Traditional retroviral or lentiviral methods, while effective, suffer from genomic integration, potentially leading to insertional mutagenesis and safety concerns. In contrast, Sendai virus (SeV) is a non-integrating RNA virus belonging to the Paramyxoviridae family. It replicates exclusively in the cytoplasm and never enters the host genome, ensuring safe, transgene-free reprogramming.
At Creative Biolabs, we are passionate about providing researchers with innovative, safe, and efficient stem cell solutions that truly make a difference in advancing science. In this protocol, we outline every step to help researchers understand how SeV powerfully drive stem cell reprogramming.
Key features that make SeV ideal for reprogramming include:
By leveraging this advanced platform, researchers can obtain high-quality iPSCs that closely resemble embryonic stem cells in morphology, gene expression, and differentiation potential.
Fig.1 Sendai virus-based reprogramming protocol.1,2
Before diving into the step-by-step workflow, it's important to understand the guiding principles behind SeV-mediated iPSC generation.
| Category | Reagents |
|---|---|
| Somatic cells | Human dermal fibroblasts, PBMCs, keratinocytes |
| Reprogramming kits | Sendai virus reprogramming kit encoding OCT4, SOX2, KLF4, c-MYC |
| Fibroblast culture medium | DMEM + FBS + supplements |
| iPSC culture medium |
mTeSR1 or Essential 8 medium Matrigel- or vitronectin-coated culture plates |
| ROCK inhibitor | Enhance survival of single cells |
Expand fibroblasts or PBMC-derived cells to ~70% confluence. Ensure cells are healthy, free of mycoplasma contamination, and not over-passaged. Plate cells in viral infection medium one day prior to transduction.
Add Sendai virus particles containing the four reprogramming factors to the somatic cells at the recommended MOI (multiplicity of infection). Incubate overnight to allow efficient infection. Replace medium the next day with fresh fibroblast culture medium.
Maintain cells in fibroblast medium. Monitor daily for signs of cytopathic effects (minimal under optimized conditions). Ensure even cell growth; avoid over-confluency.
Replace fibroblast medium with iPSC culture medium. Feed cells daily. Around day 10, early iPSC-like colonies with high nuclear-to-cytoplasmic ratios appear.
Colonies with clear borders, compact morphology, and large nucleoli are indicative of pluripotency. Pick individual colonies using a fine pipette or colony picking tool. Transfer to new Matrigel-coated plates with ROCK inhibitor for expansion.
Expand selected colonies into stable iPSC lines. Confirm pluripotency by immunostaining (e.g., OCT4, NANOG, TRA-1-60, SSEA4). Assess clearance of Sendai virus RNA using RT-PCR. Validate differentiation potential through embryoid body formation or directed differentiation assays.
Below, we have compiled an expanded troubleshooting and optimization guide based on our extensive experience at Creative Biolabs.
| Problem | Possible Cause | Solution |
|---|---|---|
| Low transduction efficiency |
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| Delayed or sparse colony emergence |
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| Residual SeV after several passages |
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| High cell death after picking colonies |
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| Poor differentiation capacity |
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To truly empower your projects, we provide an end-to-end portfolio of services that seamlessly connects reprogramming with downstream applications. Our integrated solutions help you save valuable time, reduce costs, and maintain high quality at every step.
Not every project requires the same method. We provide multiple integration-free technologies for generating iPSCs, ensuring flexibility and safety.
Ensuring pluripotency and genetic stability is critical before applying iPSCs in downstream studies. Our characterization portfolio includes pluripotency marker analysis, embryoid body (EB) formation assays, and teratoma formation assays.
Transforming iPSCs into specialized cell types opens new research opportunities. We design tailored differentiation protocols to meet your exact needs.
We combine iPSC reprogramming with precise genome engineering to create powerful research models.
A: No. The Sendai virus replicates exclusively in the cytoplasm and never integrates into host DNA, which means genomic stability is preserved. With our stringent quality control, including karyotyping and viral clearance validation, Creative Biolabs ensures that the iPSC lines you receive are integration-free, genetically stable, and reliable for both basic and translational research.
A: Residual Sendai virus can be detected by RT-PCR assays targeting viral RNA. Typically, the virus is lost after 10–15 passages in dividing cells. We perform validated viral clearance testing as part of our iPSC characterization package, ensuring your cells are fully free of viral remnants before moving into differentiation or downstream applications.
A: Sendai virus is replication-competent and requires BSL-2 conditions for handling. Researchers should follow biosafety guidelines, including personal protective equipment and sterile technique.
A: Initial colony formation typically occurs within two to three weeks after viral transduction, and stable iPSC lines can be established within four to six weeks. At Creative Biolabs, our optimized workflows shorten timelines without compromising quality, delivering fully characterized iPSCs to clients quickly, so you can move forward with downstream applications sooner.
References
Created September 2025
For Research Use Only. Not For Clinical Use.
