So What About Three-Person Embryos?


In 2013, Deiter Egli’s group at Harvard University successfully transferred chromosomes that were in the process of dividing and segregating (known as an incompletely assembled spindle-chromosome complex) from one human egg into another egg whose nucleus had been removed (Nature 493, 632–637 (31 January 2013) doi:10.1038/nature11800). They prevented the eggs from prematurely re-entering meiosis by cooling the chromosome/spindle complex to room temperature. This allowed normal polar body extrusion, efficient development to the blastocyst stage, and, eventually, the derivation of normal stem cells.

3-person-ivf

Egli’s technique allows the genome of one egg to initiate development in the cytoplasm of another egg. Why is this significant? Because within out cells is a bean-shaped vesicle called a mitochondrion. Mitochondria make the energy for our cells. To do this, mitochondria use a variety of proteins encoded on genes found in the nuclear genome. However, mitochondria also have their own genome that encodes some crucial mitochondrial proteins and RNAs. The human mitochondrial genome is a small, circular DNA molecule that encodes 37 different genes.

C:TypesetSBG36-32013-1302013-130.vp

Mutations in genes encoded by the mitochondrial genome tend to have rather catastrophic consequences for the fertility of women. When the egg undergoes fertilization, the vast majority of the mitochondria of the sperm are degraded and their mitochondrial DNA is eliminated (Katsumi Kasashima, Yasumitsu Nagao, and Hitoshi Endo. Reprod Med Biol. 2014; 13(1): 11–20). Research has shown that the father’s mitochondrial genome can make some very small contribution to the embryo, a phenomenon known as “paternal leakage,” but it is usually pretty small (Kuijper B1, Lane N, Pomiankowski A. J Evol Biol. 2015 Feb;28(2):468-80). Therefore, if the mother carries a deleterious mutation in her mitochondrial DNA, her eggs will usually not be able to progress through fertilization successfully and support the growth and development of the embryo. Consequently, the mother will be infertile.

This new technique by Egli, however, allows mothers who are infertile because of mutations in their mitochondria DNA, to have children who are genetically related to them. All that is needed are eggs from a healthy donor, and a laboratory that has the know-how and will to do this procedure. The mother’s eggs are harvested by standard IVF technologies, fertilized by the father’s spermatozoa, and after fertilization has ended, the chromosome-spindle complex is lifted from the young embryos and transferred into enucleated donor eggs that contain mitochondria with normal genomes. Development will then ensue without a hitch. Right?

Well not so fast. As it turns out, this procedure has been carried out in several different animal species, and the results are decidedly mixed (see Reinhardt and others, Science 2013;341:1345).

If we begin with insects, we can move new mitochondrial genomes into embryos by standard genetic techniques. If we do so in the fruit fly, Drosophila melanogaster, such mitochondrial transfer produces fly embryos that develop normally, but the animals show altered juvenile viability, adult male animals show accelerated aging and reduced fertility. Genetically, it is clear that transferring new mitochondria into an egg messes up the expression of nuclear genes. Identical experiments in the seed beetle causes altered development and metabolic rates, reduced fertility in males and reduced survival in females. Similar studies in copepods (Tigriopus californicus) causes reduced juvenile viability, and reduced mitochondrial function and energy production in adults.

If mice are subjected to these same experiments, the animals develop normally and survive to adulthood, but these adult mice show reduced growth and exercise ability and reduced learning ability in males.

The above-mentioned experiments used standard genetic breeding techniques to generate animal strains that had a mismatch between the nuclear and mitochondrial genome.  Such techniques are demonstrably non-invasive.  However, the technology applied in Egli’s laboratory were invasive, and included removing chromosome/spindle complexes and transferring them to donor eggs that had been enucleated. Therefore, the effects of these invasive procedures had to be tested as well. If such invasive procedures were tested in cultured mouse cells, the hybrid cells showed altered cellular respiration and growth. In short, their mitochondria worked poorly inside their new homes.

If Egli’s technique was used in non-human primates, macaques in particular, the animals developed to the juvenile stage and appeared normal.

On the strength (or weakness) of these experiments, some reproductive specialists in countries where such techniques can be performed without fear of prosecution have used mitochondrial transfer in human embryos. Again the results are quite mixed. Healthy children have been born by this procedure, but several others have not. Helen Pearson reported in Nature News on the 14th of October, 2005 about two Chinese babies that were made with mitochondrial transfer that died in utero at 24 and 29 weeks. Other outcomes include a miscarriage, an abortion of a fetus that had Turner Syndrome, at least two children with mixed mitochondria that studies linked with cognitive dysfunction and obesity, and a child born with a severe developmental disorder. I do not call these hopeful results.

Another experiment that gives me pause was published in the journal Cell Reports in June of 2014 by Joerg Patrick Burgstaller and others. This paper showed that even small amounts of diseased mitochondrial DNA in an embryo would spread throughout the organism. The amount of spread is wide and varied, but even small amounts of variant mitochondrial DNA did spread. This significance of this is stark for this debate. You see, Egli’s original paper in Nature showed that very small amounts of the original mitochondrial DNA are transferred to the donor egg. Granted it below 1% of the total mitochondrial DNA in the embryo, but it is still detectable. Burgstaller and others have shown that even with this small amount of mitochondrial DNA, it will still spread throughout the developing baby and given them a body with some cells that have most the diseased mitochondrial DNA, and others that have the normal mitochondrial DNA, and other cells that have a mixture of the two. Therefore, Egli’s technique is NOT a cure for conditions linked to mitochondrial DNA mutations. Let me repeat this for every one – Egli’s technique is NOT a cure for conditions linked to mitochondrial DNA mutations.

No vertebrates have yet been studied who have gone through mitochondrial replacement and survived to reproductive age. Given the decidedly mixed record of this technology in a variety of animal models and the paucity of data so far, this technology is clearly not ready for use in humans.

However, that has not stopped scientists and politicians in the United Kingdom from pushing this technology forward as a fertility treatment for infertile women who harbor mitochondrial DNA mutations.  Some in the scientific community warned about the potential dangers of this technology.  Their concerns were largely ignored and in many cases severely criticized.  Even worse, some thought that three-person embryos could grease the slippery slope in which this technology or similar ones like cloning would be applied as generalized treatments for infertility.  That concern was labeled ridiculous. No longer.

Science magazine reported that cloning magnate Shoukhrat Mitalipov has formed a partnership with disgraced fraudster Woo Suk Hwang.  The two are teaming up to form a joint commercial venture to use Mitalipov’s cloning techniques as a way to treat infertility and perhaps other diseases.  Mitalipov’s commercial venture Mitogenome Therapuetics and Hwang along with the company BoyaLife, which will reportedly put up more than $90 million into the effort.  Mitalipov has also generated news reports by asking FDA approval to use so-called 3-person IVF “mitochondrial transfer” technology, which shares some technical elements with cloning, to treat infertility. This surprised some in the UK, including members of Parliament who were hoodwinked into voting to approve the three-person embryo procedure by being told that this technology would only be used to treat mitochondrial diseases.

The slippery slope is real and unless citizens rise up and make noise, we are going to be dragged where angels fear to tread by over-zealous scientists who are willing to sacrifice young children for the sake of their own fame and success.  This technology is not ready for use in humans.  The approval of this technology in the UK is a very bad idea.  It will also spread to the use of cloning in general as a treatment for diseases, and we will then move to fetus farming.  May God give us the strength to say enough is enough.

The United States FDA’s Cellular, Tissue and Gene Therapies Advisory Committee will be holding a public hearing to “discuss considerations for the design of early-phase clinical trials of cellular and gene therapy products” including the three-parent IVF method. The public has until October 15 to send in written comments. If you are interested in making your views known, go here.

Cells Made From Embryonic Stem Cells Derived from Cloned Embryos Are Rejected by the Immune System


Researchers from Stanford University have shown that genetic differences in mitochondria found in cells made from pluripotent stem cells that were originally derived from cloned embryos can prompt rejection by the immune system of the host animal from which they were made, at least in mice.

According to a study in mice by researchers at the Stanford University School of Medicine and colleagues in Germany, England and at MIT, cells and tissues in mice made from cloned embryos are rejected by the body because of a previously unknown immune response to the cell’s mitochondria. These findings reveal a likely hurdle if such therapies are ever used in humans.

Regenerative therapies that utilize stem cells have the potential to repair organs, replace dead or dying tissues, and treat severe diseases.  Many stem cell scientists think that pluripotent stem cells, which can differentiate into any kind of cell in the body, show the most promise for regenerative medical applications in the clinic.  One method for deriving pluripotent stem cells that have the same genetic composition as that of the patients is called somatic cell nuclear transfer (SCNT) or cloning.  This method takes the nucleus of an adult cell and injects it into an egg cell from which the nucleus has been removed.

SCNT can potentially make pluripotent stem cells that can repair a patient’s body. “One attraction of SCNT has always been that the genetic identity of the new pluripotent cell would be the same as the patient’s, since the transplanted nucleus carries the patient’s DNA,” said cardiothoracic surgeon Sonja Schrepfer, MD, PhD, who was the co-senior author of the study, which was published online Nov. 20 in Cell Stem Cell.

“The hope has been that this would eliminate the problem of the patient’s immune system attacking the pluripotent cells as foreign tissue, which is a problem with most organs and tissues when they are transplanted from one patient to another,” added Schrepfer, a visiting scholar at Stanford’s Cardiovascular Institute, and Heisenberg Professor of the German Research Foundation at the University Heart Center in Hamburg, and at the German Center for Cardiovascular Research.

Several years ago, Stanford University professor of pathology and developmental biology, Irving Weissman, MD, chaired a National Academies panel on SCNT cells.  At this time, he raised the possibility that the immune system of a patient who received the cells derived from stem cells made from cloned embryos might still generate an immune response against proteins from the cells’ mitochondria.  Mitochondria are the energy factories for cells, and they have their own genetic system (a DNA chromosome, protein-making structures called ribosomes, and enzymes for expressing and replicating DNA).  This reaction could occur because cells created through SCNT contain mitochondria from the egg donor and not from the patient, and therefore could still appear as foreign tissue to the recipient’s immune system.

There were other indications that Weisman was probably correct.  An experiment that was published in 2002 by William Rideout in the laboratory of Rudolf Jaenisch at the Whitehead Institute for Biological Research in the journal Cell derived embryonic stem cells from cloned mouse embryos and then differentiated those embryonic stem cells into bone marrow-based blood making stem cells. These blood making stem cells were then used to reconstitute the bone marrow of a mouse that had a mutation that prevented their bone marrow from forming normal types of disease-fighting white blood cells. However, even though the recipient mouse was genetically identical to the embryonic stem cells that had been used to derived the blood-making stem cells, the immune systems of the recipient mouse still rejected the implanted cells after a time.  Weissman, however, was not able to directly test this claim himself at that time.  Weissman directs the Stanford Institute for Stem Cell Biology and Regenerative Medicine, and now, in collaboration with Schrepfer and her colleagues, he was able to test this hypothesis.

“There was a thought that because the mitochondria were on the inside of the cell, they would not be exposed to the host’s immune system,” Schrepfer said. “We found out that this was not the case.”

Schrepfer, who heads the Transplant and Stem Cell Immunobiology Laboratory in Hamburg, used cells that were created by transferring the nuclei of adult mouse cells into enucleated eggs cells from genetically different mice. When transplanted back into the nucleus donor strain, the cells were rejected although there were only two single nucleotide substitutions in the mitochondrial DNA of these SCNT-derived cells compared to that of the nucleus donor. “We were surprised to find that just two small differences in the mitochondrial DNA was enough to cause an immune reaction,” she said.

“We didn’t do the experiment in humans, but we assume the same sort of reaction could occur,” Schrepfer added.

Until recently, researchers were able to perform SCNT in many species, but not in humans.  However, scientists at the Oregon Health and Science University announced the successful derivation of human embryonic  stem cells from cloned, human embryos.  This reignited interest in eventually using SCNT for human therapies. Although many stem cell researchers are focused on a different method of creating pluripotent stem cells, called induced pluripotent stem cells, some believe that there are some applications for which SCNT-derived pluripotent cells are better suited.

The immunological reactions reported in the new paper will be a consideration if clinicians ever use SCNT-derived stem cells in human therapy, but Weissman thinks that such reactions should not prevent their use.  “This research informs us of the margin of safety that would be required if, in the distant future, we need to use SCNT to create pluripotent cells to produce the tissue stem cells to treat someone,” he said. “In that case, clinicians would likely be able to handle the immunological reaction using the immunosuppression methods that are currently available.”  I find such a statement somewhat cavalier given that the nature of the immunological rejection might be robust enough to endanger the patient regardless of the anti-rejection drugs that are used.

In the future, scientists might also lessen the immune reaction by using eggs from someone who is genetically similar to the recipient, such as a mother or sister, Schrepfer added.  Except that now you have added the dangers of egg retrieval to this treatment regimen, which not only greatly jacks up the price of this type of treatment, but now endangers another person just to treat this one patient.  Add to that the fact that you are making a cloned human embryo (a very young person) for the sole purpose of dismembering it, and now you have added a degree of barbarism to this treatment as well.

So if we some SCNT-based treatments for patients we have an added danger for the patient (immunological rejection), danger for the egg donor, the homicide of the young embryo, and a prohibitively expensive procedure that no insurance company in their right mind would fund. I say we abandon this mode of treatment for the morally-bankrupt option that it is and pursue more ethical ways of treating patients.

Embryonic Stem Cells From Cloned Embryos Vs Induced Pluripotent Stem Cells: Let the Debate Begin


In May of 2013, Shoukhrat Mitalipov and his coworkers from the Oregon Health and Science University, reported the derivation of human embryonic stem cells from cloned human embryos. Other stem cell scientists have confirmed that Mitalipov’s protocol works as well as he says it does.

Mitalipov and others have also examined the genetic integrity of embryonic stem cells made from cloned human embryos and induced pluripotent stem cells made from mature adult cells through genetic engineering and cell culture techniques. This paper was published in Nature in June 2014 and used genetically matched sets of human Embryonic Stem cells made from embryos donated from in vitro fertilization clinics, induced Pluripotent Stem cells and nuclear transfer ES cells (NT-ES cells) derived by somatic cell nuclear transfer (SCNT). All three of these sets of stem cells were subjected to genome-wide analyses. These analyses sowed that both NT-ES cells and iPS cells derived from the same somatic cells contained comparable numbers of genetic variations. However, DNA methylation, a form of DNA modification for regulatory purposes and gene expression profiles of NT-ES cells corresponded closely to those of IVF ES cells. However, the gene expression provide of iPS cells differed from these other two cell types and iPS cells also retained residual DNA methylation patterns typical of the parental somatic cells. From this study, Mitalipov stated that “human somatic cells can be faithfully reprogrammed to pluripotency by SCNT (that means cloning) and are therefore ideal for cell replacement therapies.”

Now a new study by Dieter Egli of the New York Stem Cell Foundation (NYSCF) in New York City, which included Mitalipov as a collaborator, has failed to demonstrate significant genetic differences between iPS cells and NT-ES cells. This is significant because Eglin has long been a rather vigorous proponent of cloning to make patient-specific stem cells. Egli gave an oral preview of his forthcoming paper on October 22nd, at the NYSCF annual conference. Egli told his audience, “This means that all of you who are working on iPS cells are probably working with cells that are actually very good. So I have good news for you,” he told them, eliciting murmurs and chuckles. “What this exactly means for the SCNT program, I don’t know yet.”

Egli and colleagues used skin cells from two people—a newborn and an adult—to create both stem cells from cloned embryos (using donor eggs) and iPS cells. Then they compared the genomes of these two types of cell lines with the genomes of the original skin cells in terms of genetic mutations, changes in gene expression, and differences in DNA methylation. Both methods resulted in about 10 mutations compared with the average genome of the mature source cells. These changes didn’t necessarily happen during reprogramming, however, Egli says, since many of these mutations were likely present in the original skin cells, and some could have arisen during the handling of cells before they were reprogrammed.

Both types of stem cells also carried a similar amount of methylation changes. Overall, the method didn’t seem to matter, Egli and his team concluded. Because he is a longtime proponent of SCNT, Egli says it would have been “more attractive” to reveal significant differences between the two kinds of stem cells. “This is simply not what we found.”

Now it would be premature to conclude that iPS cells are as good as NT-ES cells for regenerative purposes, but this certainly seems to throw a monkey wrench in the cloning bandwagon. Cloning would be quite complicated and expensive and also requires young, fertile women to donate their eggs. These egg donors must undergo potentially risky procedures to donate their eggs. Jennifer Lahl’s documentary Eggsploitation provides just a few of some of the horror stories that some women experienced donating their eggs. The long-term effects of this procedure is simply not known and asking young women to do this and potentially compromise their health or future fertility seems beyond the pale to me.

Alternatively, iPS technology keeps improving and may come to the clinic sooner than we think. Also, is a cloned embryo essentially different from one made through IVF or “the old-fashioned way.?” This whole things seems to me to involved the creation of very young human beings just so that we can dismember them and use them as spare parts. Such a practice is barbaric in the extreme.

For those who are interested, please see chapters 18 and 19 of my book The Stem Cell Epistles to read more about this important topic.

Mesenchymal Stem Cells Derived from Induced Pluripotent Stem Cells are Epigenetically Rejuvenated


Earlier this year, Miltalipov and his research group published a paper in Nature that compared the genetic integrity of embryonic stem cells made from embryos, to induced pluripotent stem cells and embryonic stem cells made from cloned embryos.  All three sets of stem cells seemed to have comparable numbers of mutations, but the induced pluripotent stem cells had “epigenetic changes” that were not found in either stem cell line from cloned or non-cloned embryos.

Genetic characteristics have to do with the sequence of the DNA molecules that make up the genome of an organism.  Epigenetic characteristics have nothing to do with the sequence of DNA, but instead are the result of small chemicals that are attached to the DNA molecule.  These small chemical tags affect gene expression patterns.  Every cell has a specific epigenetic signature.

During development, the cells that will form our eggs and sperm in our bodies, the “primordial germ cells,” begin their lives in the outer layer of the embryo.  During the third week of life, these primordial germ cells or PGCs move like amoebas and wander into the yolk sac wall and collect near the exit of a sac called the “allantois.”  The PGCs are outside the embryo at this time or extraembryonal.  Incidentallyyolk sac is a terrible name for this structure, since it does not produce yolk proteins.  Therefore other textbooks have renamed it the “primary umbilical vesicle,” which is a bit of a mouthful, but it probably better than “yolk sac.”

 

1 - Primordial germ cells 2 - Allantois 3 - Rectum 4 - Ectoderm 5 - Foregut 6 - Primordial Heart 7 - Secondary yolk sac 8 - Endoderm 9 - Mesoderm 10 - Amniotic cavity
1 – Primordial germ cells
2 – Allantois
3 – Rectum
4 – Ectoderm
5 – Foregut
6 – Primordial Heart
7 – Secondary yolk sac
8 – Endoderm
9 – Mesoderm
10 – Amniotic cavity

The embryo around this time undergoes a bending process as a result of its growth and the head bends toward the tail (known as the cranio-caudal curvature) and then the sides of the embryo fold downwards and eventually fuse (lateral folding).  This bending of the embryo allows the PGCs to wander back into the embryo again between the fourth and sixth week.  The PGCs move along the yolk sac wall to the vitelline and into the wall of the rectum.  After crossing the dorsal mesentery (which holds the developing intestines in place) they colonize the gonadal or genital ridge (which is the developing gonad). During their journey, and while in the gonadal ridge, the PGCs divide many times.

1 - Rectum 2 - Vitelline 3 - Allantois 4 - Nephrogenic cord (pink) 5 - Gonadal ridge (green) 6 - Primordial germ cells (red dots) 7 - Heart prominence
1 – Rectum
2 – Vitelline
3 – Allantois
4 – Nephrogenic cord (pink)
5 – Gonadal ridge (green)
6 – Primordial germ cells (red dots)
7 – Heart prominence

When the PGCs move into the developing gonad, the chemical tags on their DNA are completely removed (rather famous paper – Lee, et al., Development 129, 1807–1817 (2002).  This epigenetic erasure proceeds in order for the PGCs to develop into gametes and then received a gamete-specific set of epigenetic modifications.  These epigenetic modifications also extend to the proteins that package the DNA into chromosomes – proteins called histones.  Specific modifications of histone proteins and DNA lead to gamete-specific expression of genes.  Once fertilization occurs, and the embryological program is initiated, tissue-specific epigenetic modifications are conveyed onto the DNA and histones of particular cell populations.

This is a long-winded explanation, but because many cancer cells have abnormal epigenetic modifications, these epigenetic abnormalities in induced pluripotent stem cells (iPSCs) have been taken with some degree of seriousness.  Although, there is little evidence to date that links the cancer-causing capabilities of iPSCs with specific epigenetic modifications, although it certainly affects the ability of these cells to differentiate into various cell types.

A paper has just come from the laboratory of Wolfgang Wagner from the Aachen University Medical School, in Aachen, Germany that derived iPSCs from mesenchymal stem cells from human bone marrow, and then in a cool one-step procedure, differentiated these cells into mesenchymal stem cells (MSCs).  These  iPS-MSCs looked the same, and acted the same in cell culture as the parent MSCs, and had the same gene expression profiles as primary MSCs.  However, all age-related and tissue-specific epigenetic patterns had been erased by the reprogramming process.  This means that all the tissue-specific, senescence-associated, and age-related epigenetic patterns were erased during reprogramming.  Another feature of these iPS-MSCs is that they lacked but the ability to down-regulate the immune response, which is a major feature of MSCs.

Thus, this paper by the Wagner lab shows that MSCs derived from iPSCs are rejuvenated by the reprogramming process.  Also, the donor-specific epigenetic features are maintained, which was also discovered by Shao and others last year.  This suggests that epigenetic abnormalities are not an inherent property of the derivation of iPSCs, and that this feature is not an intractable characteristic of iPSCs derivation and may not prevent these cells from being successfully and safely used in the clinic.  However, this might be a cell type-specific phenomenon.  Also, the loss of the immune system regulatory capabilities of these iPS-MSCs is troubling and this requires further work.

iPS-MSCs

Chicken Induced Plurpotent Stem Cells Made With Minicircles


The safety of induced pluripotent stem cells (iPSCs) haws been debated in several studies and publications.  Original studies of the genetic differences between the cellular sources of iPSCs and the iPSCs derived from them tended to show a whole gaggle of new mutations that seemed to not appear in the original cells.  Therefore, several commentators warned about the “dark side of pluripotency.”. However, other studies that utilized higher-resolution techniques showers that many of these mutations that occurred in iPSCs did exist in the original cells before their reprogramming, but that these mutations occurred at low frequencies, but became amplified during the culturing of reprogrammed cells.

One feature that has received less attention in these discussions of the safety of iPSC derivation is that the method by which iPSCs are made has distinct consequences for the stem cells that are made.  Typically, methods that utilize gene vectors that do not integrate into the genomes of the host cells are inherently safer than those vectors that do integrate.  PiggyBac transposon vectors integrate, but self-excise soon after their integration, and, therefore, do not leave a trace or their previous integration.  Minicircles also do not integrate and tend to produce safer iPSCs.  For this reason, this present paper is of interest to us.

Franklin West and his colleagues at the University of Georgia have made chicken iPSCs using minicircles to reprogram adult cells.  West was interested in using iPSCs to make recombinant chickens, since chickens are a rather primary food source and major component of economic development in several countries.  Making transgenic or recombinant chickens by means of stem cell technology makes it possible to make animals with improved meat and egg production or disease resistance.

To this end, West and his group made chicken (c) iPSCs from skin fibroblast cells by means of a nonviral minicircle reprogramming method.  This resulted in ciPSCs that showed excellent stem cell appearance and expressed key stem cell marker genes (alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1).  These cells also showed very rapid growth in culture and expressed high levels of the enzyme telomerase, which is an enzyme that is vital for the maintenance of chromosomes.

When West and his research group transplanted late-passage ciPSCs into stage X chicken embryos, the cIPSCs successfully integrated into the growing embryo and contributed to tissues derived from all three primary germ layers (ectoderm, mesoderm, and endoderm).  These ciPSCs also contributed to the gonads, which means that the ciPSCs could make gametes that could contribute to the production of a new generation of chicken.

These ciPSCs provide an exciting new tool to create transgenic chickens and has broad and exciting implications for agricultural and transgenic animal fields at large.  However, it also demonstrates that iPSCs can be safely produced and used for agricultural purposes.  This means that if non-integration-based or non-viral-based techniques are used to make iPSCs it should be possible to make them safely for therapeutic purposes also.

Making Better Induced Pluripotent Stem Cells


On July 2nd of this year, a paper appeared in the journal Nature that performed complete genomic analyses of embryonic stem cells derived from embryos or cloned embryos, and induced pluripotent stem cells (iPSCs), which are made from reprogrammed adult cells.  They found that both embryonic stem cells made from cloned embryos and iPSCs derived from the same types of adult cells contained comparable numbers of newly introduced mutations.  However, when it came to the epigenetic modification of the genome (the small chemical tags attached to specific bases of DNA that gives the cell hints as to which genes to turn off), the epigenetic pattern of the embryonic stem cells made from cloned embryos more closely resembled that from embryonic stem cells.  The iPSCs still had some similarities with the adult cells from which they were derived whereas the embryonic stem cells made from cloned embryos were more completely reprogrammed.  From this the authors claimed that making embryonic stem cells by means of cloning is ideal for cell replacement therapies.

There is a big problem with this conclusion:  This was tried in animals and it did not work because of immunological rejection of the products from the stem cells.  For more information on this, see my book, The Stem Cell Epistles, chapter 18.

Despite this “bad news” for iPSCs, two recent papers have actually provided some good news for stem cells that can heal without destroying embryos.  The first paper comes from Timothy Nelson’s laboratory at the Mayo Clinic in Rochester, Minnesota.  Differentiation of iPSCs is, in some cases, rather efficient and the isolation procedures fail to effectively isolate the differentiated cells from potentially tumor-causing cells.  However, in other cases, the differentiation is inefficient and the isolation procedures are also rather poor, which leaves a large enough population of undifferentiated tumor-causing cells.

Nelson’ group has discovered that treating iPSCs and their derivatives with anti-cancer drugs like etoposide (a topoisomerase II inhibitor for those who are interested) increases engraftment efficiency and decreases the incidence of tumors.  My only problem with Nelson’s paper is that he and his colleagues used lentiviral vectors to make their iPSCs.  These vectors tend to produce iPSCs that are rather good at causing tumors.  I would have rather that he tried making iPSCs with other methods that do not leave permanent transgenes in the cells.  Nelson and his group transplanted their iPSC-derived cells into the hearts of mice where they could use high-resolution imaging to determine the number of cells that integrated into the heart and the presence of cell masses that were indicative of tumors.  None of the ectoposide-treated cell transplants caused tumors whereas 4 of the 5 transplants not treated with ectoposide caused tumors.  This paper appeared in Stem Cells and Development.

The second “good news” paper for iPSCs comes from Junji Takeda at the University of Osaka and Ken Igawa from the Tokyo Medical and Dental University, Japan.   In their paper from Stem Cells Translational Medicine, the Japanese groups collaborated to make iPSCs from skin based fibroblasts and then differentiate them into skin cells (keratinocytes).  However, they made the iPSCs in two different ways.  The first protocol utilized the piggyBac transposon system to make iPSCs.  The piggyBac system comes from moths, but it is highly active in mammalian cells.  It can deliver the genes to the cells, but the segment of DNA is then easily excised from the host cells without causing any mutations.  This system, therefore, will generate iPSCs that do not have any transgenes in them.  The second protocol used a system based on cytomegalovirus that leaves the transgenes in the cells but gradually inactivates their expression.

When these two types of iPSCs were compared, they seems to be essentially identical when grown in culture.  Thus in the pluripotent state, the cells were equivalent for the most part.  But once the iPSC lines were differentiated into skin cells, the transgene-free iPSCs formed skin cells that looked, behaved and had the same gene expression profile as normal human skin cells.  The transgene-containing iPSCs differentiated into skin cells, but they did not look quite like skin cells, did not have the same gene expression profile as normal human skin cells, and did not behave like normal human skin cells.

The moral of this story is that not all iPSC lines are created equally and the way you derive them is as important as the cell type from which they were derived.  Also, even incomplete differentiation does not need to be an obstacle for iPSCs, since the cancer-causing cells can be removed by means of specific drugs.  Finally, not all that glitters is gold.  Cloned embryos may give you stem cells that look more like embryonic stem cells, but so what.  These might still suffer from many of the same set backs.  Add to that the ethical problems with getting women to give up their eggs for research and cures (see Jennifer Lahl’s movie Eggsploitation for more disturbing information about that), and you have a losing combination.

Scientists Make Cloned Stem Cells from Adult Cells


For the first time, stem cell scientists have derived stem cells from cloned human embryos that were made from adult cells.  This brings them closer to developing patient-specific lines of cells that can be used to treat a whole host of human maladies, but at a cost.  This research was described in the April 17th online edition of the journal Cell Stem Cell.

In May of last year, Shoukhrat Mitalipov from the Oregon Health and Science University, reported the derivation of human embryonic stem cells from cloned human embryos.  However, these cloned were made using cells that came from infants.  Miltalipov worked out a new protocol for cloning human embryos by using nonhuman primate embryos, in particular those from a Rhesus monkey.

In this study, the donor cells came from two men, a 35-year-old and a 75-year-old.  By using the protocol developed by Mitalipov and his group, Robert Lanza, Young Gie Chung, and Dong Ryul Lee and their colleagues made personalized embryonic stem cells from these two men.

Stem cell biologist Paul Knoepfler, an associate professor at the University of California at Davis who runs the widely read Stem Cell Blog, called the new research “exciting, important, and technically convincing.”  He continued: “In theory you could use those stem cells to produce almost any kind of cell and give it back to a person as a therapy.”

In their paper, Young Gie Chung from the Research Institute for Stem Cell Research for CHA Health Systems in Los Angeles, Robert Lanza from Advanced Cell Technology in Marlborough, Mass., and their co-authors pointed out the potential promise of this technology for new regenerative therapies.  However, their work is also an important discovery for human cloning, since it shows that age-associated changes are not necessarily an impediment to SCNT-based nuclear reprogramming of human cells.

Even though it was the intent of Chung and others to gestate these cloned embryos to form cloned children, this work could be the first step toward creating a baby with the same genetic makeup as a donor.  Thus, this technology presents a so-called “dual-use dilemma.”

Marcy Darnovsky, executive director of the Berkeley, Calif.-based Center for Genetics and Society, explained that many technologies developed for good can be used in ways that the inventor may not have intended and may not like.

“This and every technical advance in cloning human tissue raises the possibility that somebody will use it to clone a human being, and that is a prospect everyone is against,” Darnovsky said.

This paper represents a collaboration between members of academic laboratories and industry.  Funding for this work came from a private medical foundation and South Korea’s Ministry of Science.

Technically, the somatic-cell nuclear transfer protocols used in paper are still somewhat inefficient.  Chung’s team had to attempt 39 times to produce only two blastocyst-stage embryos.  Their first attempts were complete failures, but when they modified the Mitalipov protocol and activated the cloned embryos 2 hours after fusion rather than 30 minutes after fusion, the embryos grew successfully.

“We have reaffirmed that it is possible to generate patient-specific stem cells using [this] technology,” Chung said.

Shoukhrat Mitalipov, director of the Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University, who developed the method that Chung’s group built upon, said that this work involves eggs that have not been fertilized.

“There will always be opposition to embryonic research, but the potential benefits are huge,” Mitalipov said.

Yes, there will be opposition to destructive research on embryos because they are the youngest among us.  No they do not have the right to vote, drive a car, or buy a hunting license, but they have the right to not be harmed.  To deny them that right because they cannot presently exercise particular capacities assumes that the embryo undergoes essential changes as it develops.  But human embryos develop into the kinds of entities they become because of their intrinsic human nature that drives them to do so.  Yes development is a progressive program that causes the embryo to acquire new structures and capabilities that it previously did not have, but what kind of entity can develop into a human adult that is not itself human?  It takes a human embryo to make a human fetus, which makes a human new-born baby, which makes a human toddler, and do on.  This continuum or development and change occurs throughout or lives and this continuum begins at the end of fertilization.

Cloned embryos begin this continuum at the completion of somatic cell nuclear transfer (SCNT).  SCNT works as a stand-in for fertilization, but the result is still the same – a human embryo.  It also should have the right not to be harmed, but instead she is being produced solely for the purpose of being dismembered.  Is this the way we should treat the smallest and most defenseless among us? surely not.  All this talk about, “well we did not form a fully human being” is a crock.  Yes you did.  You formed a fully formed human embryo.  We were all human embryos at one time and these embryos developed into you and me.  We were inarticulate and incapable at the time, but we gained those capacities over time.  Again, how can something that gives rise to a human child not be human?  The embryo is a human being, but it is a very young human being.  Youth should not disqualify it from being able to live.

Seventeen years ago, when Ian Wilmut from the Roslin Institute in Edinburgh, Scotland announced news about the birth of the first sheep cloned from somatic cells named Dolly, several legislators called for a ban on human cloning.  Several countries took measures to limit or outlaw such work, but in the United States.  The cloning issue was obfuscated by dividing it into “reproductive cloning” for the purposes of making cloned children, and “therapeutic cloning” for the development of new therapies.  Unfortunately, this dichotomy is slightly disingenuous since the techniques for both of these procedures are exactly the same except that reproductive cloning uses a surrogate mother to gestate the cloned embryo and bring her to term.  Both of these procedures produce human embryos, but one uses them to make a baby and the other destroys them before they can do so.

President George W. Bush tried to split the difference by restricting federal funding for stem cell research that harms to a human embryo.  This led to talk of Bush’s “embryonic stem cell ban,” which was inaccurate and was used unfairly used to paint Bush as an idiot.  However, some 15 states have laws addressing human cloning, and about half of them ban both reproductive and therapeutic cloning.

Embryonic stem cell research has typically used embryos that are left over from the fertility industry.  However, some religious groups such as the U.S. Conference of Catholic Bishops and others as well  objected to this, since it destroys a very young human being.

However, about seven years ago, Shinya Yamanaka and his colleagues discovered a way to make induced pluripotent stem cells from mature adult cells.  Genetic engineering techniques could convert ordinary cells into pluripotent stem cells without the need for human eggs.  While this technique did not present the same ethical issues, some induced pluripotent stem cells lines contain significant genetic abnormalities and there is still debate over how safe these cells are for clinical use.

The research conducted by Mitalipov and Chung provides a second way of producing pluripotent cells through laboratory techniques that is, in my view, far less ethical and will almost certainly also have unintended consequences as well.

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.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

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!!

The Archbishop of Denver Speaks Out: Cloning Kills the Smallest Among Us and the Next Victims WIll be Us


Samuel Aquila is archbishop of the Archdiocese of Denver, Colorado and has weighed in with regards to the cloning of human embryos. I am not a Roman Catholic, and Fr. Aquila is not a person whose religious authority I am obligated to accept de fide. Nevertheless, his stance on this subject is reasoned and was published on the National Review Online website here. It is well worth reading.

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.

One Embryo – Three Parents?


The web is alive with reports that scientists at the Oregon Health & Science University have managed to make embryos that contained genetic material from two mothers and one father. There has been a certain amount of “creepiness” applied to this experiment, but there are various reasons why this experiment was done. I will fully admit that there is a degree of creepiness to this experiment and the destruction of these embryos is also deplorable. However, this is a strategy to cure some genuinely nasty genetic diseases. Therefore, the research is not for nothing.

Deoxyribonucleic acid or DNA is the molecule all living organisms use to store genetic information, with the exception of some RNA viruses, but there is a debate as to whether or not viruses are actually alive. DNA is housed within the nucleus and is organized into linear molecules of DNA known as chromosomes.

However, there is another compartment in human cells that also houses DNA. The power-generation structure of the cell is called the mitochondrion. Mitochondria are enclosed by two membranes; and inner and outer mitochondrial membrane. There is also an internal network of membranes called cristae. Embedded in the membranes of the cristae are the components of the electron transport chain that are used for energy production.

Directly inside the mitochondrion is a soluble region known as the mitochondrial matrix. Soluble enzymes are found in the matrix as are metabolites and other small molecules. Another large molecule found in the mitochondrial matrix is the mitochondrial genome, which consists of multiple copies of small, circular molecule of DNA.

The mitochondrial genome encodes several genes necessary for the energy production machinery of the mitochondrion. The vast majority of the energy production machinery components are encoded by the nuclear genome, but the small number of mitochondrial components encoded by the mitochondrial genome are crucial for energy production.

Replication of the mitochondrial DNA is accomplished by a DNA replication system that is specific to the mitochondrion.  Unfortunately, this DNA replication system is less accurate than that used in the nucleus.  Therefore, mutations in mitochondrial DNA are relatively common.  Loss of function mutations in mitochondrial genes can compromise the ability of the mitochondrion to make chemical energy, and such mutations have dire consequences for several different organ systems.

The list of genetic diseases causes by mutations in mitochondrial DNA is long.  Here is a short list:

1.  Kearns-Sayre Syndrome – weakness or paralysis of the eye muscles, impaired eye movement and  drooping eyelids, loss of vision, abnormalities of the electrical signals that control the heartbeat, coordination and balance problems, abnormally high levels of protein in the fluid that surrounds and protects the brain and spinal cord, muscle weakness in their limbs, deafness, kidney problems, or a deterioration of cognitive functions (dementia). Affected individuals often have short stature and suffer from diabetes mellitus.

2.  Leber hereditary optic neuropathy – first sign is blurring and clouding of vision, and over time, vision worsens with a severe loss of sharpness and color vision.

3.  Leigh Syndrome – first signs are seen in infancy and are usually vomiting, diarrhea, and difficulty swallowing, eating problem, an inability to grow and gain weight at the expected rate, severe muscle and movement problems, weak muscle tone, involuntary muscle contractions, and problems with movement and balance, loss of sensation and weakness in the limbs.

4. MELAS – mitochondrial encephalomyopathy lactic acidosis, stroke-like episodes – signs and symptoms appear in childhood and may include muscle weakness and pain, recurrent headaches, loss of appetite, vomiting, and seizures. Stroke-like episodes beginning before age 40, and often involve temporary muscle weakness on one side of the body, altered consciousness, vision abnormalities, seizures, and severe headaches resembling migraines.  Strokes can progressively damage the brain, leading to vision loss, problems with movement, and a loss of intellectual function.

5.  MERRF – myoclonus epilepsy and ragged-red fibers – characterized by muscle twitches (myoclonus), weakness (myopathy), and progressive stiffness (spasticity).

6.  MILS – maternally inherited Leigh syndrome – a progressive brain disorder that usually appears in infancy or early childhood.  Affected children may experience vomiting, seizures, delayed development, muscle weakness, and problems with movement. Heart disease, kidney problems, and difficulty breathing can also occur in people with this disorder.

7.  Pearson Syndrome – a fatal disorder of infants with anemia and exocrine pancreatic insufficiency.  It is now known to be a rare, multisystemic, mitochondrial genetic disease, with anemia (low red blood cell count), neutropenia (low white blood cell count), and thrombocytopenia (low platelet count), as well as variable liver, kidney, and endocrine failure. Death usually occurs early in life.

8.  Progressive external ophthalmoplegia – Weakness of the eye muscles, drooping eyelids (ptosis), weakness or paralysis of the muscles that move the eye.  Affected individuals may also have general weakness of the skeletal muscles particularly in the neck, arms, or legs that may be especially noticeable during exercise.

9.  NARP – neuropathy, ataxia, retinitis pigmentosa – Beginning in childhood or early adulthood, numbness, tingling, or pain in the arms and legs; muscle weakness; and problems with balance and coordination; also vision loss learning disabilities, developmental delay, seizures, dementia, hearing loss, and cardiac conduction defects.

None of these diseases sounds terribly pleasant, and there are no known cures or effective treatments for them.

The severity of these diseases depends upon the proportion of the mitochondria that possess the mutated version of the mitochondrial genes.  Typically, mitochondria contain multiple copies of their genomes, and mutant versions of these genomes are mixed with normal copies.  When mitochondria divide, the copies of the genomes are randomly distributed between the two daughter mitochondria.  Therefore, some mitochondria will have mainly copies of the mutant version of the genome while others will have mainly copies of the normal version of the genome.  This condition is called heteroplasmy, and how widely these mutant versions are distributed throughout the body determines the severity of the mitochondrial genetic disease.

Mitochondria are inherited from the mother.  This is due to the fact that the egg, which is supplied by the mother, contains a large quantity of mitochondria, whereas the sperm that fertilizes the egg, only has relatively a few mitochondria.  Therefore, mitochondrial genetic diseases will only be transmitted through the mother, and if a mother is known to have a mitochondrial genetic disease, she will pass that disease onto her children, regardless of the health of the father.

This is the main reason for the technology tested in this paper: Masahito Tachibana, et al., Towards germline gene therapy of inherited mitochondrial diseases, Nature (2012) doi:10.1038/nature11647.  In this paper, scientists from the Division of Reproductive & Developmental Sciences at the Oregon National Primate Research Center in Oregon Health & Science University, used a technique that extracts the nuclear genome from the egg and transplants it into the egg of a donor, after which the egg is fertilized with normal sperm.  This technique would bypass the mitochondrial mutations in the mother’s eggs and replace that genome with a new genome that does not carry such a mutation.

The technique used in this paper is called “spindle transfer.”  This technique takes an oocyte donated by a woman who carries and suffers from a mitochondrial genetic disease and isolates and transplants the chromosomes (nuclear genetic material) from the patient’s unfertilized oocyte into the cytoplasm of another donated, enucleated egg, that contains healthy mtDNA as well as other organelles, RNA and proteins.  Such a child born a result of this spindle transfer procedure will be the genetic child of the patient but will carry healthy mitochondrial genes from the egg of the donor. Prior studies in a monkey model showed not only the feasibility of the spindle transfer (ST) procedure but also that ST is highly effective and completely compatible with normal fertilization and birth of healthy offspring (see Tachibana, M. et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372 (2009)).  This strategy might have an important future as a therapy to avoid transmission of serious mitochondrial diseases.

In this paper, seven volunteers (aged 21–32 years) donated a total of 106 mature eggs, and 65 eggs were used for the ST procedure and 33 served as non-manipulated controls.  Of the 64 ST eggs, 60 of them survived intracytoplasmic sperm injection (ICSI; 94%) and 44 formed showed the early signs of successful fertilization (73%).  These results were comparable to those found in the non-manipulated eggs; 32 oocytes survived ICSI (97%) and 24 (75%) formed pronuclei .  However, when these embryos were further observed, 48% (21/44) of the ST eggs were normal, but 87% of the non-manipulated embryos were normal.  Therefore, these manipulations can decrease the efficiency of fertilization.

If fertilization occurs normally, the ST embryos seem to be able to form blastocysts as well as the normal controls.  Blastocyst formation rate in the normally fertilized ST group (13/21, 62%) was statistically similar to controls (16/21, 76%).  Embryonic stem cell derivation rates were higher in the normal embryos (56%)  than in the ST group embryos (32%).

This paper uses an ingenious technique to potentially help women with a genetic disease.  That should give us some hope.  However, what I find reprehensible in this paper is the destruction of all these embryos.  These were young human lives that were snuffed out for the sake of convenience.

Wesley Smith at the Human Exceptionalism Blog has a different take on this technique.  Here are his words:  “Also note, that preventing illness is just the key that opens the door to many of these Brave New World technologies. Eventually–given the way things go these days–if the procedure ever becomes doable, it will go quickly from the “medical” to the “consumerist,” e.g., facilitating lifestyle choices and personal preferences.  That’s what happened with IVF, after all, which is no longer restricted to treating the infertile. Indeed, if we ever normalize polyamory, one could see the technique as a way for three partners to have biologially related children.”

Smith has a good point.  However, given the devastating nature of these mitochondrial genetic diseases, it seems to me that using this technique to prevent such horrific diseases from being passed on is a good thing.  However, we should certainly not let this technique be a license into another foray into experimental lifestyles.  Could we use this technique for medical purposes only?  Smith seems to think that the answer to this question is “No.”  I am certainly sympathetic to his caution, but I am also unwilling, at this point, to prevent mothers with these diseases from using this technology to have healthy babies that do not die at a young age.  If there is another way to purge such diseases from the mother’s eggs, then I am all ears, but for now this seems to be the best and only way.

Lab-Made Eggs Raise New Fertility Options


Katsuhiko Hayashi of Kyoto University is the lead author of a landmark paper that reports an achievement that has eluded scientists for decades.

In their most recent publication in Science magazine, Hayashi and his colleagues made mouse eggs from induced pluripotent stem cells in a culture dish, and then fertilized them with mouse sperm to create healthy, fertile mice.

This work is a continuation of reports published by the same core group of scientists at Kyoto University who made healthy mouse sperm in the lab from induced pluripotent stem cells and embryonic stem cells (K. Hayashi, H. Ohta, K. Kurimoto, S. Aramaki, M. Saitou, Cell 146, 519 (2011)). If this work can be applied to humans, it will revolutionize fertility treatments.

During the development of mammals, primordial germ cells (PGCs) become one of two cell types depending on the sex of the embryo. For example, if the embryo has an X and a Y chromosome, the PGCs differentiate into spermatozoa, but if the embryo is female and has two X chromosomes, they form oocytes. Sperm and eggs combine during sexual reproduction to form a single-celled embryo known as a zygote, and zygotes have the full developmental potential to grow into the adult animal.

In this paper, Hayashi used mouse embryonic stem cells and surrounded them with cells from the embryonic ovary. This creates a kind of “reconstituted ovary” which is then transplanted into a living mouse to develop. After being cultured in a mouse body for four weeks and four days, this culture system induced the embryonic stem cells to form PCG-like cells that went through all the stages of oocyte development. Fertilization of these oocytes produced by this reconstituted ovary system produced fertile, viable offspring. They also repeated this experiment with induced pluripotent stem cells and they successfully converted these stem cells into PGC-like cells that also underwent successful fertilization.

This experiment has already provided lots of fodder for bioethics bloggers all over the globe. Wesley Smith at his Human Exceptionalism blog at National Review has written the following:

“That mind-exploding point aside, the primary purpose for using this technique in humans would probably be to create mass egg quantities for use in cloning experiments. Each cloning attempt (using SCNT, the technique resulting in Dolly) requires a human egg. At present, human cloning has not been reported–primarily because of the “egg dearth” that inhibits researchers from the kind of repeated trial and error experiments necessary to perfect technique in humans.

Scientists probably need thousands of eggs to figure out human cloning, but they are in extremely short supply because the only sources currently are women of child-bearing age. Efforts are ongoing to remedy that problem–such as using eggs taken from the ovaries of aborted female fetuses or removed from women surgically. If the iPSC approach can be made to work in humans, there would be an infinite supply of eggs, meaning that human cloning would just be a matter of time.”

Smith is right on this one. Human cloning is being held back by its ridiculously low efficiency and the paucity of eggs for such research. Human cloning would be done for research purposes, but its main purpose would be to replace people who have died, or to make embryos or babies to are organ donors for sick adults.

A few years ago, there was a Michael Bay movie entitled “The Island” with Scarlett Johansson and Ewan McGregor. In this movie, McGregor and Johansson are part of a society that lives in a highly controlled environment in which they are told what to wear, what to eat, when to sleep, where to go, and what to do. The only hope they have is to win a supposed lottery that lets them go to “The Island.” Winners are announced on a daily basis, and when they are announced, they are never seen again. McGregor serendipitously discovers that they lottery winners do not go to the Island, but rather go to a surgical room where they are put to sleep and robbed of their vital organs.

McGregor returns to inform Johanssonof the elaborate ruse under which they are living just as Johansson is announced to be the recent winner of the lottery. They escape from the compound and are relentlessly pursued be those company that runs the facility where McGregor and Johansson were housed.

It turns out that McGregor and Johansson are clones of wealthy people who can afford to have a replica of themselves as “insurance policies.” The clones are known as “products” by the scientists who produce them, and the medical staff hardly thinks twice about dispatching each clone for their organs or do deliver a baby for the super-rich who do not want to go through the pain of childbirth.

In one scene, the CEO of the company that produces the clones makes a sales pitch to potential customers in which he speaks of an entity called an “agnate” that contains organs and stem cells for treatments, but has no consciousness or human structure. These patrons think that they are buying the rights to a blog that has their organs, when in fact, they are buying a clone that is a human person that was made by a manufacturing process and is genetically identical to them.

While I do not know the bioethical views of Michael Bay, his movie makes a remarkably telling case against human cloning. Cloning produces a human embryo. While it might have some developmental abnormalities, it is a human person. Farming cloned embryos for tissues is exactly the same as farming cloning human adults for body parts. The only differences are the size, age, and developmental stage of the human persons. Neither size, nor age, nor stage of development are adequate criteria for disqualifying someone from the human race. If this was the case, then six graders would be more human than fifth graders, tall people would be more human than short people, and two-year olds would be more human than one-year olds all of which are patently absurd.

If you are going to argue that the developmental abnormalities of cloned embryos should disqualify them, then you are saying that the less well endowed among us do not have the right to live, which puts you in the same ethical category as Adolph Hitler. People are people, and their identity is the same regardless of their deformities.

This research should give us pause. Human cloning should be banned regardless of whether it is called therapeutic or reproductive cloning. Both manipulate human beings and that is wrong.

Embryonic Stem Cells – Not all Genes are On


Early thinking about embryonic development and differentiation tended to view development as a matter of going from a cell with all kinds of genes on to progeny cells that have a host of these genes turned off and only a small subset of the original cache of genes turned on. If those genes were muscle-specific genes, then the cell became a muscle cell, and if they were nervous system-specific genes, then the cell became a neuron or glial cell.

Several different experiments questioned this conventional wisdom, and in particular, microarray experiments that allowed researchers to examine the gene expression pattern of the entire genome at a time showed that this was not the case. Instead of a host of genes being on in embryonic cells, a particular subset of genes were on, and as the embryo grew and aged, some cells shut one set of genes and turned on others, while a different group of cells turn off yet another set of genes and turned on a completely distinct set of genes.

With embryonic stem (ES) cells, the gene expression pattern depended on the culture system. Therefore, it was always difficult to interpret the results of such experiments.

This problem has now been largely solved, since Austin Smith at the Welcome Trust Stem Cell Institute in Cambridge (UK) has developed a culture system to standardize these conditions for embryonic stem cells. By employing this new methods, Hendrik Marks at the Nijmegen Centre for Molecular Sciences of the Radboud University Nijmegen, the Netherlands, showed that the ground state genes expression of embryonic stem cells is surprising.

There are only a few genes that are activated in embryonic stem cells. However, other genes that are not activated are not actively repressed. Instead that are ready to go and are in a kind of “on hold” status. The protooncogene (a gene that drives cells to divide and grow) c-myc, was thought to be essential for embryonic stem cell growth and division is hardly detectable.

This provides added clues as to how to keep ES cells as ES cells or how to drive them to differentiate into one cell type or another.

According to Marks, formerly researchers thought that “ES cells would subsequently differentiate by turning genes off that are not relevant for a specific specialization, to finally reach the correct combination of active genes for a particular specialization. We now see the opposite: genes are selectively turned on.”

The proteins that bind to DNA and direct gene expression, however, the so-called “epigenome,” are already prepared for action. Thus ES cells are poised to become one thing or another, and the environmental cues that they receive coaxe them into one differentiation pathway or another.

This finding also calls into question the work of Ronald Bailey who thinks that ES cell research is not immoral for the following reason: “So what about the claims that incipient therapies based on human embryonic stem cell research are immoral? That brings us to the question of whether the embryos from which stem cells are derived are persons. The answer: Only if every cell in your body is also a person.” Bailey continues: “Each skin cell, each neuron, each liver cell is potentially a person. All that’s lacking is the will and the application of the appropriate technology. Cloning technology like that which famously produced the Scottish sheep Dolly in 1997 could be applied to each of your cells to potentially produce babies.”

To support his claim, he quotes the Australian bioethicist Julian Savulescu from the 1999 Journal of Medical Ethics: “What happens when a skin cell turns into a totipotent stem cell [a cell capable of developing into a complete organism] is that a few of its genetic switches are turned on and others turned off. To say it doesn’t have the potential to be a human being until its nucleus is placed in the egg cytoplasm [i.e., cloning] is like saying my car does not have the potential to get me from Melbourne to Sydney unless the key is turned in the ignition.”

Savulescu is simply wrong. Many experiments have called this account of development into question, and now Marks’ experiments have placed the nail in the coffin. Furthermore, his analogy that Ta body cell does not have the “potential to be a human being until its nucleus is placed in the egg cytoplasm [i.e., cloning] is like saying my car does not have the potential to get me from Melbourne to Sydney unless the key is turned in the ignition,” is also flawed. The cell of our body are not undergoing development. Development is a process we know a great deal about, and our cells are not undergoing development. Embryos are undergoing development and they are unique human persons. Embryos give rise to our bodies. We are human persons and we began to assume our adult form when the embryo initiated development (i.e., at the termination of fertilization). Development also involves the hierarchical activation and inactivation of various genes. This is not a process that occurs in adult human bodies. Embryos are the beginning of a human person and they are human persons. Savulescu’s analogy would be more accurate if we say that the engine without the car would be unable to get him to Sydney, Australia: It needs a frame, tires and so on. They also all need to be properly connected and integrated with each other to work. His analogy is simply inaccurate and bogus.

Likewise, what Bailey calls “the application of the appropriate technology,” during a cloning experiment is the wholesale creation of a new human being. To say that this new human being is one of your cells is to woefully misunderstand the biological nature has happened during cloning. An egg from a female has its nucleus removed and is fused with a cell from another part of your body. After appropriate manipulation, the egg starts to divide and undergo embryonic development. Even this cell has the same genetic information as the cell from your body, it will not development into an exact duplicate of yourself. There are too many random events that occur during development that cause the individual to become a unique person who may have some similarities with their genetic parent, but will not resemble them completely. Cloning is not a minor manipulation – it is the creation of a new life, and this is a process that our cells are not going through; they are not developing. Therefore, they are not “potential persons.”

Secondly, the embryo is not a potential person, it is a very young human person.  It is a potential adult person, but it is a person nonetheless.

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.

Reprogramming human oocytes to a pluripotent stage – using triploid embryos


In the October 6, 2011 edition of the journal Nature, Scott Noggle from the New York Stem Cell Laboratory, and his collaborators from the University of San Diego and Columbia University have made a remarkable observation of cloned human embryos. When human embryos are cloned, an egg has its nucleus removed and replaced with a nucleus from a body cell. After stimulation, the egg divides and begins to recapitulate the stages of early embryonic development. This technique is often called “somatic cell nuclear transfer” or SCNT. Typically, cloned human embryos fail to develop for very long. They tend to die before they develop to the blastocyst stage, and they have massive abnormalities in gene expression. Noggle and colleagues found that if the egg nucleus is not removed, and the nucleus from the body cell is added to it, the cloned embryo develops to the blastocyst stage much more easily. Apparently, in human eggs, the removal of the nucleus conveys tremendous abnormalities upon the newly formed embryo, and prevent it from regularly developing to the blastocyst stage.  Noggle and his coworkers provide reasons for the development failure of cloned human embryos.

In this paper, Noggle and others made cloned human embryos through SCNT, but all of them stop dividing at the 6-10 cell stage.  They tried a few other experiments to determine if they could activate the egg to divide without causing developmental arrest.  First they fertilized 21 eggs with frozen sperm and 16 of these 21 eggs formed embryos that developed all the way to the spherical blastocyst stage after six days.  Because 76% of the eggs formed viable embryos, whatever problem afflicts the cloned embryos, it is not due to the quality of the eggs.  Next, the artificially activated unfertilized eggs with a molecule that let Calcium ions into the egg (called calcium ionophore).  7/52 (13.5%) of these artificially activated eggs divided and formed spherical blastocyst embryos.  Such embryos are called “parthenotes: because they were made without the benefit of fertilization and only have one copy of each chromosome (a condition that is called “haploid”).  This again confirms that the problem is not with the eggs.  To determine if developmental arrest was due to the removal of the egg’s nucleus, they removed nuclei from the eggs, and transferred egg nuclei from other eggs, and then artificially activated those eggs.  Once again, 1/7 (14.3%) of these eggs divided and formed spherical blastocysts.  In another control experiment, a nucleus from a body cell was fused with the egg, and the egg was then artificially activated, after which the body-cell nucleus was removed.  4/7  (57%) embryos developed to blastocysts after 6 days.  These control experiments suggest that the experimental manipulation the eggs are experiencing is not the cause of their developmental arrest.  Instead it is the absence of the egg nucleus that causes the developmental arrest of cloned human embryos.

To overcome the developmental arrest, they fused unmanipulated eggs with body cells and artificially activated them.  These, according to the authors, generated cells with three copies of each chromosome ( a triploid).  The problem is that they report using “MII stage oocytes.”  This is a fancy way of saying that the eggs are arrested in the last stage of meiosis.  This creates a problem:  the egg completes meiosis after fertilization and becomes a haploid cell.  The other nucleus is extruded in the form or a second polar body, which is a tiny bled of cell material attached to the egg.  Without completing meiosis, the egg has two copies of each chromosome and therefore the embryo should have four copies of each chromosome (tetraploid).  Nevertheless, they authors claim that they have generated triploid embryos, and their data support their conclusion.  They should explain more completely how they generated these cells, since the procedure they have detailed does not make complete biological sense.

Of these triploid embryos, 13/63 (20.6%) formed spherical blastocyst-stage embryos and these were used to make embryonic stem cells cultures.  From these 13 blastocysts, two embryonic stem cells lines were made.  These two lines (soPS1 and soPS2) were examined for their ability to form tumors in mice with sick immune systems.  Now only did they form the right kinds of tumors, but they expressed all the types of genes embryonic stem cell lines express.  Thus, triploid embryos can form blastocysts, and embryonic stem cell lines can be made from them.

Their gene expression studies showed that the cloned embryos failed to initiate the program of gene expression that is common observed in 4-8 cell stage human embryos.  This gene expression program, which is called “zygotic gene expression” is essential for further development, and the cloned embryos fail to properly initiate zygotic expression.

The News and Views commentary on this article by George Q. Daley suggests that triploid embryonic stem cell lines might be a potential patient-specific cell line for use in regenerative medicine.  I find this unlikely for several reasons.  Triploid embryos constitute 2-3% of all human conceptions (See D E McFadden and W P Robinson, “Phenotype of Triploid Embryos,” J Med Genet 43 (2006): 609-12), and is one of the major causes of spontaneous abortion (MR Creasy, JA Crolla, and ED Alberman, Hum Genet 1976; 31: 177–196; Kajii T, Niikawa N, Cytogenet Cell
Genet
1977; 18: 109–125; Brajenovic-Milic B, et al., Fetal Diagn Ther 1998; 13: 187–191).  Triploid result from either “digyny” (extra haploid set from mother) or “diandry” (extra haploid set from father).  Diandry tends to result in fetuses whose developmental growth is either mostly normal is or shows slow growth on one side of the fetus, but the placenta is abnormally large and filled with fluid-filled cysts.  This condition is given the formidable name of “partial hydatidiform mole,” (PHM) and it is potentially deleterious for the mother, since the huge placenta can become cancerous.  The digyny fetuses showed marked asymmetric growth in the uterus, and poor development of the adrenal hypoplasia.  The placenta is also small and underdeveloped.  The poorly developed placenta can, in some cases, or can cause preeclampsia in the mother, which is a life-threatening condition for a pregnant mother in which her blood pressure becomes dangerously high (see See Clasien van der Houwen, Tineke Schukken, and Mariëlle van Pampus, Journal of Medical Case Reports 2009, 3:7311).  Additionally, triploid fetuses may have a many other congenital anomalies that include fusion (syndactyly) of the third and fourth fingers and fusion f the toes, abnormal genitals, and cardiac, urinary tract, and brain anomalies.  These abnormalities  appear in both digynic and diandric triploids.  Given the poor developmental potential of triploid fetuses, it seems quite dangerous to use triploid embryonic stem cells for regenerative medicine, since they might cause more problems than healing.

My other problem with tis paper is that they paid women of reproductive age for their eggs.  In the first place this violates the guidelines of the National Academy of Science, which state: “Women who undergo hormonal induction to generate oocytes specifically for research purposes (such as for NT) should be reimbursed only for direct expenses incurred as a result of the procedure, as determined by an IRB. Direct expenses may include costs associated with travel, housing, child care, medical care, health insurance, and actual lost wages. No payments beyond reimbursements, cash or in-kind, should be provided for donating oocytes for research purposes. Similarly, no payments beyond reimbursements should be made for donations of sperm for research purposes or of somatic cells for use in NT.” (Final Report of the National Academies’ Human Embryonic Stem Cell Research Advisory Committee and 2010 Amendments to the National Academies’ Guidelines for Human Embryonic Stem Cell Research , Appendix C, Page 27 3.4(b) Payment and Reimbursement. See http://www.nap.edu/catalog.php?record_id=12923).  Yet Jan Helge Solbakk, faculty member at the Centre for Medical Ethics at the University of Oslo say that the authors “deserve praise rather than criticism, because their approach helps to draw attention to a possible way out of th regulatory quagmire resulting from reduction of oocyte providers to ‘donors’ or ‘gift givers’ deserving mere;y compensation for their gifts.” so, let’s praise the authors even though they broke the rules.  Folks, those rules are in place for a reason.  Luring young women to donate their eggs with money will tend to attract those who need the money; that is poor college students, or poor women.  These procedures have real risks, and women will close their eyes to the risks, because the egg donation will help them make payments.  This is exploitation of women, and Jennifer Lahl made a movie about it called “Eggsploitation” that documents what happens to when women are paid for their eggs.  Who speaks for them?  This is a very disturbing trend in this paper, and Nature should have had the backbone to reject it our of hand for that alone.  Having said, that, the paper does make some very original observations, but it is doubtful that these cell lines will plays a significant in regenerative medicine.  Also, the developmental arrest problem in cloned embryos is real, and this underscores why cloned mouse embryos are not that good a model system for cloned human embryos.