Dr. Uma Lakshimipathy presents Generation of Transgene-Free Induced Pluripotent Stem Cells





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Uploaded on Dec 12, 2011

Learn more at http://www.invitrogen.com/site/us/en/...

Dr. Uma Lakshmipathy presents work on the creation of integration-free induced pluripotent stem cells at high efficiency with Sendai Virus using the CytoTune™ -iPS Sendai Reprogramming Kit.

[audio transcript] So to start with the most common method for generating iPSC is transduction of the four factors and shown here is the Yamanaka factors and after a black box in which takes anywhere between three to four weeks you end up with iPSC colonies. So the biggest bottleneck right now; one is the efficiency of iPSC formation depending on what kinds of cells you start with, the efficiency is really low.

And the second thing, the second bottleneck is how do you detect these emerging iPSC colonies depending on the expertise of the users and if you look for pluripotent stem cells, people can either pick it really easily or there is always like an issue on what clones you place your bet on.

So when it comes to efficient methods, there are several methods starting from viral non-integrating and more small molecule methods such as mRNA, microRNA and small molecules. But one of the methods we really worked on is a non-intergrating viral method based on Sendai virus. The reason why this method is superior to the other current methods is its efficiency; it's based on Sendai virus which is a RNA virus.

So again coming back to the different methods that are out there, if you were to... this is a graph I took from a published paper... if you look at the efficiency versus the safety obviously methods suggests small molecule: microRNA, RNA and protein they don't leave a footprint, they are extremely safe to use in a clinical setting; however the efficiency of generating iPSC right now is pretty low at this point of time.

The highest efficiency so far has been obtained with viral methods such as lenti and retro; more recently the CytoTune which I will show you some data. Actually excels the efficiency that you can actually get with this traditional viral system and at the same time it's relatively much safer because it's a RNA virus and it's non-integrating. Therefore it will not leave a footprint in the gene of the cells or iPSCs that are created.

So this is again a brief introduction, this was a system that was developed by a company in Japan called DNAVEC. The original paper was published; there have been several papers since then starting from generation of iPSCs not only with fiberblast but also blood cells.

So I won't go into more details there, but what I would like to show you is that using the CytoTune System that we actually sell as a product, the process of generating iPSC is extremely streamline. The four factors come in four tubes, which can me transduced overnight onto maps. Most of our protocols right now are for fiberblast, but we are developing other cell types, mainly blood lineages. After, it's a one-time contact; you don't have to do repeated transductions so in that way it is really workflow friendly.

After transduction you have to give it around three or four weeks. At the beginning of three weeks, is when you start colony formation. At the end of four weeks you have sufficient colonies to pick up and choose. There actually more colonies than you really want be cause there are way to many colonies there.

So what we've done; here this method actually shows you that its integration free. Using PCR, we were able to show that there was no viral genome left in the clones that were established. This is 10 independent clones that were generated. You can also use an antibody, although I would say the antibodies not really that great because you can see the haze in the negative cell type, and you actually can tell when it's negative only when you have a true positive control because the positive staining is so much more robust. These cells are clearly important both in their marker expression, and differentiate into different lineages, when randomly differentiated by embryonic body formation.

These clones were all generated on feeder dependent systems [...], but since then we have also been able to generate iPSC clones both under feeder free conditions using StemPro SFM media as well as Xeno-free conditions, which is basically KRS-XF media in the presence of growth factor cocktails on human feeders.

Learn more at http://www.invitrogen.com/site/us/en/...

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