Histones Might Hold the Key to the Generation of Totipotent Stem Cells


Reprogramming adult cells into pluripotent stem cells remains a major challenge to stem cell research. The process remains relatively inefficient and slow and a great deal of effort has been expended to improve the speed, efficiency and safety of the reprogramming procedure.

Researchers from RIKEN in Japan have reported one piece of the reprogramming puzzle that can increase the efficiency of reprogramming. Shunsuke Ishii and his colleagues from RIKEN Tsukuba Institute in Ibaraki, Japan have identified two variant histone proteins that dramatically enhance the efficiency of induced pluripotent stem cell (iPS cell) derivation. These proteins might be the key to generating iPS cells.

Terminally-differentiated adult cells can be reprogrammed into a stem-like pluripotent state either by artificially inducing the expression of four factors called the Yamanaka factors, or as recently shown by shocking them with sublethal stress, such as low pH or pressure. However, attempts to create totipotent stem cells capable of giving rise to a fully formed organism, from differentiated cells, have failed.  However, a paper recently published in the journal Nature has shown that STAP or stimulus-triggered acquisition of pluripotency cells from mouse cells have the capacity to form placenta in culture and therefore, are totipotent.

The study by Shunsuke Ishii and his RIKEN colleagues, which was published in the journal Cell Stem Cell, attempted to identify molecules in mammalian oocytes (eggs) that induce the complete reprograming of the genome and lead to the generation of totipotent embryonic stem cells. This is exactly what happens during normal fertilization, and during cloning by means of the technique known as Somatic-Cell Nuclear Transfer (SCNT). SCNT has been used successfully to clone various species of mammals, but the technique has serious limitations and its use on human cells has been controversial for ethical reasons.

Ishii’s research group focused on two histone variants named TH2A and TH2B, which are known to be specific to the testes where they bind tightly to DNA and influence gene expression.

Histones are proteins that bind to DNA non-specifically and act as little spool around which the DNA winds.  These little wound spools of DNA then assemble into spirals that form thread-like structures.  These threads are then looped around a protein scaffold to form the basic structure of a chromosome.  This compacted form of DNA is called “chromatin,” and the DNA is compacted some 10,000 to 100,000 times.  Histones are the main arbiters of chromatin formation.  In the figure below, you can see that the “beads on a string” consist of histones with DNA wrapped around them.

DNA_to_Chromatin_Formation

There are five “standard” histone proteins: H1, H2A, H2B, H3, and H4.  H2A, H2B, H3 and H4 form the beads and the H1 histone brings the beads together to for the 30nm solenoid.  Variant histones are different histones that assemble into beads that do not wrap the DNA quite as tightly or wrap it differently than the standard histones.  Two variant histones in particular, TH2A and TH2B, tend to allow DNA wrapped into chromatin to form and more loosely packed structure that allows the expression of particular genes.

When members of Ishii’s laboratory added these two variant histone proteins, TH2A/TH2B, to the Yamanaka cocktail (Oct4, c-Myc, Sox2, and Klf4) to reprogram mouse fibroblasts, they increased the efficiency of iPSC cell generation about twenty-fold and the speed of the process two- to threefold. In fact, TH2A and TH2B function as substitutes for two of the Yamanaka factors (Sox2 and c-Myc).

Ishii and other made knockout mice that lacked the genes that encoded TH2A and TH2B. This work demonstrated that TH2A and TH2B function as a pair, and are highly expressed in oocytes and fertilized eggs. Furthermore, these two proteins are needed for the development of the embryo after fertilization, although their levels decrease as the embryo grows.

Graphical Abstract1 [更新済み]

In early embryos, TH2A and TH2B bind to DNA and induce an open chromatin structure in the paternal genome (the genome of sperm cells), which contributes to its activation after fertilization.

These results indicate that TH2A/TH2B might induce reprogramming by regulating a different set of genes than the Yamanaka factors, and that these genes are involved in the generation of totipotent cells in oocyte-based reprogramming as seen in SCNT.

“We believe that TH2A and TH2B in combination enhance reprogramming because they introduce a process that normally operates in the zygote during fertilization and SCNT, and lead to a form of reprogramming that bears more similarity to oocyte-based reprogramming and SCNT” explains Dr. Ishii.

Misrepresentation of the Embryological Facts of Cloning by Reporters


Wesley Smith at National Review Online has been keeping tabs on the reporting of the Cell paper by Shoukhrat Miltalipov from the Oregon Health and Science University. The misrepresentation has been extensive but it’s not really all that surprising given the ignorance and lack of clear thinking on this issue. Nevertheless, Smith has kept up his yeoman’s work, cataloging the factual errors for reporters in multiple publications.

For his first example, see here, where Loren Grush on Fox News.com wrote:

Through a common laboratory method known as somatic cell nuclear transfer (SCNT), ONPRC scientists, along with researchers at Oregon Health & Science University, essentially swapped the genetic codes of an unfertilized egg and a human skin cell to create their new embryonic stem cells…The combination of the egg’s cytoplasm and the skin cell’s nucleus eventually grows and develops into the embryonic stem cell.

Grush, as Smith points out, is quite wrong. Introducing a nucleus from a body cell into the unfertilized egg and inducing it does not turn the egg into embryonic stem cells, but turns it into a zygote. The zygote them undergoes cleavage (cell division) until it reaches the early/mid blastocyst stage 5-6 days later, then immunosurgery is used to isolated the inner cell mass cells, after which they are cultured. Somatic cell nuclear transfer is a stand-in for fertilization. It produces an embryo and all the redefinition in the world will not change that.

Next comes my favorite newspaper, the Wall Street Journal, which normally has decent to pretty good scientific reporting, but this one story from Gautam Naik contains a real howler:

Scientists have used cloning technology to transform human skin cells into embryonic stem cells, an experiment that may revive the controversy over human cloning. The researchers stopped well short of creating a human clone. But they showed, for the first time, that it is possible to create cloned embryonic stem cells that are genetically identical to the person from whom they are derived.

As Smith points out, Miltalipov and others did not stop short of creating a human clone, then explicitly made a cloned human embryo and therefore made a cloned young human being.

Then there is this humdinger from an online Australian news report:

US researchers have reported a breakthrough in stem cell research, describing how they have turned human skin cells into embryonic stem cells for the first time. The method described on Wednesday by Oregon State University scientists in the journal Cell, would not likely be able to create human clones, said Shoukhrat Mitalipov, senior scientist at the Oregon National Primate Research Center. But it is an important step in research because it doesn’t require the use of embryos in creating the type of stem cell capable of transforming into any other type of cell in the body.

Oh my gosh, folks the paper describes the production of cloned embryos expressly for the purpose of dismembering them and destroying them. This “doesn’t require the use of embryos” crap reveals a very basic ignorance of how the experiment was done. See Smith’s excellent post for more details.

Then there is this story from one of my least favorite papers, the LA Times:

Some critics continue to argue that it’s unethical to manipulate the genetic makeup of human eggs even if they’re unfertilized, and others warn about potential harm to egg donors. The biggest ethical issue for the OHSU team, though, is that it artificially created a human embryo, albeit one that was missing the components needed for implantation and development as a fetus.

Come on people! The cloned embryo does not have the components needed to implant because there is no womb into which it can be implanted. Dolly was made the same way. Surely Dolly had the components required to implant.  The problem here is one of will, since these embryos were made to be destroyed. Not capacity. What was done to those embryos was dismemberment. Would we object if they were toddlers?

Just to show that obfuscation is not wholly an American news feature, there is this story from the German newspaper Deutche Welle:

Scientists, for the first time, have cloned embryonic stem cells using reprogrammed adult skin cells, without using human embryos…The process used by Mitalipov is an important step in research because it does not require killing a human embryo–that is, a potential human being–to create transformative stem cells.

As Smith points out, this research made a human embryo that was then killed to make embryonic stem cells. Calling this research humane is to redefine humane to the point of absurdity.

Finally this jewel of blithering ignorance from bioethicist Jonathan Moreno in his column in the Huffington Post:

Despite some confused media reports, the Oregon scientists did not clone a human embryo but a blastocyst that lacks some of the cells needed to implant in a uterus.

And you wonder why people like me have lost all faith in American bioethics. As a developmental biologist, this one just grates on me.  A blastocyst has two cell populations; an outer trophectoderm composed of trophoblast cells that will form the placenta and the inner cell mass cells on the inside of the embryo, which will form the embryo proper and a few placental structures. To be a blastocyst is to have the equipment to implant.

To drive the nail into the coffin, Smith quotes the father of embryonic stem cells James Thomson from an MSNBC interview:

See, you are trying to redefine it away…If you create an embryo by nuclear transfer, if you gave it to somebody who didn’t know where it came from, there would be no test you could do on that embryo to say where it came from. It is what it is. By any reasonable definition, at least as some frequency, you are creating an embryo. If you try to redefine it away, you are being disingenuous.

Check out Smith’s posts. They are all worth reading. Maybe the press will learn some embryology, but I doubt it.

Postscript:  Brendan P. Foht writes at the Corner on National Review Online that in 2010 Shoukhrat Mitalipov, the leader of the Oregon cloning team, reported that he had achieved a single pregnancy using cloned monkey embryos that were made with exactly the same technology as was employed with human eggs in his 2013 Cell paper.  The fetus developed long enough to have a heartbeat detectable through ultrasound. Although the pregnancy failed after 81 days (about half the normal gestation period for that species), the fact that a pregnancy would develop so far indicates that reproductive cloning of primates is in principle possible.  This definitively shows that all this talk about the embryos made in Mitalipov’s lab not being able to implant is pure drek.

Wesley Smith and Cloning


My favorite bioethicist, Wesley Smith said this about human cloning in his prescient book: A Consumer’s Guide to A Brave New World:

We can pursue biotechnology to treat disease and improve the human condition, while retaining sufficient humility and self-restraint to keep ourselves from endangering the intrinsic value of human life. Or, we can hubristically rush onto the very anti-human path warned against by Aldous Huxley, driven by our thirst for knowledge, vast profits, and obsession with control and vastly expanded life spans.

These issues are too important to be “left to the scientists.” Nor can we afford to allow the marketplace to determine what is right and what is wrong. The stakes are too high, the potential impact on each and every one of us too profound, to remain passive and indifferent to the decisions that are to be made. It is our duty to participate in the crucial cultural and democratic debates over biotechnology. The human future, quite literally, depends on it.

Prophetic and poignant – and DEAD RIGHT!!

Human Stem Cells From Cloned Embryos: What Horrors WIll Follow?


First the news, then the commentary. Here’s the news:

In the May 14th edition of the international journal Cell, Shoukhrat Miltalipov from the Oregon Health and Science University, reported the derivation of human embryonic stem cells from cloned human embryos. This is the first time this has been successfully reported. In 2004, a South Korean researcher, Woo Suk Hwang, reported that his laboratory had succeeded in making patient-specific human embryonic stem cells from cloned embryos, but his papers were later shown to be completely fraudulent, and Hwang, in a word, walked. For more on this sad, sordid event, see my “Catastrophic Cloning Caper” here.

Many laboratories have tried and failed to get cloned human embryos to live long enough to get embryonic stem cells from them. The cloning procedure produces a very abnormal embryo that dies very early during development.

How did Mitalipov succeed when so many others before him had failed? Mitalipov honed his cloning protocol in work with early embryos from Rhesus macaques, and during this work, Mitalipov and his coworkers discovered that including caffeine with the mix of chemicals used during donor removal and transplantation into the host egg prevents the oocytes that have just had their nuclei removed from dividing prematurely, and if these oocytes are used in a cloning experiment, they survive longer than oocytes treated with standard cloning techniques.

“It was a huge battery of changes to the protocols over a number of different steps,” said Mitalipov. “I was worried that we might need a couple of thousand eggs to make all these optimizations, to find that winning combination.”

The procedure used in this paper, cloning, is more technically known as “somatic cell nuclear transfer” or SCNT. SCNT requires human eggs that are extracted from female volunteers of reproductive age who are given several drugs to hyperstimulate their ovaries, which then ovulate several eggs at a time. The eggs are harvested by means as aspiration, and used in SCNT.

For SCNT, the egg nucleus is removed by means of a micropipette. The egg is ever so gently squeezed until the nucleus, which is usually off to one side in the egg, protrude through the cell membrane, and the nucleus is sucked off with the micropipette. Then a body cell; in this paper, fibroblasts from the skin were used, is laid next to the nucleus-less egg, and an electric current is pulsed through the two cells, which causes them to fuse. This fusion converts the egg, which used to have one set of every chromosome, into a cell that now has two sets of every chromosome, and the egg cell, begins to divide and recapitulate the events of early development. This is also referred to as cloning.

Somatic_cell_nuclear_transfer-image

Sperm and eggs have chromosomes that have been modified in specific ways. When the sperm and egg fuse, the process of fertilization begins, and the modifications to the chromosomes serve their purpose during the early stages of development, but those modifications and gradually undone as development proceeds. This phenomenon is known as genetic imprinting and it is very common in mammals. For a good paper on genetic imprinting see Wood AJ, Oakey RJ (2006) Genomic Imprinting in Mammals: Emerging Themes and Established Theories. PLoS Genet 2(11): e147. doi:10.1371/journal.pgen.0020147.

Since cloned embryos have a genome that is not properly imprinted, its development is hamstrung to one degree or another. Most researchers were unable to get cloned human embryos to survive past the 8-cell stage. However, by including caffeine in the SCNT medium during egg nucleus removal and transplantation of the donor nucleus into the host egg, enough of the cloned embryos survived to the 150-cell blastocyst stage to allow for the derivation of embryonic stem cells. Even though SCNT is an exceedingly inefficient process, Mitalipov was able to derive six embryonic stem cells lines from 128 eggs, which is about a 4% success rate.

George Daley of Boston Children’s Hospital and the Harvard Stem Cell Institute, who was not involved in the research, said of it: ““I think it is a beautiful piece of work.” He continued: “This group has become remarkably proficient at a very technically demanding procedure and [has] shown that SCNT-ESCs may in fact be a practical source of cells for regenerative medicine.”

Mitalipov and his group analyzed four of the cloned embryonic stem cell lines and found that their NT-hESCs could successfully differentiate into beating heart cells in culture dishes. Also, they could differentiate into a variety of cell types in teratoma tumors when transplanted into live, immunocompromised mice. These stem cells also had no chromosomal abnormalities, and displayed fewer problematic epigenetic leftovers from parental somatic cells than are typically seen in induced pluripotent stem cells (although, for the life of me, no one has shown that these epigenetic holdovers are a big problem for regenerative medicine). Mitalipov said more comparisons are required, however.

“We are now left to analyze the detailed molecular nature of SCNT-ES cells to determine how closely they resemble embryo-derived ES cells and whether they have any advantages over iPS cells,” added Daley. “iPS cells are easier to produce and have wide applications in research and regenerative medicine, and it remains to be shown whether SCNT-ES cells have any advantages.”

Mitalipov, however, did point out one fundamental difference between NT-ESCs and iPSCs: their nuclear genomes come from the donor cell, but NT-hESCs contain mitochondrial DNA (mtDNA) from the host egg cell. Therefore, SCNT reprograms the cell but also corrects any mtDNA mutations that the donor may carry. Thus, patient-specific NT-hESCs could be used to treat people with diseases caused by mitochondrial mutations. “That’s one of the clear advantages with SCNT,” Milatipov said.

The cells used for this cloning experiment came from infants.  It still remains for cloning to succeed with adult cells as the donor cells.

Now for the commentary:

Regular readers of this blog will already know that I am deeply opposed to human cloning in any form.  It is the equivalent of making people for spare parts.  This is immoral and barbaric.  I predicted some time ago (OK not so long ago, 4 years to be exact), that the technical problems with human cloning would be solved and scientists would one day clone a human embryo.  Now that it is here, I hope that people are as horrified by it as I am.

“Get over it.  It’s an embryo and a cloned one at that.” you might say.  But what if the malady that doctors want to cure is poorly served by embryonic stem cells made from cloned embryos and a cloned fetus is a better source of cells?  Do we allow gestation of the cloned embryo to the fetal stage so that we can dismember it and take its tissue?  Let’s bring this home.  What if the cells needed to come from a five-year old?  Can we justify that because the kid was cloned?

“But wait, that’s a five-year old and this is an embryo,” you say.  But you were once a blastocyst.  You did not pass through the blastocyst stage, you WERE a blastocyst.  The only difference between the blastocyst and you now is time, environment, degree of dependence, and size.  Are any of these differences morally significant when it comes to whether or not we can kill you?  Can we kill all the short people?  Can we kill all the younger people because they are not as well-developed?  Can we kill people who are dependent on others (that includes everyone mate, so put your hand down)?  Can we kill those in a different location (genocide anyone)?  None of these categories constitutes a good reason for terminating someone’s life.  Likewise, none of these changes renders you essentially different from who and what you are.  To kill someone at the earliest stages for their tissue is simple murder, and we use size, location, extent of development, location and degree of dependence to salve of consciences for doing it, but that won’t define what we are doing.

People will go on and on about the great advances that could lead to.  Sorry, I’m not buying that one.  Embryonic stem cells have been promising that one for the last 15 years with pert near little to nothing to show for it.  This discovery is a great technical advance, but it opens to door to reproductive cloning – an even bigger mistake, and fetus farming, in which we destroy our own children in the womb, not because they are in inconvenience to us, but because we want their tissues to save our lives.  Now children, rather than being a blessing, are merely tissues to be harvested.  We have become like the Greek gods from the stories of old who ate their own children.  May God forgive us.

John Gurdon Embraces Human Cloning


Wesley Smith has reported that Nobel Laureate John Gurdon, who shared the Nobel Prize in Medicine this year with Japanese induced pluripotent stem cell discoverer Shinya Yamanaka, has come out in favor of human cloning.

From the story in the Daily Mail:
‘I take the view that anything you can do to relieve suffering or improve human health will usually be widely accepted by the public – that is to say if cloning actually turned out to be solving some problems and was useful to people, I think it would be accepted,’ he said. During his public lectures – which include speeches at Oxford and Cambridge Universities – he often asks his audience if they would be in favour of allowing parents of deceased children, who are no longer fertile, to create another using the mother’s eggs and skin cells from the first child, assuming the technique was safe and effective.

‘The average vote on that is 60 per cent in favour,’ he said. ‘The reasons for “no” are usually that the new child would feel they were some sort of a replacement for something and not valid in their own right. ‘But if the mother and father, if relevant, want to follow that route, why should you or I stop them?’

 

Smith then quotes from his magnificent book “Consumers Guide to a Brave New World,” which all my readers to RUN out to buy and read over and over again:

Scientists would have to clone thousands of embryos and grow them to the blastocyst stage [one week] to ensure that part of the process leading up to transfer into a uterus could be “safe,” monitoring and analyzing each embryo, destroying each one in the process. Next, cloned embryos would have to be transferred into the uteruses of women volunteers [or implanted in an artificial womb]. The initial purpose would be analysis of development, not bringing the pregnancy to a live birth. Each of these clonal pregnancies would be terminated at various points of development, each fetus destroyed for scientific analysis. The surrogate mothers would also have to be closely monitored and tested, not only during the pregnancies but also for a substantial length of time after the abortions.

Finally, if these experiments demonstrated that it was probably safe to proceed, a few clonal pregnancies would be allowed to go to full term. Yet even then, the born cloned babies would have to be constantly monitored to determine whether any health problems develop. Each would have to be followed (and undergo a battery of tests both physical and psychological) for their entire lives, since there is no way to predict if problems [associated with gene expression] might arise later in childhood, adolescence, adulthood, or even into the senior years.

 

Smith, in my view, is spot on. Therapeutic cloning will not stop at using cloned blastocysts to make patient-specific embryonic stem cell lines. The reason for this is that even though cells made from differentiated embryonic stem cells can have therapeutic value, such cells can also be rejected by the immune system of the host animal. A much more fail-safe way to do this experiment is to gestate the embryos to the fetal stage and use the fetal tissues.

Once we go down the road of cloning and destroying embryos just to make embryonic stem cell lines from them, what’s to keep us from aborting fetuses just to get their cells? This slippery slope is real and speaks volumes, none of it good, about a society that sacrifices its youngest and more vulnerable members to serve the needs of others. It cheapens human life to the nth degree and at its lowest point, it simple murder.

Gurdon, however, speaks of reproductive cloning to replace children lost through tragedy. While I can appreciate the sentiment, sentiment is an extremely poor reason basis for ethics. Folks, biology is not destiny. Cloning experiments in animals have shown us that even cloned embryos made from material taken from the same mother, that are genetically identical are neither identical to their mothers nor are they identical to each other. Random events that occur during development and the way each individual responds to their environment shapes them in a unique manner. The cloned sheep Dolly was completely unlike her cloned siblings in personality, behavior, or overall appearance. The same can be said for CC (for “Carbon Copy”), the first cloned cat, which looked unlike her mother and had a very different personality.

Yet these cloned children are asked from the second they are born to replace another child who is unlike them. The cloned child is a human person and while the right for each person to be authentically who there are in an inherent right of all human beings, this very right is denied these cloned kids – they are born for the very reason that they can be someone else. This is a violation of everything it means to be human, and it is the very reason no good thing can come from human cloning.

Gurdon is a brilliant scientist, but as we have seen before, great scientists sometimes make terrible ethicists.

Wesley Smith and the New Stem Cells in Ovaries: The Means for Mass Human Cloning


Wesley Smith, who runs a bioethics blog called “Secondhand Smoke” has blogged about the discovery of stem cells in the human ovary that can make cells that look like human eggs. He fears, and I think rightly so, that this could lead to the “Brave New World project of human self design, genetic engineering, transhumanistic tinkering, human enhancement, and using reproductive technologies to shatter the remaining vestiges of norms surrounding families.”

Human cloning can lead to nothing good. Right now it has stalled because of an acute shortage of human eggs (oocytes). The risk of the egg procurement procedure, and the work of people like my friend Jennifer Lahl and her film Eggsploitation, has dried up the supply of eggs. Thus cloning work has greatly slowed. TIlley’s work, however, could change that. Oh, sure, it will be sold as providing eggs for the infertile, which is a laudatory thing by the way. However, the use of huge numbers of eggs for human cloning purposes is a bridge too far.  A cloned  embryo is still a human embryo and is therefore still a human person.  Cloning for manipulation and enhancement research is a grotesque abuse of human rights.

You can read Smith’s article here.