ml final concentration) and Bovine Serum Albumin (1% final concentration) into Dulbecco's Phosphate Buffered Saline (Calcium and Magnesium free).
   2. Sterilize the buffer through a 0.22 µm filter prior to using.
9. Cell Sorting Recovery Medium
   1. Modify 50 ml of Fibroblast Medium by adding Penicillin/Streptomycin (100 units/ml final concentration) and Rock Inhibitor Y-27632 (10 µM final concentration). Pre-warm this medium to 37 °C prior to seeding the sorted cells.

2. Sendai Mediated Reprogramming of Human Fibroblasts

1. 24 hr prior to transduction with the Sendai viruses, seed the fibroblasts at a density of 2 × 10⁵ cells per well into the desired wells of a 6-well TC plate.
2. Perform the transduction on Day 0 as follows.
   1. Pre-warm 2 ml of Fibroblast Medium in a 37 °C water bath. Remove one set of Sendai virus tubes from -80 °C storage and thaw each tube one at a time by first immersing the bottom of the tube in a 37 °C water bath for 5 – 10 sec, and then remove the tube from the water bath and allow it to thaw at room temperature. Once thawed, briefly centrifuge the tube and place it immediately on ice.
   2. Use commercially second generation Sendai viruses by adding the appropriate volumes (as per the COA) for each of the tubes in order to achieve the following multiplicity of infection (MOI) to 1 ml of pre-warmed Fibroblast Medium per well to be transduced as follow: KOS = 5 x 10⁶ CIU, hc-Myc = 5 x 10⁶ CIU, hKlf4 = 3 x 10⁶ CIU .
   3. Thoroughly mix the viral solution by pipetting the mixture gently up and down. Complete the next step within 5 min.
   4. Aspirate the Fibroblast Medium from the well of the fibroblasts to be reprogrammed, and add 1 ml of the viral solution to each well. Place the cells in a 37 °C, 5% CO₂ incubator and incubate overnight.
3. Following overnight incubation, aspirate off the transduction medium and dispose of the old medium properly (10% bleach solution for 30 min). Replace with 2 ml of pre-warmed Fibroblast Medium and continue to culture the transduced cells.
4. Culture the cells for 6 more days, replacing the old medium with fresh Fibroblast Medium every other day.
   Note: Do not passage the reprogrammed cells until 7 days post transduction.
5. Day 6: Prepare Dishes for iPSC Generation
   1. For feeder-dependent iPSC generation, add 1.5 ml of 0.1% gelatin solution to each well of a 6-well tissue culture (TC) dish and incubate at 37 °C for 1 hr.
   2. Remove the 0.1% gelatin solution after 1 hr of incubation. Add 3 x 10⁵ mitotically-inactivated mouse embryonic fibroblasts (iMEF) in 2 ml of iMEF medium per well and incubate in the 37 °C incubator overnight.
   3. For feeder-independent iPSC generation, add 1.5 ml of diluted basement membrane matrix (1:100 dilution) to each well of a 6-well tissue culture (TC) dish and incubate at 37 °C for 1 hr.
6. Seven days after transduction, harvest the transduced fibroblasts and re-plate them onto the pre-made culture dishes for iPSC colony generation.
   1. Aspirate off the normal culture medium from the fibroblasts, and wash the cells once with 2 ml of D-PBS.
   2. Aspirate off the D-PBS wash and add 0.5 ml of pre-warmed 0.05% trypsin/EDTA. Incubate the cells for 3 – 5 min at 37 °C.
   3. When the cells have rounded up, add 2 ml of pre-warmed Fibroblast Medium into each well to stop trypsinization. Dislodge the cells from the well by gently pipetting the medium across the surface of the dish. Collect the cell suspension in a 15-ml conical centrifuge tube.
   4. Centrifuge the cells at 200 x g for 2 min. Aspirate the supernatant, and re-suspend the cells in 2 ml fresh, pre-warmed Fibroblast Medium.
   5. Count the cells using the desired method (hemocytometer or automated cell counter) and seed the pre-prepared culture dishes with 5 x 10⁴ cells for feeder-dependent conditions and 1 x 10⁵ cells for feeder-independent conditions, per well of a 6-well dish and incubate at 37 °C, 5% CO₂ incubator overnight.
   6. Following the overnight incubation, change the medium to hPSC medium, E8 medium or iMEF-conditioned media, depending on the application. Replace the old medium with fresh medium everyday thereafter.

3. Sendai Reprogramming of BGO1v hOct4-GFP Reporter Human Embryonic Stem Cells (hESC) Derived Secondary Fibroblasts

1. Derive secondary human fibroblasts from the BG01v hOct4-GFP reporter hESC line as per the procedure previously described ⁹.
2. Two days before transduction, plate BGO1v/hOG derived fibroblasts into two wells of a 6-well plate at 2 x 10⁵ cells per well, using Fibroblast Medium.
   1. On the day of transduction, pre-warm 2 ml of Fibroblast Medium to 37 °C.
   2. Remove one set of Sendai tubes from the -80 °C storage. Remove the vials from the pouch and thaw each tube one at a time by first immersing the bottom of the tube in a 37 °C water bath for 5 – 10 sec, after which, allow the tubes to thaw at room temperature. Once thawed, briefly centrifuge the tube and place it immediately on ice.
   3. Add the indicated volumes of each of the three Sendai tubes (see the CoA for the appropriate volume) to 2 ml of Fibroblast Medium, pre-warmed to 37 °C and thoroughly mix the viral solution by pipetting up and down. Complete the next step within 5 min.
      Note: For this experiment, a MOI of 5:5:6 was utilized.
   4. Aspirate the Fibroblast Medium from the wells of fibroblasts to be transduced. Add 1 ml of the viral suspension to each of two wells to be transduced. Place the cells in a 37 °C, 5% CO₂ incubator and incubate overnight.
   5. Following overnight incubation, remove the transduction medium and dispose of it properly (10% bleach solution for 30 min). Replace each well with 2 ml of pre-warmed Fibroblast Medium and continue to culture the transduced cells.
3. Culture the cells for 6 more days, changing the old medium with fresh Fibroblast Medium every other day.
   1. Seven days post transduction, harvest the transduced fibroblasts and re-plate them onto the pre-made culture dishes for iPSC colony generation.
   2. Aspirate off the normal culture medium from the fibroblasts, and wash the cells once with 2 ml of D-PBS.
   3. Aspirate off the D-PBS wash and add 0.5 ml of pre-warmed 0.05% trypsin/EDTA. Incubate the cells for 3 – 5 min at 37 °C.
   4. When the cells have rounded up, add 2 ml of pre-warmed Fibroblast Medium into each well to stop trypsinization. Dislodge the cells from the well by gently pipetting the medium across the surface of the dish. Collect the cell suspension in a 15-ml conical centrifuge tube.
   5. Centrifuge the cells at 200 x g for 2 min. Aspirate the supernatant, and re-suspend the cells in an appropriate amount of fresh, pre-warmed Fibroblast Medium.
   6. Count the cells using the desired method (hemocytometer or automated cell counter).
   7. Seed the transduced cells at a density of 1 x 10⁵ cells per well of a basement membrane matrix -coated 6-well plate and incubate in a 37 °C, 5% CO₂ incubator overnight.
4. Following the overnight incubation, change the medium to iMEF Conditioned Medium, supplemented with 10 ng/ml of b-FGF. Replace the old medium with fresh iMEF CM everyday thereafter.
5. Three weeks post transduction, mechanically dissect and transfer the desired colonies for clonal expansion or use for analysis.

4. Live Cell Imaging of Fibroblast Reprogramming

1. Real-Time Monitoring
   1. 7 days post transduction with the Sendai reprogramming kit, seed the desired amount of cells onto 6-well dishes with the desired medium and matrix systems as previously described. Place the seeded dishes inside the imager, located in a humidified incubator set at 37 °C, and 5% CO₂.
   2. Set the imaging software to capture whole well images of each individual well at a set interval (every 6 – 8 hr) of continuous image capturing for the next 14 days according to manufacturer's protocol.
      Note: Phase contrast image settings are selected for normal reprogramming to evaluate colony progression. In the case of BG01v/hOG derived fibroblast reprogramming, it is best to capture images in phase contrast and in the green fluorescent channel as the re-activation of the endogenous OCT4-GFP reporter can be tracked throughout the reprogramming process.
   3. Change the medium daily, it can be hPSC medium, E8 medium, or iMEF-CM, depending on the experiment, as previously described from Day 8 to 21 post-transduction. The wells can be further analyzed for colony formation using indirect immunofluorescence.
   4. At the end of the experiment on Day 19 to 21, remove the culture medium from each well to be probed with antibodies for fibroblast and stem cell markers of choice.
   5. Wash each well once with 2 ml of pre-warmed DMEM/F-12 medium.
   6. Prepare aliquots of each positive/negative primary antibody pairs in 1 ml of DMEM/F-12 medium as follows: anti-mouse SSEA4 (1:200) and anti-rat CD44 (1:50), anti-mouse TRA-1-60 (1:200) and anti-rat CD44 (1:50), and Alkaline Phosphatase Live Stain (1:50) and anti-rat CD44 (1:50). Add the 1 ml primary antibody solutions to each corresponding well. Incubate at 37 °C for 45 min.
   7. Aspirate off the primary antibody solution and wash twice with pre-warmed DMEM/F-12 medium.
   8. Prepare secondary antibody solutions (1 ml for each well) using goat-anti-mouse Alexa Fluor 488 conjugated secondary (1:500) for green fluorescence and goat-anti-rat Alexa Fluor 594 conjugated secondary (1:500) for red fluorescence in DMEM/F-12 medium. Add to each well after the final wash. Incubate the secondary antibodies for 30 min at 37 °C.
      Note: Due to Alkaline Phosphatase Live Stain's (APLS) transient signal, the anti-CD44 primary incubation should be first performed by itself and the APLS should be added at the time of incubation with the secondary antibodies
   9. Following the secondary antibody incubation, aspirate off the solution and wash twice with DMEM/F-12 medium. Add fresh pre-warmed DMEM/F-12 medium to each well prior to final image capture.
   10. Set the imager software to acquire whole well images of each well using all three scanning parameters: phase contrast, green fluorescence and red fluorescence. Create images at various magnifications and create time-lapse movies with the software to monitor growth and marker expression. Use manufacturer's protocol.
   11. Utilizing the analysis software, evaluate confluence of the well over time in phase contrast and in green fluorescent units for the BG01v/hOG reprogramming.
2. Monitoring Reprogramming Intermediates using Cell Surface Markers
   1. Monitor reprogramming events at different time points by probing the reprogramming dishes at various time points with antibodies against different stem cell positive/negative markers in order to monitor the reprogramming process. Probe cultures every 2 to 3 days depending on availability of replicates. Probe the reprogramming dishes at day 19 to 21 using the dual staining strategy to identify fully reprogrammed iPSC colonies and partially reprogrammed colonies based on the staining patterns.
   2. At the desired time point, aspirate off the normal growth medium from each well to be probed with antibodies for the markers of choice.
   3. Wash each well once with 2 ml of pre-warmed DMEM/F-12 medium.
   4. Prepare aliquots of each positive/negative primary antibody pairs in 1 ml of DMEM/F-12 medium as follows: SSEA4 conjugated to the green fluorescent fluorophore (1:200), TRA-1-60 conjugated to the green fluorescent fluorophore(1:50), Alkaline Live Stain (1:500), CD24 conjugated to a green fluorescent fluorophore (1:50), B2M conjugated to a green fluorescent fluorophore (1:50), EpCAM conjugated to a green fluorescent fluorophore(1:50), mouse CD73 (1:100) which was probed with anti-mouse secondary antibody conjugated to a green fluorescent fluorophore(1:500), and rat CD44 (1:50) which is probed with anti-rat secondary antibody conjugated to the red fluorescent fluorophore(1:500). Add the 1 ml primary antibody solutions to each corresponding well. Incubate at 37 °C for 45 min.
   5. Aspirate off the primary antibody solution and wash twice with pre-warmed DMEM/F12 medium.
   6. For unconjugated antibodies, proceed to using a 2-step method by preparing the appropriate secondary antibody solutions and incubate for 30 min at 37 °C. Confirm that secondary antibodies are to the primary hosts and do not cross react. Perform additional wash steps as previously described.
   7. After all appropriate washes, add fresh pre-warmed DMEM/F-12 medium to each well prior to final image capture.
   8. Capture the fluorescent images under the appropriate filter at 100X magnification using a fluorescent microscope and analyze the images using available analysis software.

5. Measurement of Reprogramming Kinetics Using Flow Cytometry

1. Quantitative Kinetic Measurement of Reprogramming using Cell Surface Antibodies
   1. Prior to reprogramming, set up the appropriate number of reprogramming experiments for each time point. For measuring kinetics of reprogramming for the first 7 days of reprogramming, individual transductions are performed for each desired time point. For time points after Day 7, seed sufficient feeder-independent time points to continue to measure the reprogramming events. A single well of a 6-well plate will yield sufficient amounts of cells for performing flow cytometry analysis.
   2. Measure all desired reprogramming time points from individual wells, as this is an end point assay. Aspirate off the Fibroblast Medium from the desired well. Wash once with D-PBS.
   3. Aspirate off the D-PBS wash. Add 1 ml of 0.05% trypsin-EDTA solution to the well to be tested. Incubate at room temperature for 5 min.
   4. Following the incubation, dissociate the cells into single cells by flushing the 1 ml cell suspension against the surface of the well and transfer the suspension into a 15 ml conical tube. Use 3 ml of D-PBS to wash off any remaining cells from the well and add them to the 15 ml conical. Use 10 ml of additional D-PBS to further dilute the cell solution in the conical tube.
   5. Centrifuge the cells at 200 x g for 2 min. Aspirate the supernatant and re-suspend the pellet into 1 ml of DMEM/F-12 medium. Perform a cell count as needed to create a standard cell suspension that is less than 1 x 10⁶ cells/ml.
   6. Prepare each aliquot of positive/negative directly-conjugated antibodies of choice in 1 ml of DMEM/F-12 medium for less 1 x 10⁶ cells/ml using the following combinations: SSEA4 conjugated to a green fluorescent fluorophore (1:200) with CD44 conjugated to PE-Cy5, or CD44 conjugated to a green fluorescent fluorophore and SSEA4 conjugated to a far- red fluorescing fluorophore. Incubate the antibodies with the cell suspension for 45 min at room temperature.
   7. Aspirate the primary antibody solution and wash twice with pre-warmed DMEM/F-12 medium.
   8. Aspirate the final wash and re-suspend the cell pellet in 1 ml of D-PBS. Pipet the content of each suspension in individual FACS tubes.
   9. Using the appropriate negative controls (unstained and isotype controls), confirm the proper forward and side scatter profile and voltages on the cytometer. Set the voltages for the corresponding fluorophores according to initial single stain parameters, such that the green fluorophores are activated by the Blue Laser and signals acquired in the BL1 channel, while far red fluorescing fluorophore and PE-Cy5 fluorophore are activated by the Red Laser and signals acquired in the RL1 channel. Acquire the dual-stained population for each time point.
   10. Analyze the FCS files for each time point using the appropriate analysis software and use the editor to arrange data to observe the population expression shift over time.
   11. Follow steps 5.1.1 to 5.1.6, if cell sorting is desired at different time points. Use the appropriate fluorophore combinations according to the laser configuration of the cell sorter to be used.
   12. Following the final wash, re-suspend the cell pellet in 1 ml of the cell sorting buffer. Set up the cell sorter to the proper forward and side scatter settings using an unstained control. Use the single stained controls to set up the proper settings for each fluorophore.
   13. Use a small sample of the double-stained cell suspension; select the 2 populations to be sorted and assign the respective charge to ensure proper collection of the sorted cells.
   14. Add 200 µl of the cell recovery medium into the collection tubes to ensure that the sorted cell suspension is cushioned and protected at the time of sorting. Harvest the desire number of cells and transfer onto new iMEF dishes containing freshly prepared recovery medium.
   15. Change the medium following overnight incubation with the recovery medium. The cultures must be subsequently fed and routinely passaged using hPSC medium containing Penicillin/Streptomycin (100 units/ml final concentration).

6. Endpoint Analysis

1. Terminal Alkaline Phosphatase (AP) Staining
   1. Use terminal chromogenic AP staining to correlate observed kinetic and morphological events with final reprogramming efficiencies.
   2. Following 21 days of reprogramming, remove the culture medium from the well to be stained with terminal AP stain and wash once with 200 mM Tris-HCl (pH 8.0).
   3. Prepare the appropriate amount of staining solution as directed by the manufacturer's manual in 200 mM Tris-HCl.
   4. Aspirate the wash and add 2 ml of the prepared staining solution to each well. Incubate for 20 to 30 min at room temperature and away from the light.
   5. Aspirate the staining solution and wash once with 200 mM Tris HCl. Remove the wash and add distilled water prior to visualization.
   6. Capture the colorimetric signal using a normal picture camera. Count the number of positively stained colonies by hand. Visualize the stain using fluorescent analysis in the Trit-C channel, as the stain is also fluorescent in nature and perform whole-well imaging and analyzed using the whole-well imager. Use manufacturer's protocol.