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.

A New Way to Mend Broken Hearts

Salk Institute researchers have discovered a way to heal injured hearts by reactivating long dormant molecular machinery found in the heart cells. This significant finding could open the door to new therapies for heart disorders in humans.

These new results were published in the November 6th, 2014 edition of the journal Cell Stem Cell. Although adult mammals don’t normally regenerate damaged tissue, they seem to retain a latent ability to do so. When the Salk team inhibited four different molecules that suppress genetic programs that lead to organ regeneration, they observed a dramatic improvement in heart regeneration and healing in laboratory mice.

These experiments provide proof-of-concept for a new type of clinical treatment in the fight against heart disease, which, according to the US Centers for Disease Control and Prevention, kills about 600,000 people each year in the United States alone.

“Organ regeneration is a fascinating phenomenon that seemingly recapitulates the processes observed during development. However, despite our current understanding of how embryogenesis and development proceeds, the mechanisms preventing regeneration in adult mammals have remained elusive,” says the study’s senior author Juan Carlos Izpisua Belmonte, holder of the Roger Guillemin Chair and primary investigator in the Gene Expression Laboratory and the Salk Institute.

We have within every cell of our bodies, the genes for organ regeneration. For several years, Izpisua Belmonte and his coworkers have attempted to clarify the genes that organism uses during embryonic development and during tissue healing highly regenerative organisms such as the zebrafish.

An injured zebrafish heart showing proliferating cells in the wounded area of the heart (red) and cardiac muscle cells (green).
An injured zebrafish heart showing proliferating cells in the wounded area of the heart (red) and cardiac muscle cells (green).

In 2003, Izpisua Belmonte’s laboratory first identified the signals that precede zebrafish heart regeneration, which they followed-up with a 2010 Nature paper, in which scientists from Izpisua Belmonte’s laboratory described how regeneration occurred in the zebrafish. Rather than stem cells invading injured heart tissue, the cardiac cells themselves reverted to a precursor-like state (a process called ‘dedifferentiation’). Dedifferentiation allowed the cells to proliferate within the damaged tissue.

n a dish, heart muscle cells return to a precursor-like state after pro-regenerative treatment with microRNA inhibitors. Green shows a disorganized cardiomyocyte cytoskeleton indicative of cell dedifferentiation; red shows mitochondrial organization.
In a dish, heart muscle cells return to a precursor-like state after pro-regenerative treatment with microRNA inhibitors. Green shows a disorganized cardiomyocyte cytoskeleton indicative of cell dedifferentiation; red shows mitochondrial organization.

They next determined if mammals retained any of the molecular players responsible for this kind of regenerative reprogramming. However, such an experiment came with some risks, recalls Ignacio Sancho-Martinez, a postdoctoral researcher in Izpisua Belmonte’s lab.

“When you speak about these things, the first thing that comes to peoples’ minds is that you’re crazy,” he says. “It’s a strange-sounding idea, since we associate regeneration with salamanders and fish, but not mammals.”

What are the things that cause a heart to regenerate in these smaller animals? Extensive work on the regenerating hearts of fish and salamanders failed to reveal anything concrete. Therefore, the laboratory changed its tack. “Instead, we thought, ‘If fish know how to do it, there must be something they can teach us about it,’” says the study’s first author Aitor Aguirre, a postdoctoral researcher in Izpisua Belmonte’s group.

The team focused on microRNAs, which control the expression of many genes. They used an extensive genetic screen for microRNAs that changed their expression levels during the healing of the zebrafish heart and that were found in the mammalian genome.

Their studies uncovered four molecules in particular–MiR-99, MiR-100, Let-7a and Let-7c–that fit their criteria. All were heavily repressed during heart injury in zebrafish and they were also present in rats, mice and humans.

However, in studies of mammalian cells in a culture dish and studies of living mice with heart damage, the group saw that the levels of these molecules were high in adults and failed to decline after the heart experienced injury. Therefore, Izpisua Belmonte’s team used adeno-associated viruses that could specifically infect the heart to target each of those four microRNAs and experimentally suppress their expressing levels.

Injecting these inhibitors into the hearts of mice that had suffered a heart attack triggered the regeneration of cardiac cells, and improved numerous physical and functional aspects of the heart, such as the thickness of its walls and its ability to pump blood. The scarring caused by the heart attack was significantly reduced with treatment compared to controls.

The improvements were still obvious three and six months after treatment–a long time in a mouse’s life. “The good thing is that the success was not limited to the short-term, which is quite common in cardiac regenerative biology,” Sancho-Martinez says.

The new study focused only on a handful of 70 some microRNA candidates that turned up in their initial screen. These other molecules might also play a part in heart cell proliferation, healing scars and promoting the formation of new blood vessels–all processes critical for heart repair, Sancho-Martinez says. The data are available so that other research groups can focus on molecules that interest them.

The next step for Izpisua Belmonte’s team is to move into larger animals and see whether “regenerative reprogramming” can work in larger hearts, and for extended periods after treatment, says Sancho-Martinez. And, although the virus packaging disappeared from the animals’ bodies by 2 weeks after treatment, the scientists are working on a new way to deliver the inhibitors to avoid the need for viruses altogether.

Human Amniotic Epithelial Cells Modulate Tooth Socket Restoration in Rats

Human amniotic epithelial cells have the capacity to differentiate into several different cell types. To that list, we can now add bone.

A study from Steve G.F. Shen at his colleagues at the Shanghai Jiao Tong University School of Medicine, Shanghai, China has used human amniotic epithelial cells to regenerate the tooth sockets in laboratory animals.

The first set of experiments examined the ability of human amniotic epithelial cells (hAECs) to form bone under controlled laboratory conditions. Then hAECs were loaded into artificial scaffolds that were then placed into the mouths of rats with tooth socket defects.

In culture, hAECs expressed bone-specific genes 10-14 days after induction. The cells also changed shape and made bone-specific proteins. When implanted into rat tooth sockets, the hAECs were embedded in a scaffold imbued with growth factors known to induce bone differentiation. These implants improved bone regeneration by directly participating in bone repair of the tooth socket defect. They also had an additional benefit in that they modulated the localized immune response against the implanted scaffolds. This immune response modulation augmented regeneration of the tooth sockets and allowed the implanted cells to get on with the job of fixing the surrounding bone without dealing with insults from the immune system.

This study has provided the first evidence that hAECs exhibit direct involvement in new bone regeneration and a localized modulatory influence on the early tissue remodeling process. These cells indirectly contributed to the bone-making process in the alveolar defect. Altogether, these results imply the potential clinical use of hAECs as an alternative stem cell-based for restoring tooth socket deformities.

Engineering Stem Cells to Fight Cancer

Advanced brain tumors are typically treated by surgical removal. However, it is difficult in the extreme to extirpate an entire tumor and therefore, tumor relapse is a perennial problem. A special group of small proteins known as ‘cytotoxic proteins” can target and destroy remaining cancer cells, but these proteins have short half-lives in the body and recent clinical trial called the PRECISE trial was not able to demonstrate that administered cytotoxic proteins had any efficacy against glioblastoma multiforme (GBM) brain tumors.

A new study, however, published online from the journal Stem Cells, a research group led by Khakid Shah from the Harvard Stem Cells Institute, have devised a new strategy designed around these engineered cytotoxic proteins has shown that neural stem cells (NSCs) can be genetically engineered to express these proteins and help treat GBM tumors.

So how did Shah and his colleagues design this novel strategy? They engineered NSCs to not only express specific cancer cell-killing toxins, but also have resistance to these toxins. Secondly, they designed cytotoxins that have the ability to enter cancer cells and target proteins known to be over-expressed by GBM tumors. Then these neural stem cells were encapsulated, they were transplanted into the space left after the bulk of the tumor was surgically removed.

In a mouse model of GMB, the implanted engineered stem cells survived and mediated an increase in long-term survival. This therapy was also effective against multiple patient-derived GBM cancer cell lines, which demonstrated their potential clinical relevance and applicability.

Shah and his coworkers want to bring these results to human trials within the next five years. They hope that this strategy can be successfully deployed in combination with surgical removal of the tumor mass. Shah also hopes that this approach can be adapted to treat other tumor types by using tissue-specific stem cells that express tumor-specific cytotoxins.

See Stuckey DW, Hingtgen SD, Karakas N, et al. Engineering toxin-resistant therapeutic stem cells to treat brain tumors. Stem Cells 2014.

Stem Cells in Breast Milk Might Help Baby Grow Up Strong

We have all probably heard about the benefits of breast milk for your baby as opposed to some other source of nutrition. This list of benefits is extensive: antibodies, better microfloral adaptation throughout the gastrointestinal tract, it helps you get your figure back, lower rates of illness in breast-fed babies, better for the environment, and so on. However, this long litany of benefits, and do not get me wrong, I am not knocking the benefits of breast-feeding, does not include one other benefit, and that includes a dose of breast-specific stem cells. Preliminary evidence has shown that mouse pups take in stem cells from their mother during breastfeeding, suggesting that the same thing might happen in humans.

Several years ago, it became clear that human breast milk contains a breast-specific kind of stem cells. This remarkable finding however did not answer the question of whether these cells coincidentally leaked into breast milk or they do anything useful with respect to the breast-feeding infant.

A presentation at the National Breastfeeding and Lactation Symposium in London last week presented data that suggests that, in mice at least, breast milk stem cells cross into the offspring’s blood from their stomach and play a functional role later in life.

Foteini Hassiotou from the University of Western Australia and her coworker used genetically modified mice whose cells contain a gene called tdTomato, which glows and intense shade of red under fluorescent light. The red-glowing females were mated and gave birth to mouse pups, but they were then presented with mouse pups from mothers who were genetically unmodified. Thus any red-glowing cells in these unmodified pups must have come to them from their mother’s milk.

When the these mouse pups that had been suckled by the tdTomato-expressing mothers grew to adulthood, assays of their tissues showed that red-glowing cells were found in their blood and the brain, thymus, pancreas, liver, spleen and kidneys. Hassiotou’s team also discovered that the breast-specific stem cells had differentiated into mature cells. Those red-glowing cells in the brain had the characteristic shape of neurons, those cells in the liver made the liver protein albumin, and those in the pancreas made insulin. According to Hassiotou, “They seem to integrate and become functional cells.”

What, precisely, is the role of these cells in the life of mice? Do they play a role in normal growth and development, or could they help to make the offspring tolerant to its mother’s cells and proteins, to reduce chances of an allergic reaction to her breast milk? “There must be some evolutionary advantage,” says Hassiotou.

According to Hassiotou, since her work and that of her colleagues clearly shows that these breast milk stem cells can differentiate into several different types of tissues makes it more likely they could be used for therapeutic applications. Chris Mason of University College London adds: “If these intriguing cells are functional, they could be a novel option for producing future cell therapies.”

Breast milk stem cells seem to have less capacity for unlimited cell division than embryonic stem cells. “But that’s actually a good thing,” says Hassiotou, because they do not form tumors when injected into mice. Therefore they may be less likely to trigger cancer if used to treat people.

Hassiotou points out that this kind of work cannot be done in humans, but she is planning to repeat it in a non-human primate species known as macaques.