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.

Gene Editing Does not Increase Mutation Rate in Stem Cells

Substituting one gene for another in cultured cells was once the stuff of science fiction, but with the ability to grow cells from our own bodies in culture and even convert them into embryonic-like stem cells, gene replacement has moved from the realms of science fiction to reality. However, the introduction of any all new technology comes with risks and trade-offs. In the case of gene replacement, there is the promise of fixing genes with mutations in them that cause genetic diseases. Unfortunately, any manipulation of the human genome runs the risk of adding new mutations to the genome whose side effects are unknown. Thus the cure might end up being worse than the disease itself.

New work from scientists at the Salk Institute in La Jolla, California has shown that new gene replacement techniques in stem cells does not increase the overall occurrence of mutations in those cultured cells. These new results were published the July 3 edition of the journal Cell Stem Cell.

“The ability to precisely modify the DNA of stem cells has greatly accelerated research on human diseases and cell therapy,” says senior author Juan Carlos Izpisua Belmonte, professor in Salk’s Gene Expression Laboratory. “To successfully translate this technology into the clinic, we first need to scrutinize the safety of these modified stem cells, such as their genome stability and mutational load.”

Introducing new genes into cells can occur by one of two methods. Engineered viruses can deliver new genes to a cell, which is then integrates the new DNA sequence in place of the old one. Alternatively, scientists can use custom targeted nucleases, such as TALEN proteins, which cut DNA at any desired location. Such proteins will extirpate the gene that needs to be replaced and then the new (potentially improved version of the gene) is simply added to the mix. The cell’s natural repair mechanisms will paste the new gene in place.

Belmonte’s lab has pioneered the use of modified viruses known as helper-dependent adenoviral vectors (HDAdVs) to fix genetic mutations that cause sickle-cell anemia. Sickle cell anemia is one of the most severe blood diseases found in the world. Belmonte and his collaborators have used HDAdVs to replace the mutant version of the globin gene in a stem cell line with a mutant-free version. This generated stem cells that could be theoretically be infused into patients’ bone marrow where they would create healthy blood cells.

Before such technologies are applied to humans, though, researchers must ascertain the risks of editing genes in stem cells. Even though both common gene-editing techniques have been shown to be accurate at altering the right stretch of DNA, concerns remain that the editing process could make the cells more unstable and prone to mutations in unrelated genes.

“As cells are being reprogrammed into stem cells, they tend to accumulate many mutations,” says Mo Li, a postdoctoral fellow in Belmonte’s lab and an author of the new paper. “So people naturally worry that any process you perform with these cells in vitro—including gene editing—might generate even more mutations.”

To test the safety of gene editing techniques, Belmonte’s research group, collaborated with BGI and the Institute of Biophysics, Chinese Academy of Sciences in China. They originally used a stem cell line that contains mutations in the beta-globin gene, which cause sickle-cell anemia. Belmonte then used HDAdV to edit the beta-globin genes of some cells, and edited the beta-globin genes of other cells by means of one of two TALEN proteins. Other cells were grown without any gene editing. Then, with the help of their Chinese collaborators, they fully sequenced the entire genome of each cell from the four edits and control experiment.

While all of the cells gained a low level of random gene mutations during the experiments, the cells that had undergone gene-editing—whether through HDAdV—or TALEN-based approaches—had no more mutations than the cells kept in culture.

“We were pleasantly surprised by the results,” Keiichiro Suzuki, a postdoctoral fellow in Belmonte’s lab and an author of the study, says. “People have found thousands of mutations introduced during iPSC reprogramming. We found less than a hundred single nucleotide variants in all cases.”

According to Li, this does not necessarily mean that there are no inherent risks to using stem cells with edited genes. However, it does mean that the editing process does not make stem cells that have undergone gene replacement are any less safe.

“We concluded that the risk of mutation isn’t inherently connected to gene editing,” he says. “These cells present the same risks as using any other cells manipulated for cell or gene therapy.” He adds that two other papers published in the same issue support their results (one by Johns Hopkins University and one from Harvard University and collaborators).

The next step for the Belmonte group is to determine if gene-repair in other cell types might be more likely to increase the mutation rate or if targeting other genes can cause unwanted mutations. They also hope that their findings will encourage those in the field to keep pursuing gene-editing techniques as a potential way to treat genetic diseases in the future.

Myriad Genetics Hordes Breast Cancer Data

Kathleen Sloan the president of the National Organization of Women has a troubling article at the Center for Bioethics and Culture website. It tells the story of a biotechnology company called Myriad Genetics and it BRCA1 & 2 test.

What the heck is BRCA1 & 2?  BRCA stands for “breast cancer” and mutations in BRCA 1 or 2 predispose females to breast and ovarian cancer. Mutations in BRCA genes also increase the risk of colon, prostate and pancreatic cancer.  Approximately 7% of breast cancer and 11 – 15% of ovarian cancer cases are caused by mutations in the BRCA genes.  If someone carries a mutation in either BRCA 1 or 2, they have a syndrome called Hereditary Breast and Ovarian Cancer (HBOC) syndrome.

The BRCA genes encode proteins that help repair DNA when it is damaged. Even though BRCA 1 & 2 work with several other proteins to accomplish this repair, mutations in the BRCA genes that compromise the quality of the proteins they encode can diminish the ability of cells to repair their DNA. Loss of efficient DNA repair systems leads to greater numbers of mutations in cells, some of which cause either loss of tumor suppress genes that normally put the brakes of cell proliferation, or activation of proto-oncogenes, which encode proteins that promote cell proliferation. Loss of tumor suppressor genes and activation of proto-oncogenes produces a cancer cell, and mutations in BRCA 1 or 2 and accelerate the onset of cancer cell formation (this is a highly simplified explanation and I apologize to the aficionados out there, but I am trying put the cookies on a nice low shelf).

Myriad Genetics came along and developed a genetic test for cancer-causing mutations in BRCA 1 & 2. This is good news, but Myriad Genetics is presently with holding their data from patients. This is not good news. Myriad Genetics wants to generate a database of mutations found in BRCA 1 and 2 genes from women all over the world. Some of these mutations do not affect the function of the encoded protein and do not predispose the patient to breast cancer, but some do. Which ones are harmful and which ones are not?

At this point things get sticky. Myriad has complied its sequence data on BRCA in order to construct a “variants of unknown significance” or VUS. Such a compilation would be invaluable, since it would help physicians correctly interpret the results of a breast cancer test. According to its present data archive, Myriad Genetics claims that only 3% of its tests fall into the VUS unknown category. However, other testing services report a 20% VUS rate. Who’s right? hard to say, given that Myriad Genetics will not release its data. Apparently they feel that their data has commercial value.

The problem is that lots of outfits that provided data to Myriad Genetics free of charge in order for them to develop their test. These other outfits have all their data available on public databases. What about Myriad Genetics – nope.

According to Ms. Sloan, “Myriad Genetics, producer of the world’s biggest-selling gene test for breast and ovarian cancers, has become synonymous with corporate greed. In an egregious breach of bioethics, the company refuses to share groundbreaking knowledge that could benefit cancer patients.”

Myriad worked hard to develop this test – I do not think anyone is contesting that. Myriad Genetics has every right to make money off their test, but when they start hoarding potentially life-saving data, I think Ms. Sloan is right that they have crossed the line.

Myriad Genetics is also being sued because of their attempts to patent the BRCA genes. An impressive consortium of researchers, genetic counselors, women patients, cancer survivors, breast cancer and women’s health groups, and scientific associations representing 150,000 geneticists, pathologists and laboratory professionals are all plaintiffs in this lawsuit against the U.S. Patent Office, Myriad Genetics and the University of Utah Research Foundation, which hold the patents on the genes.

The lawsuit avers that patents on human genes violate the First Amendment because genes are “products of nature.” Therefore, such things cannot be patented. Such an argument has a strong intuitive appeal, and is almost certainly correct.

Read Ms. Sloan’s article here and see what you think.