Episomal Expression of iPS Inducing Genes -- No Trace of Transgenes Afterwards

The potential use of induced pluripotent stem cells (iPS) in basic research and therapeutics is still mostly on the level of imagination. However, few doubt that this field will be one of the most actively contested and fastest evolving research topics in recent history. It reminds me of the early days of RNAi discovery, when 5 papers on DNA-based shRNA/RNAi appeared within a span of a few days (one draft from the Allele team was considered a few days too late to catch up with Nature Biotech by Science, but in the end we were the only recipient of patents on the subject).

The latest big news is a publication in Science by Junying Yu et al in the Thomson lab, who induced human iPS by using OriP/EBNA1 plasmid vector [1]. This method avoids integration of transgenes into the genome, thus reducing the risk of causing mutations.

A bit about the background: OriP/EBNA1 system originated from Epstein-Bar virus, which allows the establishment of stable episomes at 5-20 copies per cell, and duplication occurs once per cell division.

There are very few suppliers of vectors with the OriP/EBNA1, because of low demand (I was told so by the only supplier at the time, which explains why it was terminated altogether). The Phoenix™ Retrovirus system actually has the complete episomal cassette on the packing vector pBMN, which if not used in packaging Eco or Ampho cells, will behave as a regular plasmid. Therefore, the Retrovirus based iPS product within Allele’s iPS product group will provide two systems in one: a retroviral vector as published by Takahashi et al, and a OriP/EBNA1 system by Yu et al. They will also contain the brightest green fluorescent protein, mWasabi.

1. Yu, J., et al., Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science, 2009.

Allele Announces New Policy Encouraging Customer Participation on Products and Services

People at Allele have realized the importance of staying open when it comes to interacting with our customers, colleagues, contributors, and friends. Therefore, we will continue to open up more channels through network places such as FaceBook, Twitter, professional social networks, Wiki, blogs on google, yahoo. The purpose is for people who wants to know what’s going on at Allele will know, so that lab people can find new products in new fields, convenient equipment of much much lower costs; companies can find sub-licensing or outsourcing chances they would otherwise know exist; and any researchers and business people to exchange ideas with us in any way.

Of course, we welcome you whole-heartedly to post and discuss scientific ideas right here in our Blog/Forums. Feel free to ask questions about science behind our products, process of product development, test-run your idea, or simply throw stuff from your scientific mind to our wall and see what sticks. As a matter of fact, we worked on a number of products on suggestions by our customers and scientist friends through discussions over a drink or during intermission of a game.

It’d be alright and actually encouraged to criticize Allele’s products or services or behavior, even vent your anger here. Oh, we do have to delete profanities J

Induced pluripotent stem cells, or iPS (or iPSC as some call it), are differentiated cells from adult that “regained” capabilities to differentiate into all 3 germ layer specific cell types. The iPS induction process currently involves using viral vectors to introduce 3 or 4 cDNAs, which seemed surprisingly simple considering how complex it is for stem cells to go through each differentiation pathway.

The potentials of using iPS as models for research, cell assay systems, drug screening, toxicological testing, etc., seem to be tremendous at this point. However, for therapeutic use, the biggest hurdle standing in the way is the tendency of these iPS cells to form tumors once transplanted. It could be due to the oncogenic nature of the stemness inducing cDNAs themselves, or the retrovirus or lentivirus used for bring the cDNAs into the cells. A number of labs like that of Sheng Ding at Scripps, San Diego (Li et al., 2009), and Doug Melton at Harvard (Huangfu et al., 2008a; Huangfu et al., 2008b), are screening chemicals that would replace the use of some or maybe eventually all of the cDNAs. Such advances may help mitigate the oncogenic effects possibly associated with the inducing genes. Using non-integrating vectors as carriers would be preferred method for gene transfer if the retroviral or lentiviral vectors are the cause of tumors from iPS.

Today is the day that Allele launches its iPS product line, officially in this exciting field as one of the very first companies that produce products to make iPS research easier for everybody. New products in the pipeline include those for iPS induction and detection, stem cell culturing, differentiation tracking, and safer, novel delivery methods. It is just the beginning!

Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A.E., and Melton, D.A. (2008a). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature biotechnology 26, 795-797.
Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., and Melton, D.A. (2008b). Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature biotechnology 26, 1269-1275.
Li, W., Wei, W., Zhu, S., Zhu, J., Shi, Y., Lin, T., Hao, E., Hayek, A., Deng, H., and Ding, S. (2009). Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell stem cell 4, 16-19.

Tags: antibodies, cancer stem cells, CSCs, iPS, lentivirus, Nanog, Oct3, oncogenic, primer set, reprogramming, retrovirus, stem cells, tumor