Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells that exhibit a pluripotent stem cell-like state similar to embryonic stem cells (Meissner et al., 2007; Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007). While these artificially generated cells are not known to exist in the human body, they show qualities remarkably similar to those of embryonic stem cells (ESCs); thus, they are an invaluable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
There are multiple methods to generate iPSCs, including retrovirus-mediated gene transduction and chemical induction. While retroviral vectors require integration into host chromosomes to express reprogramming genes, DNA-based vectors, such as adenovirus, adeno-associated virus, and plasmid vectors exist episomally and do not require integration; however, they may still be integrated into host chromosomes at certain frequencies. Unlike these vectors, the CytoTune® reprogramming vectors do not integrate into the host genome or alter the genetic information of the host cell (Fusaki et al., 2009; Li et al., 2000; Seki et al., 2010).
CytoTune®-iPS Sendai Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune® Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins.
The CytoTune®-iPS Sendai Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety.
Gelatin coating culture vessels
- Cover the whole surface of each new culture vessel with Attachment Factor (AF) solution and incubate the vessels for 30 minutes at 37°C or for 1 hour at room temperature.
- Using sterile technique in a laminar flow culture hood, completely remove the AF solution from the culture vessel by aspiration just prior to use. Coated vessels may be used immediately or stored at room temperature wrapped in Parafilm® sealing film for up to 24 hours.
Note: It is not necessary to wash the culture surface before adding cells or medium.
Thawing Gibco® MEFs (Irradiated)
- Remove the cryovial containing inactivated MEFs from the liquid nitrogen storage tank.
- Briefly roll the vial between hands to remove frost, and swirl it gently in a 37°C water bath.
- When only a small ice crystal remains in the vial, remove it from water bath. Spray the outside of the vial with 70% ethanol before placing it in the cell culture hood.
- Pipet the thawed cells gently into a 15-mL conical tube.
- Rinse the cryovial with 1 mL of pre-warmed MEF medium. Transfer the medium to the same 15-mL tube containing the cells.
- Add 4 mL of pre-warmed MEF medium dropwise to the cells. Gently mix by pipetting up and down.
Note: Adding the medium slowly helps the cells to avoid osmotic shock.
- Centrifuge the cells at 200 × g for 5 minutes.
- Aspirate the supernatant and resuspend the cell pellet in 5 mL of pre-warmed MEF medium.
- Remove 20 μL of the cell suspension and determine the viable cell count using your method of choice (e.g., Countess® Automated Cell Counter).
- Centrifuge the remaining cell suspension (step 9, Thawing Gibco® MEFs) at 200 × g for 5 minutes at room temperature.
- Aspirate the supernatant. Resuspend the cell pellet in MEF medium to a density of 2.5 × 106 cells/mL.
- Aspirate the gelatin solution from the gelatin coated culture vessel.
- Add the appropriate amount of MEF medium into each culture vessel (refer to Table 1, below).
- Into each of these culture vessels, add the appropriate amount of MEF suspension (refer to Table 1, below).
Note: The recommended plating density for Gibco® Mouse Embryonic Fibroblasts (Irradiated) is 2.5 × 104 cells/cm2.
- Move the culture vessels in several quick back-and-forth and side-to-side motions to disperse the cells
across the surface of the vessels.
- Incubate the cells in a 37°C incubator with a humidified atmosphere of 5% CO2.
- Use the MEF culture vessels within 3–4 days after plating.
Table 1. Amount of Inactivated MEFs Needed
| Vessel size || Growth area || Volume of media || Number of MEFs || Volume of MEF suspension |
|96-well plate||0.32 cm2/well||0.1 mL||1.0 × 104/well||4 μL|
|24-well plate||2 cm2/well||0.5 mL||5.0 × 104/well||20 μL|
|12-well plate||3.8 cm2/well||1 mL||1.0 × 105/well||40 μL|
|6-well plate||9.6 cm2/well||2 mL||2.5 × 105/well||0.1 mL|
|60-mm dish||19.5 cm2||5 mL||5.0 × 105||0.2 mL|
|100-mm dish||58.95 cm2||10 mL||1.5 × 106||0.6 mL|
|25-cm2 flask||25 cm2||5 mL||6.3 × 105||0.25 mL|
|75-cm2 flask||75 cm2||15 mL||1.9 × 106||0.75 mL|
The following protocol has been optimized for human neonatal foreskin fibroblast cells (strain BJ; ATCC no.
CRL2522). We recommend that you optimize the protocol for your cell type.
Day –2: Prepare the cells for transduction
- 2 days before transduction, plate human neonatal foreskin fibroblast cells into two wells of a 6-well plate at the appropriate density to achieve 5 × 105 cells per well on the day of transduction (Day 0).
Note: We recommend about 80–90% confluency on the day of transduction. Because overconfluency results in decreased transduction efficiency, we recommend replating your cells to achieve 80–90% confluency if your cells have become overconfluent during culturing.
- Culture the cells for two more days, ensuring the cells have fully adhered and extended.
Day 0: Perform transduction
- On the day of transduction, warm 2 mL of fibroblast medium in a water bath.
- Remove one set of CytoTune® Sendai tubes from the –80°C storage. Thaw each tube one at a time by first immersing the bottom of the tube in a 37°C water bath for 5–10 seconds, and then removing the tube from the water bath and allowing it to thaw at room temperature. Once thawed, briefly centrifuge the tube and place it immediately on ice.
- Add the indicated volumes of each of the four CytoTune® Sendai tubes (3 × 106 CIU each; see the CoA for the appropriate volume) to 2 mL of fibroblast medium, pre-warmed to 37°C. Ensure that the solution is thoroughly mixed by pipetting the mixture gently up and down. Complete the next step within 5 minutes.
- Aspirate the fibroblast medium from the cells, and add one half of the solution prepared in Step 5 to each of the two wells. Place the cells in a 37°C, 5% CO2 incubator and incubate overnight.
Day 1: Replace medium and culture cells.
- 24 hours after transduction, replace the medium with fresh fibroblast medium.
Note: Depending on your cell type, you should expect to see some cytotoxicity 24–48 hours post-transduction, which can affect >50% of your cells. This is an indication of high uptake of the virus. We recommend that you continue culturing your cells and proceed with the protocol.
- Culture the cells for 6 more days, changing the spent medium with fresh fibroblast medium every other day.
Note: Depending on your cell type, you may observe high cell density before Day 5. We do not recommend passaging your cells onto MEF culture dishes before 7 days post-transduction.
Day 5 or 6: Prepare MEF culture dishes
- One to two days before passaging the transduced fibroblasts onto MEF feeder-cells, prepare 100-mm MEF culture dishes.
Day 7: Plate transduced cells on MEF culture dishes
- Seven days after transduction (Step 6), fibroblast cells are ready to be harvested and plated on MEF culture dishes. Remove the medium from the fibroblasts, and wash cells once with DPBS.
- To remove the cells from the 6-well plate, use 0.5 mL of TrypLE™ Select reagent or 0.05% trypsin/EDTA following the procedure recommended by the manufacturer and incubate at room temperature. When the cells have rounded up (1–3 minutes later), add 2 mL of fibroblast medium into each well, and collect the cells in a 15-mL conical centrifuge tube.
Note: Because the cells can be very sensitive to trypsin at this point, minimize trypsin exposure time and incubate the cells at room temperature.
- Centrifuge the cells at 200 × g for 4 minutes, aspirate the medium, and re-suspend the cells in an
appropriate amount of fibroblast medium.
- Count the cells using the desired method (e.g., Countess® Automated Cell Counter), and seed the MEF culture dishes with 5 × 104–2 × 105 cells per 100-mm dish and incubate at 37°C, 5% CO2 incubator overnight.
Note: We recommend plating 5 × 104, 1 × 105, and 2 × 105 cells per 100-mm dish. Depending on your cell type, you may need to plate most of your cells on the same plate to ensure sufficient numbers of colonies.
Note: Set aside any remaining cells for RNA extraction to be used as a positive control in the RT-PCR detection of the SeV genome.
Day 8 to 28: Feed and monitor the cells
- 24 hours later, change the medium to iPSC medium, and replace the spent medium everyday thereafter.
- Starting on Day 8, observe the plates every other day under a microscope for the emergence of cell clumps indicative of transformed cells (see Figure 1).
Note: For BJ fibroblasts, we normally observe colony formation on Day 12 post-transduction. However,
depending on your cell type, you may need to culture for up to 4 weeks before seeing colonies.
- Three to four weeks after transduction, colonies should have grown to an appropriate size for transfer. The day before transferring the colonies, prepare MEF culture plates using Attachment Factor-coated 12- or 24-well plates.
Note: We typically harvest colonies closer to three weeks to avoid differentiation.
- When colonies are ready for transfer, perform live staining using Tra1-60 or Tra1-81 for selecting
- Manually pick colonies and transfer them onto prepared MEF plates.
By Day 21 post-transduction, the cell colonies on the MEF culture dishes will have become large and compact, covering the majority of the surface area of the culture dish. However, only a fraction of these colonies will consist of iPSCs, which exhibit a hESC-like morphology characterized by a flatter cobblestone-like appearance with individual cells clearly demarcated from each other in the colonies (see Figure 1). Therefore, we recommend that you perform live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated hESCs.
Note: Although colonies of “transformed” cells may emerge as early as 7 days after transduction, most of these colonies will not be correctly “reprogrammed” cells. iPSCs usually emerge a little later (around day 14 posttransduction), resemble embryonic stem cells in morphology, and express the cell surface markers Tra1-60 and Tra1-81.
Figure 1. Human neonatal foreskin fibroblast cells (strain BJ) were transformed using the CytoTune®-iPS Sendai Reprogramming Kit and allowed to proliferate on MEF feeder layers in fibroblast medium. The images were obtained using a 5X or a 10X objective, as indicated.
One of the fastest and most reliable methods for selecting a reprogrammed colony is live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated iPSCs and enable the identification of reprogrammed cells from a variety of human cell types.
Note: Other methods of identifying iPSCs (such as alkaline phosphatase staining) are also acceptable. 1.
- Aspirate the medium from the reprogramming dish.
- Wash the cells once with 1X KnockOut™ DMEM/F-12.
- Add the diluted primary antibody to the cells (6 mL per 100-mm dish).
- Incubate the primary antibody and the cells at 37°C for 60 minutes.
- Remove the primary antibody solution from the dish.
Note: The primary antibody solution can be stored at 4°C for 1 week and re-used up to 2 times.
- Wash cells three times with KnockOut™ DMEM/F-12.
- Add the diluted secondary antibody to the cells (6 mL per 100-mm dish).
- Incubate the secondary antibody and the cells at 37°C for 60 minutes.
- Remove the secondary antibody solution from the dish.
Note: The secondary antibody solution can be stored at 4°C for 1 week and re-used up to 2 times.
- Wash cells three times with KnockOut™ DMEM/F-12 and add fresh KnockOut™ DMEM/F-12 to cover the surface of the cells (6 mL per 100-mm dish).
- Visualize the cells under a standard fluorescent microscope and mark the successfully reprogrammed colonies for picking and expansion. Successful antibody staining can very specifically distinguish
reprogrammed colonies from just plain transformed counterparts, and can be detected for up to 24–36 hours. This is particularly useful because it helps identifying and tracking of candidate iPS colonies before picking and the day after they are transferred into a new culture dish for expansion.
Figure 2. Human neonatal foreskin fibroblast cells (strain BJ) were transformed using the CytoTune®-iPS Sendai Reprogramming Kit and allowed to proliferate on MEF feeder layers in fibroblast medium. On Day 21, the cells were analyzed by live staining using the antibody against the cell surface markers Tra1-60 and Tra1-81. The images were obtained using a 10X objective.
Guidelines for generating vector-free iPSCs
- The time needed to derive vector-free iPSCs may vary depending on culture and passage conditions. In the case of human neonatal foreskin fibroblast cells (strain BJ), it takes about 2 months after gene transduction to obtain iPSCs free of CytoTune® Sendai reprogramming vectors.
- To obtain virus-free clones faster, we recommend that you perform single colony subcloning for the first few passages (minimum 5) instead of bulk or pooled-clone passaging.
- To perform single colony subcloning, pick from a single colony to transfer to another 6-well plate (Passage 1). From Passage 1, pick a single colony and transfer to another 6-well plate (Passage 2) and so forth. We recommend subcloning for 5 passages and then testing for virus free iPSCs.
Protocol for generating vector-free iPSCs
- When passaging iPSC colonies, prepare duplicate plates; one for immunostaining and one for further
- Perform immunostaining on one plate using anti-SeV antibodies (see below).
- If any colonies stain positive, perform cell cloning on the other duplicate plate.
- Repeat immunostaining with anti-SeV antibodies on the cloned colonies until all colonies in a plate are negative.
- If all colonies are negative for anti-SeV antibodies, passage the cells and confirm the absence of the
CytoTune® Sendai reprogramming vectors by RT-PCR.
Immunocytochemistry with Anti-SeV Antibodies
- Wash cells once with DPBS
- Fix the cells in 4% paraformaldehyde for 5 minutes at room temperature.
- Wash cells twice with DPBS.
- Add the anti-SeV antibody (MBL, Cat. no PD029) diluted in 0.1% Triton® X-100 in DPBS to the cells and
incubate for 1 hour at 37°C.
- Remove the antibody solution. Wash the cells 3 times with DPBS.
- Add the secondary antibody diluted in 0.1% Triton® X-100 in DPBS and incubate for 1 hour at 37°C.
- Remove the secondary antibody solution from the dish. Wash the cells 3 times with DPBS.
- Visualize the cells under a fluorescence microscope.
RT-PCR Protocol for detecting the SeV genome and transgenes
- Extract the total RNA from 5 × 106 iPSCs using the TRIzol® Reagent following the instructions provided with the reagent. As a positive control, use cells set aside at the last step of the reprogramming procedure.
- Carry out a reverse transcription reaction using 1 μg of RNA (from step 1, above) and the SuperScript® VILO™ cDNA Synthesis Kit following the instructions provided with the kit.
Note: Because the CytoTune™ Sendai reprogramming vectors are based on SeV, which is an RNA virus, reverse transcription is required for detecting the presence of the SeV genome in your reprogrammed cells.
- Carry out the PCR using 10 μL of cDNA from the reverse transcription reaction (step 2, above) and
AccuPrime™ SuperMix I with the parameters below. For the RT-PCR primer sequences and the expected product size, refer to the Table 2.
| Step || Temperature || Time || Cycles |
- Analyze the PCR products using 2% agarose gel electrophoresis.
Note: If you still detect CytoTune® Sendai virus in your iPSC lines after more than 10 passages, and have performed RT-PCR to show that Oct4, Sox2, and Klf4 (these vectors do not have the temperature sensitive mutations) are absent from your cells, then you can perform temperature shift to remove the cMyc gene. CytoTune® Sendai hc-Myc tends to persist in the cells longer than the other CytoTune® Sendai reprogramming vectors. However, because this vector contains a temperature sensitivity mutation, you can enhance its removal and obtain complete absence of Sendai virus by incubating your cells at 38–39°C for 5 days.
Table 2. RT-PCR primer set used for detecting the SeV genome and transgenes in cells reprogrammed using the CytoTune® Sendai reprogramming vectors
| Target || Primer sets || Product size |
|SeV||Forward: GGA TCA CTA GGT GAT ATC GAG C* |
Reverse: ACC AGA CAA GAG TTT AAG AGA TAT GTA TC*
|Sox2||Forward: ATG CAC CGC TAC GAC GTG AGC GC |
Reverse: AAT GTA TCG AAG GTG CTC AA*
|Klf4||Forward: TTC CTG CAT GCC AGA GGA GCC C |
Reverse: AAT GTA TCG AAG GTG CTC AA*
|cMyc||Forward: TAA CTG ACT AGC AGG CTT GTC G* |
Reverse: TCC ACA TAC AGT CCT GGA TGA TGA TG
|Oct3/4||Forward: CCC GAA AGA GAA AGC GAA CCA G |
Reverse: AAT GTA TCG AAG GTG CTC AA*
* Primer contains SeV genome sequences. Pairing of these primers with transgene-specific primers allows specific detection of transgenes carried by the CytoTune® Sendai reprogramming vectors. Note that the same reverse primer is used for detecting Sox2, Klf2, and Oct3/4.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.