Generating Virus-free and Integration-free Induced Pluripotent Stem Cells (iPSCs)
by David Welch - 10/12/12
In our last post about induced pluripotent stem cells, we discussed iPSC generation with the Sendai virus based technology in the CytoTune® -iPS Sendai Reprogramming Kit. Sendai virus is the best option when generating iPSCs from hard-to-reprogram cells such as peripheral blood mononuclear cells (PBMCs) and when high efficiency is a requirement.
If you are unable to use a viral method for iPSC generation, episomal vectors are your next best choice. With episomal vectors, good reprogramming efficiency with multiple somatic cell types can be achieved with a single application.
Episomal iPSC Reprogramming Vectors are a mixture of three vectors designed to provide the optimal system for generating transgene-free and virus-free iPSCs in a feeder-free environment. The optimized mixture of three vectors has the oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone that delivers six reprogramming factors: Oct4, Sox2, Nanog, Lin28, Klf4 and L-Myc. The vectors also contain SV40LT.
Schematic of the plasmid set successfully used for reprogramming. From Mack et al. (2011) Generation of Induced Pluripotent Stem Cells from CD34+ Cells across Blood Drawn from Multiple Donors with Non-Integrating Episomal Vectors. PLoS ONE 6(11): e27956. doi:10.1371/journal.pone.0027956
How do the episomal induced pluripotent stem cell reprogramming vectors work?
The episomal vectors are introduced into the cell by electroporation. These non-integrating vectors replicate only once per cell cycle, with activation of replication achieved by binding of multiple EBNA-1 homodimers to oriP within the nucleus (1).
In the video below, Dr. Emile Nuwaysir, Chief Operating Officer of Cellular Dynamics International (CDI), discusses the advantages of reprogramming with episomal vectors and how CDI uses them for routine iPSC production.
What are the advantages of using oriP/EBNA vectors?
oriP/EBNA vectors have been successfully used to deliver the reprogramming genes. High transfection efficiency due to oriP/EBNA-1 mediated nuclear import and retention of vector DNA allows iPSC derivation in a single transfection (2). Silencing of the viral promoter driving EBNA-1 expression and the loss of the episomes at a rate of ~5% per cell cycle, allows for the removal of episomal vectors from the iPSCs without any additional manipulation (3).
1. Thyagarajan B., Scheying K., Xue H., Fontes A., Chesnut .J, Rao M., Lakshimipathy U. (2009) A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells. Regenerative Medicine 4(2): 239–250.
2. Yu, J., Chau, K. F., Vodyanik, M. A., Jiang, J., and Jiang, Y. (2011) Efficient Feeder-Free Episomal Reprogramming with Small Molecules. PLoS One 6, e17557.
3. Nanbo, A., Sugden, A., and Sugden, B. (2007) The coupling of synthesis and partitioning of EBV’s plasmid replicon is revealed in live cells. EMBO J 26, 4252–4262.