Integration-Free iPS Cells with a Single Transduction & Streamlined Workflow
by Jennifer Hornstein - 05/01/12
Dr. Uma Lakshmipathy a Sr. Staff Scientist, Biology presented work on the creation of integration-free induced pluripotent stem cells with the CytoTune™ -iPS Reprogramming Kit. The presentation; summarized in this blog post, was recorded for viewing and placed on the Life Technologies website (watch the video).
Fast and Easy to Use
The CytoTune™ - iPS Reprogramming Kit, a product sold by Life Technologies, provides an extremely streamlined process for the generation of induced pluripotent stem cells. The four Yamanaka factors (Oct3⁄4, Sox2, Klf4, and cMyc) included in the kit can be transduced overnight creating a simple efficient workflow.
A. Single Transduction iPSCs
Traditional reprogramming methods such as Lentivirus or Retrovirus can require transduction multiple times before the creation of iPSCs will occur. With the Cytotune™ -iPS Reprogramming Kit the generation of induced pluripotent stem cells is accomplished in 3-4 weeks after a single transduction, while mRNA requires 17 sequential days of transduction.
B. Fast & Substantial iPS Cell Colony Formation
After only three weeks post-transduction, iPS cell colony formation will start. At the end of four weeks, sufficient colonies will be available to select, characterize and expand. When reprogramming fibroblasts, we frequently observe more iPS cell colonies than required for downstream applications.
C. Easy to Use
iPS cell generation with the Cytotune™ –iPS Reprogramming kit is very simple and efficient. While most of our work has focused on CytoTune™ mediated iPSC generation from fibroblasts, recent data from collaborators indicates that CytoTune™ can reprogram a variety of blood lineage cells.
The following iPS cell protocol timeline for iPSC generation from human fibroblasts using Cytotune™ demonstrates the streamlined process that the kit provides.
Day -2 - Plate fibroblasts in fibroblast culture medium (DMEM + 10% FBS) into two wells of a 6-well plate to achieve 5 x 105 cells/well on day 3.
Day 0 - Add CytoTune™ -iPS Reprogramming Kit contents to your cells.
Day 1-7 - Replace the fibroblast culture medium (DMEM + 10% FBS) with fresh fibroblast culture medium on day 4, and then every other day thereafter.
Day 7 - Harvest cells with trypsin, then plate the cells onto MEF feeder-coated plates in fibroblast culture medium.
Day 8 - Replace the fibroblast medium with complete KnockOut™ Serum Replacement medium.
Day 9-25 - Observe the cells and replace the spent medium daily. Look for the emergence of cell clumps indicative of iPSCs.
3-4 weeks post-transduction - Perform live staining with Tra1-60 or Tra1-81 to select reprogrammed colonies. Manually pick colonies and transfer them to fresh MEF plates.
Finally, this method is integration free. Using PCR we can demonstrate that there was no viral genome remaining in the iPS cell colonies generated using the CytoTune™-iPS Reprogramming Kit. In addition, an anti-Sendai virus antibody can also be used to show the absence of Sendai virus in iPS cell colonies.
Note that the generation of induced pluripotent stem cell colonies can be done on feeder dependent systems using KnockOut™ SR and also under feeder free conditions using StemPro® hESC SFM media and xeno-free conditions using KnockOut™ SR xeno-free CTS™.
- Cytotune iPS Cell Reprogramming Kit
- Uma Lakshmipathy's Presentation (video)
- The Stem Cell Research Community
- Pluripotent Stem Cell Protocols