The development of induced pluripotent stem cells (iPSCs) allows differentiated somatic cells to be reprogrammed back into a pluripotent state. One of the earliest and still widely used strategies involves genome-integrating reprogramming methods, primarily based on retroviruses and lentiviruses. These viral vectors introduce key transcription factors—OCT4, SOX2, KLF4, and c-MYC (OSKM)—into somatic cells, where they stably integrate into the host genome and drive reprogramming.
At Creative Biolabs, we provide comprehensive genome-integrating reprogramming solutions tailored for researchers who require efficient and reproducible generation of iPSCs. This protocol outlines the complete workflow, optimization strategies, and related services we provide to accelerate your stem cell projects.
Genome-integrating systems, such as retroviruses and lentiviruses, introduce reprogramming factor cDNAs into the host cell genome. Once integrated, these transgenes are stably expressed, ensuring persistent transcription factor activity to reset the epigenetic state of somatic cells and drive them into a pluripotent state. Because integration is permanent, the introduced factors continue to influence cell behavior until silenced epigenetically, providing strong and reliable induction pressure.
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While we provide cutting-edge integration-free reprogramming services, we also maintain optimized protocols, custom vector systems, and expert support for genome-integrating methods, ensuring our clients can choose the right tool for the right research stage.
Seed cells at 70% confluence. Co-transfect cells with vector plasmids, packaging plasmids, and envelope plasmids. Replace media after 6–8 hours. Collect viral supernatant at 48 and 72 hours. Filter through filter; concentrate virus by ultracentrifugation if needed.
Plate target somatic cells at ~50% confluence. Add viral supernatant with polybrene. Incubate overnight. Replace with fresh medium the next day. Perform 2–3 rounds of infection for optimal efficiency.
Maintain cells in fibroblast medium during initial post-transduction days. Monitor cell survival; supplement with ROCK inhibitor if needed. Change media daily.
Switch to iPSC medium supplemented with bFGF. Replace media every day. Observe for morphological changes, such as increased nuclear-to-cytoplasmic ratio.
Colonies with tight borders and ES-like morphology typically appear within 2–3 weeks. Select and manually pick colonies between days 18–25. Transfer colonies to coated plates for expansion.
Expand colonies under feeder-free, xeno-free culture conditions. Use EDTA or enzyme-free passaging to preserve pluripotency. Avoid over-confluence to prevent spontaneous differentiation.
Confirm pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60). Perform qPCR to verify endogenous OSKM expression. Test silencing of integrated viral transgenes. Conduct karyotyping and embryoid body differentiation assays.
We leverage decades of expertise to provide troubleshooting support and tailored optimization strategies. Below, we outline common problems and practical solutions.
| Problem | Possible Cause | Solution |
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| Low viral titer or inefficient packaging |
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| Poor transduction efficiency |
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| High cell death after transduction |
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| Delayed or absent colony formation |
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| Abnormal colony morphology |
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| Persistent transgene expression |
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| Loss of pluripotency during expansion |
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Genome-integrating reprogramming provides researchers with a powerful and efficient route to generating iPSCs. Our mission is to empower your stem cell research by providing end-to-end solutions that cover every stage—from viral vector design to downstream differentiation and organoid development.
We offer comprehensive reprogramming services, generating genome-integrated iPSCs from your starting materials. Each project is tailored to maximize efficiency and colony quality, with full documentation provided.
Every iPSC line requires careful validation. We provide a multi-tier QC package to confirm pluripotency and genomic stability.
Once genome-integrating iPSCs are established, they can serve as versatile platforms for lineage-specific studies. We offer directed differentiation services.
With us, you gain not only high-quality iPSCs but also the confidence that your project is supported by a team with over 20 years of CRO experience.
A: Retroviruses can only infect dividing cells, making them highly effective for fibroblasts and similar sources. Lentiviruses, in contrast, infect both dividing and non-dividing cells, broadening their utility to PBMCs, keratinocytes, and other somatic types. Creative Biolabs offers both systems, guiding clients to the most suitable platform depending on the target cells and research goals.
A: Colony formation usually begins within 10–14 days post-transduction, with stable iPSC lines established and validated within 6–8 weeks. The exact timeline depends on cell type, viral titer, and culture conditions. Creative Biolabs provides streamlined workflows with integrated QC steps to reduce variability and accelerate delivery of validated iPSC lines.
A: Persistent transgene activity indicates incomplete silencing. Possible solutions include extended passaging, clone selection based on silencing markers, and screening multiple colonies. If reactivation persists, integration-free systems may be better suited. Creative Biolabs provides transgene clearance assays and expert consultation to help identify stable clones.
A: Absolutely. We provide a full suite of reprogramming solutions, including retroviral, lentiviral, Sendai virus, episomal, and mRNA-based systems. This flexibility allows us to design the most appropriate workflow for each project, whether efficiency, safety, or translational readiness is the primary goal.
Created September 2025
For Research Use Only. Not For Clinical Use.
