Induced Pluripotent Stem Cells – Addressing Safety Concerns


In 2012, John B. Gurdon and Shinya Yamanaka won the Nobel Prize in Physiology or Medicine “for the discovery that mature cells can be reprogrammed to become pluripotent.” Since that time, induced pluripotent stem cells (iPSCs) have largely taken the stem cell scene by storm. Because of the ease with which iPSCs can be made from just about any mature cell type, and because they can be made so more cheaply and faster than embryonic stem cells, they are the perfect pluripotent stem cell for laboratory use. The additional advantage to iPSCs is that can instantly reflect the genetic defect of the patient from whom they are made. Therefore, they are provide excellent model systems for a variety of genetic diseases and provide a kind of “disease in a dish” system by which the cellular and molecular characteristics of a disease can be modeled in cell culture.

In addition to their experimental utility, many scientists have sought to promote iPSCs for clinical purposes. However, before iPSCs can be used in the clinic, their safety must be established beyond question. Despite their success in many animal models (most in rodents), the long-term safety of iPSC derivatives has yet to be firmly demonstrated.

To that end, three different experiments have added to our concerns about the safety of iPSCs. For these and other reasons, several scientists have hypothesized that if iPSCs derivatives are going to be used in a clinical setting, they will need to come from young, healthy donors. In particular, blood cells from umbilical cord blood can be matched to just about any tissue and can be easily converted into iPSCs. Therefore, allogeneic iPSC derivatives seem to be the best way to go about treating particular diseases.

That being said, there are three studies about the safety of iPSC derivatives that make important contributions to the debate.

The first study comes from the laboratory of Shoukhrat Mitalipov at the Oregon Health and Science University. Mitalipov and his team have examined the mitochondrial genomes of iPSCs made from older patients.

Mitochondria are small, vesicles surrounded by two membranes, within cells that are the energy-production structures of most cells (not bacteria). Mitochondria also contain their own DNA molecules that express a variety of mitochondrial-specific genes and their own bacterial-like ribosomes that synthesized the mRNAs made from those genes into proteins. However, the vast majority of mitochondrial proteins are encoded on genes housed in the nucleus.

Mutations in genes encoded by the mitochondrial genome are rather devastating and are responsible for several really nasty (albeit rare) genetic diseases. These mitochondrial genetic diseases include: Mitochondrial myopathy, diabetes and deafness, Leber’s hereditary optic neuropathy (includes visual loss beginning in young adulthood, progressive loss of central vision due to degeneration of the optic nerves and retina), Leigh syndrome subacute sclerosing encephalopathy (disease usually begins late in the first year of life, although onset may occur in adulthood; a rapid decline in function occurs and is marked by seizures, altered states of consciousness, dementia, ventilatory failure), neuropathy, ataxia, retinitis pigmentosa, and ptosis (progressive symptoms as described and dementia), Myoneurogenic gastrointestinal encephalopathy (gastrointestinal pseudo-obstruction and neuropathy), Myoclonic epilepsy with ragged red fibers (progressive myoclonic epilepsy, “Ragged Red Fibers” or clumps of diseased mitochondria accumulate in the muscle fiber, short stature, hearing loss, lactic acidosis, exercise intolerance), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS).

Mitochondrial DNA mutations have long been thought to be a driving force in aging and age-onset diseases. Therefore, if iPSCs are made from older patients, will their starting cells have these mitochondrial mutations?

Taoseng Huang from Cincinnati Children’s Hospital Medical Center said: “If you want to use iPS cells in a human, you must check for mutations in the mitochondrial genome. Every single cell can be different. Two cells next to each other could have different mutations or different percentages of mutations.”

In this study from Mitalipov’s laboratory, his team derived and sequenced 10 iPS clones from each patient tissue sample to get a better understanding of mitochondrial DNA mutations rates. They took samples of blood and skin samples from healthy subjects and patients with degenerative diseases, who ranged in age from 24-72 years old. In these pools of these sampled cells, the rate of mitochondrial DNA mutations was low.

20 iPS cell lines per patient were profiled. Ten of these lines were derived from skin cells and the other 10 were derived from blood cells. Sequencing of the mitochondrial genomes of the iPSC lines revealed higher numbers of mitochondrial DNA mutations, particularly in cells from patients older than 60 years old. Of the 130 iPSC lines analyzed, 80 percent of them showed mitochondrial mutations and higher percentages of the mitochondria per cell contained mutations.

Such mitochondrial mutations can seriously compromise the ability of derivatives of these iPSC lines to carry out their desired function. Mitalipov in his paper, which was published in Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.2016.02.005, that all iPSC lines for use in human patients should be screened for mitochondrial mutations.

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One feature not addressed by Mitalipov and his colleagues is whether or not cells that may not show the signs of aging should be used to derived iPSCs, such as particular bone dormant marrow stem cells.

If mitochondrial mutations aren’t bad enough Jennifer E. Phillips-Cremins and her coworkers at the University of Pennsylvania School of Engineering and Applied Science have found that the chromatin structures of iPSCs might prevent them from properly differentiating into particular derivatives.

As previously mentioned in other blog posts, the DNA in the nuclei of our cells is packaged into a compact structure known as chromatin. Chromatin helps cells express those genes it needs to express and shut down other genes whose expression is not needed.

Occasionally, iPSC lines show an inability to differentiate into particular cell types while others have the ability to differentiate into many cell types. According to this study by Phillips-Cremins and her team, defects in DNA packaging might explain these disparities in iPSC lines.

By using experimental and computational techniques, Phillips-Cremins and her graduate student Jonathan Beagan identified chromatin conformations in a variety of iPSC lines. The DNA topology of embryonic stem cells and neural stem cells were also analyzed as comparisons.

“We know there is a link between the topology of the genome and gene expression,” Jennifer Phillips-Cremins, said in a press release. “So this motivated us to explore how the genetic material is reconfigured in three dimensions inside the nucleus during the reprogramming of mature brain cells to pluripotency. We found evidence for sophisticated configurations that differ in important ways between iPS cells and embryonic stem cells.”

The three-dimensional DNA conformations of pluripotent stem cells are reorganized during differentiation. Phillips-Cremin and others discovered that when mature cells are reprogrammed to pluripotent cells, most pluripotency genes reconnect to their enhancers (which are crucial for their expression). However, when these same iPSCs are differentiated into neural progenitor cells, the interactions between pluripotency gene and their enhancers remain in some lines, which should not occur.

“We found marked differences among the heatmaps we generated for each cell type,” said Jonathan Beagan, a graduate student in Phillips-Cremin’s laboratory at the University of Pennsylvania. “Our observations are important because they suggest that, if we can push the 3D genome conformation of cells that we are turning into IPSCs to be closer to that of embryonic stem cells, then we can possibly generate IPSCs that match gold-standard pluripotent stem cells more rapidly and efficiently.”

This paper was published in Cell Stem Cell (2016), 18(5): 611–624. Therefore, the chromatin structure of iPSCs is also important.

Finally, another paper reports some good news for iPSCs. Research from the Wellcome Trust Sanger Institute tracked the genetic mutations acquired by iPSCs when they are made in the laboratory. These cells came from the blood of a 57-year-old male subject.

This research, led by Allan Bradley, showed that mutations arise 10 times less often in iPSCs than they do in cultured laboratory-grown blood cells. Furthermore, non of the iPSC-acquired mutations were in genes known to cause cancer.

Bradley and his colleagues were able to trace the history of every mutation that each cell acquired from its extraction from the body to its reprogramming in the laboratory and propagation in culture.

The techniques utilized in the Bradley laboratory can surely help scientists evaluate the genetic integrity of laboratory-derived iPSCs.

This work was published in PLOS Genetics, 2016; 12(4): e1005932 DOI: 10.1371/journal.pgen.1005932.

All in all, it seems that it is possible to make sound iPSC lines, but those lines must be properly screened before they can be used in a clinical setting to treat live patients. These three papers provide new ways to screen iPSC lines for ensure high levels of safety and efficacy.

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.

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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.

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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.