You guys might have noticed I've been quiet lately, that's because I've scheduled a thesis defense and am under deadlines. However, I couldn't let these two (1) papers(2) on reprogramming of human adult cells into stem cells slip by without some comment (NYT piece here)
These reports are a follow-up on landmark animal studies that we discussed previously that showed that expressing 4 genes in cells obtained from adult animals you could induce them to form embryonic stem cell (ESC) like cells that researchers dubbed induced pluripotent stem cells (iPS cells). At the time we noted several obstacles to the practicability of this technology, and these papers represent success in overcoming the first - transferring the technique into human cells.
I admit I'm surprised they were able to do so so quickly. But this turned out to be a nice example of the discovery of common exploitable pathways between humans and other animals.
Below the fold I'll go over the differences between these studies and the previous animal studies, the evidence of the pluripotent nature of these cells, unresolved problems with this technology and why this isn't a victory for the anti-ES cell crusaders.
Both papers performed similar studies to demonstrate the pluripotent nature of the iPS cells, however, in humans, a slightly different set of genes could be used to transform the adult cells into pluripotent cells. Animal studies used Oct4, Sox2, c-Myc, and Klf4, and these were the same genes used by the Yamanaka group (2). However the Thomson group(1) showed that the transformation could also be induced by Oct4, Sox2, Nanog, and Lin28. So why did one group stick with the same set of genes and the other try different ones, and why did it still work? Well the Thomson group started by identifying genes that are expressed in ESC but not in slightly less pluripotent myeloid (blood) precursors. They then screened these genes by using retroviruses to insert them into a cell line that has the Oct4 promoter driving a survival gene. The result was a screening tool that when pluripotency is achieved, the Oct4 promoter activates thus allowing the cells to survive treatment with a toxin. This was, I think, a smart move, as it essentially repeated the screening process used in mice, and the Thomson group didn't assume that identical factors in mice could accomplish this goal. They were wrong, but as a result they still managed to generate iPS cells and probably have discovered some additional interesting biology. Namely that the inclusion of Nanog and Lin28, while not required for generating pluripotent lines, greatly increased the efficiency of the cloning process.
The other neat difference between the two studies is that they both used different cell lines to generate iPS lines. The Thomson group tested fetal fibroblasts and neonatal foreskin fibroblasts successfully, while the Yamanaka group used human skin cells or dermal fibroblasts to achieve the transition. The Thomson group also had a more elegant system for identifying iPS clones (humans don't have Oct4 promoters driving survival genes) using a tissue culture trick, they simply used conditions conducive to ESC growth but inhibitory to fibroblast growth, as a result ESC colonies popped up more quickly and efficiently, while the Yamanaka group had to sort through more noise - lots of non iPS cells grew in their cultures.
One should note that the likely reason the Yamanaka group achieved higher rates of iPS induction is that they also inserted the mouse-retroviral receptor gene into the cells, thus increasing their infection efficiency. It's likely the two techniques are otherwise equivalent.
Both groups then tested the function of these cells through various techniques like embryoid body formation, directed differentiation, etc., and the gold-standard (at least the ethical gold standard for human cells) injection into nude mice to generate teratomas - a tumor comprised of all three germ cell layers. Here is figure 7 from the Yamanaka group (2).
The cells, in vivo were capable of generating cells from the three primary germ cell types - endoderm, mesoderm, and ectoderm. This is as about as good as it gets for demonstration of their pluripotent potential. The only better measure - chimera formation - is simply unethical for human cells, and teratomas are adequately convincing, especially combined with their in vitro embryoid body analysis.
So what are the remaining problems? The most critical is, of course, the use of retroviruses to transduce the cells. The problem is that previous attempts to use these vectors in humans, to treat severe-combined immunodeficiency (bubble boys), resulted in an unacceptable rate of oncogenic transformation of the treated cells causing the FDA to stop the trials. The kids got leukemias. This is terrible. The reason is that the retroviruses incorporate their genomes into ours. The advantage of this is that the genes are permanently expressed - long enough to transform the cells and maintain them until they are differentiated. The draw back is that the retroviruses prefer to insert themselves randomly in the genome in areas that are transcriptionally active. The result is that the transgenic promoters - powerful viral sequences that drive gene expression - may get stuck next to an oncogene, that can then cause cancer. Or cause epigenetic changes in the region of an oncogene that should be silenced. Or even interrupt a tumor-suppressor gene. All very bad outcomes that have been shown to cause cancer in humans using these vectors.
There is a solution though, and I would humbly suggest that the researchers in this field either use a conditional promoter - one that only expresses in the presence of a drug like tetracycline or tamoxifen. Or that they use a non-viral integrase system. Now, these integrase systems have also been shown to cause chromosomal aberrations. However, mutagenesis of the proteins has been shown previously to increase their specificity of action, and the technology, I believe, can be improved to the point that it can be used in humans. Further, they can be engineered for the integration of the genes into one location, rather than randomly throughout the genome, which would be a problem even if one used conditional promoters.
It's also possible that tagging the 4 proteins required for the transformation with an HIV-protein that allows them to cross the cell membrane (tat-tagging), may provide a viral-free approach for generating and maintaining the iPS cells, without any gene manipulation at all.
Finally, as far as the anti-ESC types are concerned, they should not consider this a victory for their anti-science agenda or the policies of George Bush. First, these discoveries would not have been possible without ESC research. I also believe these cells would have been discovered in the same amount of time, with or without the political interference in science. Not only because somatic cell reprogramming was hotly studied long before this became a political issue but also because they represent an ideal stem cell - one that can be genetically matched to the donor, yet is still pluripotent. SCNT has been an incredibly difficult technology to make practical, as human eggs are difficult to obtain, and the process is very inefficient. We've also lost valuable years of study of pluripotent human stem cells in this idiotic debate, that would directly translate to our understanding of how to apply these cells in studies of disease and for clinical practice.
Further, the precedent that has been set - that a minority of people can inflict their religious beliefs onto science policy - is atrocious, and damaging, and will likely have long-term consequences for science. Because, now they've learned that not only can they get away with it, but they can delude themselves into believing it is the right thing to do for scientific progress.
1. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Junying Yu, Maxim A. Vodyanik, Kim Smuga-Otto, Jessica Antosiewicz-Bourget, Jennifer L Frane, Shulan Tian, Jeff Nie, Gudrun A. Jonsdottir, Victor Ruotti, Ron Stewart, Igor I. Slukvin, and James A. Thomson (20 November 2007) Science [DOI: 10.1126/science.1151526]
2. Takahashi et al., Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined
Factors, Cell (2007), doi:10.1016/j.cell.2007.11.019
I've scheduled a thesis defense.
Congratulations. Fantastic news.
Finally, as far as the anti-ESC types are concerned, they should not consider this a victory for their anti-science agenda or the policies of George Bush.
I'm not sure this is correct. Your given reasons for this claim are perfectly sensible, but sanity has nothing to do with politics. Many people will leap at even the hint that there may be an alternative to a practice they found uncomfortable, and in their minds 'perhaps' and 'maybe' will quickly morph into 'will'.
Depending on what you mean by 'inflicting,' I think you might be overstating the case when you refer to a minority of people inflicting their religious beliefs onto science policy. The minority certainly haven't managed to effect a ban on ESCR, just limited the scope of federal funding. And even there, to permit federal funding of _any_ ESCR was really a compromise on their part, since they were _still_ providing financial support to research they believe is immoral. So just what is the bad precedent that has been set here?
Right; this is somatic cell reprogramming technique made possible by the findings of embryonic stem cell research. This is neither purely somatic cell nor embryonic cell research. Too often, ESC proponents and anti-ESC activists alike have dismissed advances in either embryonic or somatic stem cell research and overhyping their own side (which precipitated the South Korea fiasco). The anti-ESC side is far worse because they want to stop an entire research program rather than just being dismissive or overhyping.
There is currently a danger of under-informed ESC research advocates foolishly giving credence to the notion that ESC research is ethically tenuous - and giving aid and comfort to anti-ESC activists - by saying something to the effect of "Well, now we don't have to destroy embryos, thank goodness."
Kevin Drum, who apparently has little background in biology, almost drifts into that in his response to Ramesh Ponnuru remarking (correctly, even though I totally disagree with his stance on ESC) that the battle over stem cells is probably not over.
There is much that we do not understand about cellular epigenetic programming, cell-cell interactions in development and somatic maintenance, cytokine networks and so on. So ESC research will be required for the foreseeable future.
My longtime interest in stem cells is not due to their therapeutic potential or the bioethics issues; rather, it's because of my interest in evolutionary ecology and life history theory. Somatic stem cells are crucial in plasticity, maintenance, cancer, and aging, and cell differentiation pathways represent a major trade-off.
Bob, you don't know what a pain in the ass it is to get human ESC running in a lab. It took us a year and a half just to get the lines, tons of paper work, and ultimately, it's just been a handful of lines that we could study. All of this was for a religious reason. Some people think that a soul is put into a person at conception. That's it.
So a completely religious concept, ensoulment, was the justification for restrictions on a scientific field. This is terrible. What piece of dogma will be discovered to conflict with science next that will require federal regulation?
Mark - I know that I'm in a distinctly minority position, since I believe ESCR should be pursued, but without compelling those with moral objections to support it through their tax contributions. For me, the question is how to further the pursuit of scientific truth without trampling on freedom of conscience.
I figure that researchers who want the public to bankroll their investigations are in the position of beggars, not choosers. Public monies just don't come without a few strings attached; nor should they, in a liberal polity.
Making science subject to religious dogma seems to be excessive. The objections are based on the concept of ensoulment. That life "begins" at conception. Life doesn't begin. It's a scientifically invalid position, and fundamentally religious.
As far as freedom of conscience in public funding, I'm sorry but this is totally absurd. I can't choose to not fund the Iraq war, or pay taxes that go to weapons research. Why can the religious object to ESCR? Where is my freedom of conscience?
I don't buy it. This is nothing more than the excessive obeisance to the religious right, who get to interfere simply because they are in power. This freedom of conscience is recognized for no one else, so I do not acknowledge its utility or value.
In what sense has science been "made subject" to religious dogma? Refusing to support science on religious grounds is hardly what is usually meant by that phrase.
Your remarks about freedom of conscience suggest to me that you would benefit from careful study of that notion. Public funding of war in the face of conscientious objections is usually justified on the grounds that the ability of the state to engage in war is necessary to the maintenance of a well ordered society, and a showing of compelling societal interest can trump conscientious objections. Do you think an analogous argument can be made for ESCR? And your asking "Where is my freedom of conscience?" is puzzling. Are you suggesting that by not supporting ESCR somebody is compelling you to act contrary to your moral beliefs? How does that work?
The basis of objections to ESCR is that destruction of embryos destroys a human life. This is based on the belief that human life begins at conception. Those of us who understand that life doesn't begin realize the line is fuzzier and many of us feel this demeans the value of real human life to equate real human beings to little balls of cells.
Ok, I'll give you the war, we don't have a choice. But how about research into weapons? Why can't I object to that based on freedom of conscience? There is all sorts of research that I object to, but I realize that part of living in a country with other people is that each of us doesn't get a veto on what work is done.
Mark - You ask about research into weapons, and mention "all sorts of research" to which you object, presumably on moral grounds. I happen to agree with you if your point is that there are a lot of expenditures of public monies for projects that many of us think are morally objectionable, and which can't pass the "compelling societal interest" test. But surely, you aren't suggesting that we should succumb to the "two wrongs make a right fallacy," are you?
Again, I suggest careful study of the notion of freedom of consience. It's not even close to being equivalent to giving each of us a veto on what work is done.
But many people's conscience is stupid! If we had a system where every jackass special interest group could interfere with scientific research, the ARAs will object to public money on animal research, the right wingers will object to anything that doesn't conform to "magic man done it" and "sex is dirty", people like me will object to research into more effective ways of killing people, name the crankery, people will object to it based on conscience.
In a free society freedom of conscience should mean they don't have to perform the research if they don't want to. It shouldn't mean people can line-item veto a type of research they don't like.
Think about the implications! It's a terrible idea for everybody.
MarkH: "object to research into more effective ways of killing people"
And where would DARPA be? DARPA kicks ass! (Note: Not being facetious nor sarcastic.)
So, good point.
Mark - Again, refusing to support an activity is not the same, either in ethics or law, as interfering with those who would engage in the activity in question. And again, respecting freedom of conscience is not giving anybody the power to veto actions by others. These are red herrings. In other words, the implications you think you're thinking about aren't implications at all.
But Mark, first things first. Prepare for your dissertation defense.
Good luck on your thesis defense, Mark! As far as the iPS issue goes, it looks like we're about on the same page, although I'm just a protein guy, so I can't claim any expertise.
Wrong: This is a victory for the anti-ESC campaigners. You are thinking as a scientist, not in soundbite politics. In the soundbites, the media is already describing these are ESC-substitutes, the anti-ESC side has already declared them to have rendered ESCs obsolete, and the typical person isn't going to hear otherwise.
As a scientist and atheist I completely understand why MarkH objects to dogmatic ideas of the "beginning" of life and existence of souls limiting funding of research. As a citizen of a constitutional republic I also understand that people have the right to elect representatives to congress that will prevent funding of projects they find morally objectionable.
The religious right currently has a powerful ally in the White House and substantial representation in congress. That means they get to hold the purse strings. You still have the right to pursue the research on your own dime.
I'll bet MarkH, based on his remarks about the Iraq war, is one of the people who went to the polls with the idea of electing a democratic majority that would de-fund the war. They may yet do so, although it doesn't look like they have the votes or the balls. So it would appear that he has no problem with government acting to selectively fund projects based on moral objections, as long as they are his moral objections.
Either MarkH doesn't know how the game works or he just likes to whine about losing.
Wow, way to miss every single subtlety of this argument lance.
How is it that you can't see that you're a troll? You have no insight do you?