Stem Cell Breakthrough: Toronto reprograms by jumping genes in and out
March 2, 2009 by Aaron Cheung
Toronto stem cell researchers have now, for the first time, devised a way to reprogram somatic cells from human back to a pluripotent-like state termed induced pluripotent stem (iPS) cells without the use of harmful viruses. I have blogged about the incredible feat of iPS cells recently as the scientific journal Science has named it “Breakthrough of the year”. These iPS cells are thought to hold the same regenerative potential as human embryonic stem cells but with additional advantages as it side steps the ethical issues related to the use of human embryos. Therefore, scientists now have a way of making patient-specific stem cells for uses such as disease modeling, drug screening, and hopefully one day, cell replacement therapy.
As promising as these iPS cells were, they had severe drawbacks, which included the requirement for viruses to deliver the four magical factors to induce reprogramming to happen. These viruses deliver the factors into the genome and will modify your genome in a permanent way such that it may disrupt your endogenous genes leading to tumourigenesis. Furthermore, even if these factors don’t disrupt your endogenous genes, leaving those factors in your genome can also lead to tumourigenesis as one of these four factors is the oncogenic gene, C-MYC.
For this reason, scientists have been trying to avoid the use of viruses and/or try to substitute the use of those factors with chemicals. Indeed, the use of non-integrating viruses, such as adenovirus, and the use of plasmid-based system have been succeeded in reprogramming mouse fibroblasts into mouse iPS cells (I have previously blogged about this here). However, neither of these breakthroughs have been achieved in the human system and furthermore, the efficiency is extremely low.
However, as of March 1st, Toronto scientists led by Dr. Andras Nagy at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital has now devised a method to reprogram mouse and, for the first time, human fibroblasts without the use of viruses (Reported in scientific journal Nature). Dr. Nagy and lead author, Dr. Knut Woltjen, took advantage of using transposons from Moths; these transposons are small pieces of DNA elements that hop in and out of the genome and are one of the sources of genetic variation in Moths (and many other organisms). Dr. Nagy inserted the four factors for reprogramming and inserted them into these transposons (called the piggyBac (PB) vectors). He then transfected both mouse and human fibroblasts with these PB vectors along with another enzyme called transposase which will cause the PB vectors to “jump” into the genome. Upon doing so, he was able to get mouse and human iPS colonies from mouse and human fibroblasts respectively.
This alone was a feat on its own as it was the first report of human reprogramming without the use of viruses. However, Dr. Nagy did not stop there as the true potential of the PB system is that it allows the subsequent removal of the PB vectors that were used to reprogram. Therefore, after reprogramming, Dr. Nagy added transposase again in order to mobilise the PB vectors within the genome. Sure, enough, he found that for some of the mouse iPS cells, the PB vectors jumped out and in to another part of the genome, but under certain circumstances, some of these PB vectors would jump out and never jump back in. Therefore, now we have mouse iPS cells that are genetically unmodified by any exogenous factors at all, it’s as if they reprogrammed all on their own. Currently, Dr. Nagy is trying to achieve the same feat in humans and he is confident that it shouldn’t be a problem.
Since the first report of generating human iPS cells back in the scientific journal Cell in November 2007, scientists have been sceptical whether it would be possible achieve this feat without the use of harmful viruses in humans for its potential use in therapeutic settings. Many scientists have reported the use of non-integrating methods (such as adenovirus), non-viral methods including use of plasmids and chemicals, but never were these reports in human and the reprogramming efficiency suffered drastically. This long awaited report by Dr. Nagy has now moved iPS cells one step closer to the clinics.
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Rett Syndrome and induced Pluripotent Stem cells goes “Green”
June 17, 2009 by Aaron Cheung
Stem cell researchers at The Hospital for Sick Children have now devised a method, reported in the scientific journal Nature Methods, that will facilitate the generation of induced Pluripotent Stem (iPS) cells. I have previously blogged about the promise of iPS cells (see related articles). iPS cells are cells which have been reprogrammed from a differentiated somatic cell (such as your skin cells) to a pluripotent stem cell (like an embryonic stem cell, only that you don’t need to use embryos!). The generation of iPS cells is inherently very rare which occurs at a frequency of about 0.01% meaning you can generate about one iPS cell line out of hundred thousand cells. Out of which, many lines fail to satisfy the different rigorous tests necessary to be given the prestigious name of “iPS cell”.
A group led by Dr. James Ellis, and lead author Dr. Akitsu Hotta, at The Hospital for Sick Children has generated a reporter, named EOS, that will facilitate the generation of iPS cells for both mouse and human. The reporter has DNA elements (called promoters) that will drive the expression of a fluorescent protein (GFP) and an antibiotic resistance gene (puromycin) that will only turn on in pluripotent stem cells, but remain off in differentiated cells. Therefore, scientists can now generate iPS cells from different sources and the iPS cells will literally “light up with a green colour” once reprogramming has been achieved. Furthermore, by adding puromycin to your culture, it will keep the iPS cells in a pluripotent state as iPS cells will normally spontaneously differentiate.
To demonstrate the applicability of EOS, Ellis also derived iPS cells from patients who have a neurodevelopmental disorder called Rett Syndrome. Rett Syndrome is an Autism Spectrum Disorder which affects almost exclusively females at an incidence of 1 in 10,000 and is one of the leading causes of mental retardation in girls. These Rett Syndrome iPS cells were green when placed under a fluorescent microscope and Ellis demonstrated that neurons can be generated from these Rett Syndrome iPS cells, the cell type affected in Rett Syndrome.
In conclusion, the EOS reporter will facilitate the generation of iPS and will be particularly useful for new labs going into this rapidly developing field. Furthermore, Rett Syndrome iPS cells that were also generated in this study will be invaluable for future drug screens that may be beneficial to those with Rett Syndrome.
Article:
Hotta, A. et al. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nature Methods 6, 370–376 (2009).
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A New Era for Disease Modeling and Drug Screening
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