Frequently Asked Questions

         About CytoTune® -iPS Sendai Reprogramming Kit

  1. What are induced pluripotent stem cells (iPSCs)?
  2. What is the CytoTune®-iPS Sendai Reprogramming Kit?
  3. What is Sendai virus (SeV)?
  4. Are there any publications that reference the use of the Sendai virus for reprogramming?
  5. How does the Sendai virus (SeV) reprogram somatic cells?
  6. How is Sendai virus different from lentivirus?
  7. What are the benefits of using an integration-free reprogramming method?
  8. What cell types have been successfully reprogrammed with Sendai virus?
  9. Are there protocols for reprogramming blood cells?
  10. Can you reprogram mouse cells with this kit?
  11. Are there safety concerns for Sendai virus?

    How to use the CytoTune® -iPS Sendai Reprogramming Kit

  12. How should I store the CytoTune® -iPS Sendai Reprogramming Kit?
  13. Do I need to use all four reprogramming vectors?
  14. How many wells of a 6-well plate can I reprogram with one CytoTune® -iPS Sendai Reprogramming Kit?
  15. How many cells do I need to start my reprogramming experiment with the CytoTune® -iPS Sendai Reprogramming Kit?
  16. What is the recommended medium to use for my somatic cells?
  17. What is the optimal passage number for reprogramming patient fibroblasts?
  18. How do I determine how much virus to add to my cells?
  19. What is the recommended medium to use for my reprogrammed cells?
  20. When do you replate your cells onto Human iPSC Medium?
  21. Can I grow my iPSCs feeder-free?
  22. Can I use the virus more than once?
  23. Can I thaw and refreeze the unused virus?

    Expected results with the CytoTune® -iPS Sendai Reprogramming Kit

  24. How do I confirm that my cells were transduced with the Sendai virus?
  25. How long does it take to see the iPSC colonies?
  26. What reprogramming efficiency should I expect with this product?
  27. Is Valproic Acid (VPA) required when reprogramming using the CytoTune® -iPS Sendai Reprogramming Kit?
  28. Is it normal to see cell death 1–2 days post-transduction? 
  29. How will my cells look after I use the CytoTune® -iPS Sendai Reprogramming Kit?
  30. How can I visualize the iPSCs?
  31. How do I know when to passage the iPSCs?
  32. How many passages does it take to clear the Sendai virus from the generated iPSCs?
  33. Am I able to obtain Sendai virus-free clones faster?
  34. Are temperature sensitive mutants available?
  35. What do I do if my iPSCs still contain the Sendai virus after 10 passages?
  36. Can I cryopreserve the iPSCs?

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About CytoTune® -iPS Sendai Reprogramming Kit

1. What are induced pluripotent stem cells (iPSCs)?
iPSCs are genetically reprogrammed somatic cells which exhibit a pluripotent stem cell–like state similar to embryonic stem cells. iPSCs can be derived by inducing selected gene expression via various methods including retrovirus-mediated gene transduction and chemical induction.

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2. What is the CytoTune®-iPS Sendai Reprogramming Kit?
The CytoTune® -iPS Sendai Reprogramming Kit is a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). This kit utilizes Sendai virus-based vectors each capable of expressing one of the four Yamanaka factors: Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs.

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3. What is Sendai virus (SeV)?
Sendai virus, also known as Hemagglutinating Virus of Japan (HVJ), is a respiratory virus of mouse and rat first isolated in Sendai, Japan in the early 1950s. The virus is classified as mouse parainfluenza virus type I, belonging to the Paramyxoviridae family. SeV is an enveloped virus, 150–250 nm in diameter, whose genome is a single chain of (-) sense RNA (15,384 bases). The virus infects cells by attaching to the sialic acid receptor present on the surface of many different cells and is thus able to infect a wide range of cell types of various animal species.

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4. Are there any publications that reference the use of the Sendai virus for reprogramming?
To view a list of publications citing the Sendai virus vectors which are part of the CytoTune® -iPS Sendai Reprogramming Kit, visit  the CytoTune® -iPS Sendai Reprogramming Kit publications.

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5. How does the Sendai virus (SeV) reprogram somatic cells?
The Sendai virus vectors in the CytoTune® -iPS Sendai Reprogramming Kit are based on a modified, non-transmissible form of SeV, which has the Fusion protein (F) deleted. The viral vectors maintain full infectivity to a wide range of cells, however they are no longer capable of producing infectious particles from infected cells because the viral genome lacks the F gene. The Sendai virus vectors contain transgenes that will express each of the four Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). After transduction, the viral vectors will cause the cells to express these four genes, resulting in reprogramming.

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6. How is Sendai virus different from lentivirus?
The main difference between Sendai virus and lentivirus is that, when using SeV reprogramming methods, the vectors and transgenes can be eliminated from the cells. Some viruses, like lentiviruses, require integration of viral DNA into the host genome. This can be problematic because this integration is random and can potentially disrupt the function of important genes. Sendai virus requires no integration for viral proteins to be made in the host cell. Other DNA-based viruses, like adenovirus, are non-integrating but must localize inside the nucleus for the viral proteins to be made. This means that there can still be random integration events, where the viral DNA integrates into the host genome. Since Sendai virus is an RNA virus, it does not need to enter the nucleus for transcription. This eliminates the possibility of integration of the transgenes into the host genome.

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7. What are the benefits of using an integration-free reprogramming method?
Integration-free reprogramming methods generate iPSCs that do not contain detectable vectors or transgenes. Traditional technologies used for reprogramming (e.g., lentivirus, retrovirus) integrate into the genome of the target cells. The resulting iPSCs and cells differentiated from those iPSCs will contain foreign DNA and could be unsafe and problematic for use in cell therapy and drug discovery applications. Furthermore, the integration could occur in a critical region of the genome, causing problems with unrelated developmental processes.

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8. What cell types have been successfully reprogrammed with Sendai virus?
The CytoTune® -iPS Sendai Reprogramming Kit has been validated for a wide variety of cell types, including human fibroblasts, CD34+ cord blood cells, Peripheral Blood Mononuclear Cells (PBMCs), and T cell/lymphocytes. For a current list of publications citing the cell types validated using this method visit our publications page citing the Sendai virus for iPSC generation.

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9. Are there protocols for reprogramming blood cells?
Yes. The following blood cells have been reprogrammed with the CytoTune® -iPS Sendai Reprogramming Kit:  CD34+, circulating T-cells and PBMCs.  Click here for a protocol or publication.

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10. Can you reprogram mouse cells with this kit?
This kit has not been tested on mouse cells.

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11. Are there safety concerns for Sendai virus?
Although human is not the natural host for SeV, and the virus is non-pathogenic to humans, appropriate care must be taken to prevent the potential mucosal exposure to the virus. The CytoTune® -iPS Sendai Reprogramming Kit must be used under Biosafety Level 2 (BL-2) containment with biological safety cabinet and laminar flow hood, and with appropriate personal safety equipment to prevent mucosal exposure/splash.

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How to use the CytoTune® -iPS Sendai Reprogramming Kit

12. How should I store the CytoTune® -iPS Sendai Reprogramming Kit?
Upon receipt, the kit should be stored at –80°C.

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13. Do I need to use all four reprogramming vectors?
We recommend that all four reprogramming vectors are used together. Omitting any of the vectors results in a significant drop in reprogramming efficiency.

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14. How many wells of a 6-well plate can I reprogram with one CytoTune® -iPS Sendai Reprogramming Kit?
One kit (one tube of each vector, i.e., 4 tubes) is sufficient for 2 wells of a 6-well plate (5 x 105 cells/well) at MOI = 3. The kit is available in two sizes: Cat. no. A13780-01 contains 1 tube of each vector (total 4 tubes), while Cat. no. A13780-02 includes 3 tubes of each vector (total 12 tubes).

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15. How many cells do I need to start my reprogramming experiment with the CytoTune® -iPS Sendai Reprogramming Kit?
This can vary depending on the cell type. We recommend the following for initial experiments: one to two days before transduction, plate your cells onto two wells of a 6-well plate at the appropriate density to ensure that the cells are 80–90% confluent the day of transduction. Since overconfluency results in decreased transduction efficiency, we recommend replating your cells to achieve 80–90% confluency if your cells have become overconfluent during culturing.

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16. What is the recommended medium to use for my somatic cells?
This will vary based on the specific somatic cell type being used. For fibroblasts, we recommend the following.
To prepare 100 mL of complete medium, aseptically combine the components listed below:

Component Stock Concentration Final Concentration Volume
Dulbecco’s Modified Eagle Medium (DMEM), High Glucose with GlutaMax™-1 and Pyruvate (Cat. no. 10569-010)1X89 mL
MEM Non-Essential Amino Acids Solution
(Cat. no. 11140-050)
10 mM0.1 mM1 mL
Fetal Bovine Serum (FBS), ESC-Qualified
(Cat. no. 16141-079)
10%10 mL

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17. What is the optimal passage number for reprogramming patient fibroblasts?
We recommend reprogramming patient cells at the earliest passage possible. However, it is important to have the cells growing and healthy, which can take between 1–4 weeks.  The cells are usually ready to reprogram once they have gone through a total of 3–4 passages.

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18. How do I determine how much virus to add to my cells?
An MOI of 3 is recommended for most cell types. The virus titer varies from lot to lot; the required volume for an MOI of 3 is listed on the Certificate of Analysis (CoA) for each lot of product.  The CoA can be found here. You may also choose to optimize your MOI as this may vary depending on the cell type.

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19. What is the recommended medium to use for my reprogrammed cells?
The reprogrammed cells can be grown on feeders in a KnockOut™ Serum Replacement (KSR) complete medium in a feeder-dependent culture, or feeder-free in StemPro® hESC SFM.  For KSR medium, we recommend the following.
To prepare 100 mL of Human iPSC Medium, aseptically combine the components listed below:

Component Stock Concentration Final Concentration Volume
KnockOut™ DMEM/F-12 (Cat. no. 12660-012)1X78 mL
KSR (Cat. no. 10828-028)20%20 mL
MEM Non-Essential Amino Acids Solution
(Cat. no. 11140-050)
10 mM0.1 mM1 mL
GlutaMAX™-I (Cat. no. 35050-061)100X1X1 mL
β-mercaptoethanol, 1000X (Cat. no. 21985-023)1000X1X100 µL
Penicillin-Streptomycin (optional)
(Cat. no. 15140-122)
100X1X1 mL
bFGF* (Cat. no. PHG0264)10 µg/mL4 ng/mL100 µL

*The medium can be stored at 2–8°C for up to one week.  Add bFGF when the medium is used.

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20. When do you replate your cells onto Human iPSC Medium?
We recommend replating 6– 7 days post-transduction.

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21. Can I grow my iPSCs feeder-free?
iPSCs cultured on MEF feeder layers can be adapted to feeder-free conditions in StemPro® hESC SFM. This can be done by directly thawing or splitting the iPSCs in MEF-conditioned medium (MEF-CM) and then replacing the culture medium daily with medium that contains increasing amounts of StemPro® hESC SFM. For the complete protocol, visit our web protocols.

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22. Can I use the virus more than once?
We recommend using this virus only once since viral titers decrease significantly with each freeze-thaw cycle.

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23. Can I thaw and refreeze the unused virus?
Avoid repeated freezing and thawing of the reprogramming vectors. Viral titers can decrease significantly with each freeze-thaw cycle and are not guaranteed for kits that have been refrozen or thawed.

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Expected results with the CytoTune® -iPS Sendai Reprogramming Kit

24. How do I confirm that my cells were transduced with the Sendai virus?
You can confirm the presence of the Sendai virus in your cells by several methods, including staining with anti-Sendai virus antibody or performing RT-PCR using TaqMan® -iPSC Sendai Detection Kit (Cat. No. A13640) (see page 17 of the CytoTune® -iPS Sendai Reprogramming Kit). Please check here for the latest product updates.

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25. How long does it take to see the iPSC colonies?
iPSC colonies will begin to form roughly 3 weeks post-transduction. Only one application of the virus is required for successful reprogramming, enabling selection of iPSC colonies 21–28 days after transduction.

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26. What reprogramming efficiency should I expect with this product?
The CytoTune® -iPS Sendai Reprogramming Kit provides a 100–fold increase in efficiency over lentiviral methods to generate iPSCs, allowing for reprogramming efficiencies between 0.01% and 1%. Visit the CytoTune® -iPS Sendai Reprogramming Kit  web page for more information.

Table 1. Comparison of efficiencies for various reprogramming methods

CytoTune® Kit (Sendai virus) Lentivirus/Retrovirus Adenovirus Episomal/Minicircle Protein Modified mRNAs
0.01%–1%0.001%–0.01%0.0001%–0.001% 0.0001%0.00001%>1%

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27. Is Valproic Acid (VPA) required when reprogramming using the CytoTune® -iPS Sendai Reprogramming Kit?
We don’t recommend using VPA with the CytoTune® kit..

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28. Is it normal to see cell death 1–2 days post-transduction? 
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.

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29. How will my cells look after I use the CytoTune® -iPS Sendai Reprogramming Kit?
Time-course images of the generation of iPSCs using the CytoTune® -iPS Sendai Reprogramming Kit are shown below.  The expected morphology of iPSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. If you do not observe this morphology or the number of colonies observed is low, then the MOI used for transduction may need to be increased.

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30. How can I visualize the iPSCs?
The iPSC colonies can be easily visualized using Alkaline Phosphatase Live Stain (Cat. no. A14353). Image is shown below.  In addition, reprogrammed colonies can be selected utilizing 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. For the complete protocol, visit our web protocols.

31. How do I know when to passage the iPSCs?
iPSCs must be monitored and growth medium must be replaced daily in order to maintain a healthy culture. In general, iPSC colonies should be passaged when the cells reach 70–80% confluence or when most of the colonies are larger than 700 µm. For the complete protocol, visit our web protocols.

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32. How many passages does it take to clear the Sendai virus from the generated iPSCs?
It typically takes 5–10 passages for the virus to clear from the cell.  This can be confirmed using several methods including staining with anti-Sendai virus antibody or performing RT-PCR using the TaqMan® -iPSC Sendai Detection Kit (Cat. No. A13640) (see page 17 of the CytoTune® -iPS Sendai Reprogramming Kit). The data below demonstrate the absence of the virus in iPSCs generated using the CytoTune® -iPS Sendai Reprogramming Kit.

(A) Amplification Plots to Monitor Clearance of Transgenes and Sendai Virus using the TaqMan® -iPSC Sendai Detection Kit.
(B) Immunofluorescent staining for anti-Sendai antibody showing the absence of Sendai virus in iPSC colonies generated with the CytoTune® kit.

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33. Am I able to obtain Sendai virus-free clones faster?
To obtain virus-free clones faster (5–6 passages instead of 5–10), we recommend single colony subcloning for the first few passages (3–5 passages) as indicated in the CytoTune® -iPS Sendai Reprogramming Kit manual (see page 17).

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34. Are temperature sensitive mutants available?
The CytoTune® -iPS Sendai Reprogramming Kit contains a temperature sensitive mutant of c-Myc that facilitates the clearance of this vector. It has been shown that c-Myc persists in the cells longer than the other vectors.

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35. What do I do if my iPSCs still contain the Sendai virus after 10 passages?
Of the clones tested, we have found that about 10% still have the virus after passage 10. It has been shown that c-Myc persists in the cells longer than the other vectors. The CytoTune® -iPS Sendai Reprogramming Kit contains a temperature sensitive mutant of c-Myc that facilitates the clearance of this vector.  To clear c-Myc, incubate the iPSCs at 38–39°C for 5 days (see page 18 of CytoTune® -iPS Sendai Reprogramming Kit manual).  One caveat is that given the sensitive nature of iPSCs, only perform this temperature shift if Sendai virus is in your iPSC lines after more than 10 passages, and you have performed RT-PCR to show that Oct4, Sox2, and Klf4 are absent from your cells (these vectors do not have the temperature sensitive mutations). Then you can perform temperature shift to remove the c-Myc gene.

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36. Can I cryopreserve the iPSCs?
You can cryopreserve iPSCs just as you would cryopreserve any pluripotent stem cells. Growth medium with 10% DMSO is recommended for freezing. For the complete protocol, visit our web protocols.

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Visit our CytoTune® -iPS Sendai Reprogramming Kit web page for more product information, data, protocols, and troubleshooting tips.