Solving Challenges in the Generation of Induced Pluripotent Stem Cells
by Jennifer Hornstein - 03/23/12
The CytoTune™ - iPS Reprogramming Kit is a high efficiency, integration- free, easy-to-use somatic cell reprogramming kit used in the generation of induced pluripotent stem cells. This kit utilizes Sendai Virus particles of the four Yamanaka factors, which have been shown to be critical in the successful generation of induced pluripotent stem cells.
Dr. Uma Lakshmipathy of Life Technologies presented work on the creation of integration-free induced pluripotent stem cells at high efficiency with Sendai Virus using the CytoTune™ -iPS Reprogramming Kit.
The presentation; summarized in this blog post, was recorded for viewing and placed on the Life Technologies website.
Researchers Face Two Challenges During Cell Reprogramming
The most common method for generating induced pluripotent stem cells is the transfection of the four Yamanaka factors using lentivirus or retrovirus. One of the biggest challenges for scientists right now is the low efficiency of iPSC generation. With difficult to transfect cell types or cells from older patients, efficiencies can be 0.001% or lower when using lentiviral or retroviral methods.
Expertise of Users
The second challenge is for users with little expertise that have a difficult time detecting these emerging iPSC colonies. When looking for pluripotent stem cells, people can either pick them up really easily or have trouble deciding what clones to place their bet on.
Efficiency & Safety of iPSC generation
There are several methods which improve reprogramming efficiency including viral non-integrating and small molecule methods such as mRNA, microRNA and small molecules. During the development of the CytoTune™ -iPS Reprogramming Kit the focus was on a non-integrating viral method utilizing Sendai Virus, a negative sense RNA virus. Sendai Virus is able to infect a wide variety of cell types and generates induced pluripotent stem cells at efficiencies 100-fold higher than lentiviral or retroviral methods.
When comparing efficiency vs. safety of reprogramming methods, small molecules like microRNA, RNA and protein which don’t leave a footprint are safer for cell therapy research; however, the efficiency of generating induced pluripotent stem cells with these methods is pretty low at this point in time.
The highest efficiency so far has been achieved with viral methods such as Retrovirus and Lentivirus. More recently the CytoTune™ -iPS Reprogramming Kit actually exceeds the efficiency that can be obtained with these traditional viral systems and at the same time it is much safer because it is a non-integrating RNA virus. Therefore it will not leave a footprint in the iPSCs that are created.
The CytoTune™ -iPS Reprogramming Kit will:
- Reduce hands on time - enables successful iPS reprogramming in one simple transduction
- Generate more cells - high efficiency reprogramming offers more iPS cells from a single experiment
- Use in a broad range of experiments - lack of genomic integration and viral remnants allows use from basic to clinical research
Ease of Use
The CytoTune™ -iPS Reprogramming Kit provides a simple system for somatic cell reprogramming. For most cell types, the CytoTune™ -iPS Reprogramming Kit requires only one application of the virus for successful cell reprogramming, unlike other methods such as Lentivirus and mRNA which can require multiple rounds of transduction to produce iPS cells. Selection of colonies is also easier with the CytoTune™ –iPS Reprogramming Kit due to the lower number of non-induced pluripotent stem cells that are generated.