Induced Pluripotent Stem Cells Slow to Grow Tumors in Monkeys

One of the major concerns that dogs the use of pluripotent stem cells in human clinical trials is the risk of tumor formation. Embryonic stem cells and induced pluripotent stem cells have an inherent ability to form special tumors known as teratomas. Teratomas are a rather strange group of tumors that develop from cells early in the developmental program of cells, before they have become committed to mature, adult cell types. Therefore, they contain a mixture of cell types organized to a greater or lesser extent into recognizable structures such as muscles or nerve tissue. In bizarre cases partial teeth may be found.

Embryonic stem cells and their derivatives have a distinct disadvantage in that they are rejected by the immune system of the patient. However, induced pluripotent stem cells (iPSCs), which are made from the patient’s own mature, adult cells, possess the same array of cell surface proteins as the patient’s own cells. Therefore, they are not rejected by the patient’s immune systems. Unfortunately, iPSCs can harbor cancer-causing mutations that were induced during the reprogramming process, and these mutations can seriously compromise their clinical usefulness and safety. Having, not all iPSC lines are the same. Some appear to be safer than others and screening methods that have been developed by stem cell scientists seem to be able to detect unsafe iPSC lines over others.

Now, a new study has shown that it takes a lot of effort to get iPSCs to form tumors after transplantation into a monkey. These findings will bolster the prospects of one day using iPSCs human patients.

Making iPSCs from an animal’s own skin cells and then transplanting them back into the creature also does not trigger an inflammatory response as long as the cells have first been differentiated into a more mature, specialized cell type.

“It’s important because the field is very controversial right now,” says Ashleigh Boyd, a stem-cell researcher at University College London, who was not involved in the work. “It is showing that the weight of evidence is pointing towards the fact that the cells won’t be rejected.”

Pluripotent stem cells have the ability to differentiate into many specialized cell types in culture. Therefore, they have been held out as potential sources of treatments for regenerative therapies for diseases such as Parkinson’s and some forms of diabetes and blindness. iPSCs, which are made by reprogramming adult cells, have an extra advantage because transplants made from them could be genetically matched to the recipient. Also, iPSC derivation is cheaper than cloning procedures and does not destroy a young embryo.

Globally, stem cells researchers are pursuing a variety of iPSCs-based therapies. For example, a group in Japan began enrolling patients for an iPSC-based human clinical trial last year. Experiments in mice from 2011 suggested that even genetically matched iPSCs can elicit an immune response, and pluripotent stem cells can also form slow-growing tumors. Both of these results have elicited deep safety concerns.

A stem-cell scientist from the National Institutes of Health in Bethesda, Maryland, named Cynthia Dunbar led this new study. She decided to evaluate both of these above-mentioned concerns in healthy rhesus macaques. The ability of pluripotent stem cells to form teratomas in laboratory mice is normally a test of their pluripotency. However, to prevent the immune systems of the mice from attacking and destroying these implanted stem cells, mice that lack the cell-mediated arm of the immune response are used. Such mice are called “nude” mice because they do have any hair.

Dunbar said, “We really wanted to set up a model that was closer to human. It was somewhat reassuring that in a normal monkey with a normal immune system you had to give a whole lot of immature cells to get any kind of tumor to grow, and they were very slow-growing.”

Dunbar and her team made iPSCs from skin and white blood cells from two rhesus macaques, and transplanted them back into the monkeys. She and her coworkers were careful to make sure that each monkey was injected with those iPSCs that had been derived from their own cells. For example, if monkey A provided cells that were used to derive iPSC cell line A1, then monkey A was only injected with iPSC line A1 cells and so on. Dunbar and others found that tumor formation required 20 times as many iPSCs as those needed for form a tumor in a nude mouse. These data are invaluable for safety assessments of potential iPSC-dependent therapies. Additionally, even though the injected iPSCs did trigger a mild immune response (white blood cells were attracted to the site of injection, which caused local but not systemic inflammation), when iPSCs were differentiated to a more mature cell types caused no such response.

Dunbar’s study is the first to examine the effects of transplanting undifferentiated iPSCs into the monkey they came from. However it is not the first primate study what happens when cells differentiated from iPSCs are transplanted into non-human primates. Scientists at Kyoto University in Japan transplanted monkey iPSCs that had been differentiated into dopaminergic neurons (the type of neuron that dies in Parkinson’s disease) into the brains of other monkeys and notes that these cells survived for months without forming tumors. Researchers at RIKEN in Kobe, Japan, observed similar results when they transplanted iPSCs that had been differentiated into retinal pigment epithelial cells, which support the photoreceptors at the back of the eye. In neither study did the implanted cells form tumors nor were they immunologically rejected when animals received their own cells. However, in both cases, the transplantation sites that were chosen tend to have a weak capacity to trigger immune responses.

In contrast, Dunbar differentiated iPSCs into bone precursor cells and placed them into small scaffolds just under the skin. Such a location can potentially elicit a robust immune response. However, the transplants did not cause irritation or inflammation, since the differentiated cells do not express embryonic proteins that are normally absent in mature tissues. By eight weeks, new bone had formed, and almost a year later no tumors had formed, and bone formation persisted.

The caveat to these studies is that some work has suggested that bone precursor cells can suppress the immune response against them. To circumvent this problem, Dunbar hopes to repeat these studies using iPSCs that have been differentiated into heart and liver cells.

Safe and Efficient Cell Reprogramming Inside a Living Animal

Research groups at the University of Manchester, and University College, London, UK, have developed a new technique for reprogramming adult cells into induced pluripotent stem cells that greatly reduces the risk of tumor formation.

Kostas Kostarelos, who is the principal investigator of the Nanomedicine Lab at the University of Manchester said that he and his colleagues have discovered a safe protocol for reprogramming adult cells into induced pluripotent stem cells (iPSCs). Because of their similarities to embryonic stem cells, many scientist hope that iPSCs are a viable to embryonic stem cells.

How did they do it? According to Kostarelos, “We have induced somatic cells within the liver of adult mice to transient behave as pluripotent stem cells,” said Kostarelos. “This was done by transfer for four specific gene, previously described by the Nobel-prize winning Shinya Yamanaka, without the use of viruses but simply plasmid DNA, a small circular, double-stranded piece of DNA used for manipulating gene expression in a cell.”

This technique does not use viruses, which was the technique of choice in Yamanaka’s research to get genes into cells. Viruses like the kind used by Yamanaka, can cause mutations in the cells. Kostarelos’ technique uses no viruses, and therefore, the mutagenic properties of viruses are not an issue.

Kostarelos continued, “One of the central dogmas of this emerging field is that in vivo implantation of (these stem) cells will lead to their uncontrolled differentiation and the formation of a tumor-like mass.”

However, Kostarelos and his team have determined that the technique they designed does not show this risk, unlike the virus-based methods.

“[This is the ] only experimental technique to report the in vivo reprogramming of adult somatic cells to plurpotentcy using nonviral, transient, rapid and safe methods,” said Kostarelos.

Since this approach uses circular plasmid DNA, the tumor risk is quite low, since plasmid DNA is rather short-lived under these conditions. Therefore, the risk of uncontrolled growth is rather low. While large volumes of plasmid DNA are required to reprogram these cells, the technique appears to be rather safe in laboratory animals.

Also, after a burst of expression of the reprogramming factors, the expression of these genes decreased after several days. Furthermore, the cells that were reprogrammed differentiated into the surrounding tissues (in this case, liver cells). There were no signs in any of the laboratory animals of tumors or liver dysfunction.

This is a remarkable proof-of-principle experiment that shows that reprogramming cells in a living body is fast and efficient and safe.

A great deal more work is necessary in order to show that such a technique can use useful for regenerative medicine, but it is certainly a glorious start.


Also involved in this paper were r, , and .