Induced Pluripotent Stem Cells Used to Make New Bone In Monkeys

Cynthia Dunbar, MD and her colleagues at the National Heart, Lung, and Blood Institute, which is a division of the National Institutes of Health (NIH) in Bethesda, Maryland have shown for the first time that it is possible to make new bone from induced pluripotent stem cells that are derived from a patient’s own skin cells.

This study, which was done in monkeys, shows that there is some risk that induced pluripotent stem cells (iPSCs) can form tumors, but that the risk of tumor formation is less than what was shown in immuno-compromised mice.

iPSCs are made from adult cells by means of a process called “reprogramming.” To reprogram adult cells, genetic engineering techniques are used to introduce specific genes into adult cells. These introduced genes drive the adult cells to de-differentiate into a less mature state, until they eventually become pluripotent, much like embryonic stem cells.

Originally, discovered by Nobel-prize winner Shinya Yamanaka, reprogramming was initially done with genetically engineered viruses that insert genes into the genome of cells. Even though these viruses do a passable job of reprogramming cells, they also introduce insertion mutations. Yamanaka and others originally used four transcription factors (Oct4, Sox2, Klf4, c-Myc) to reprogram adult cells. Several of these genes are overexpressed in a variety of tumors, and therefore, the use of these genes does create a risk of forming cells that overgrown and become tumorous. Secondly, The reprogramming process does put cells under the types of stresses that increase the mutation rate, and these mutations can also increase the risk of forming tumor cells. However, it is clear that not all reprogramming protocols cause the same rate of mutations, and that the mutation rate of iPSCs was originally overestimated. What is required is a good way to screen iPSC lines for mutations and for safety, especially since not all iPSC lines are equal when it comes to their safety.

The advantage of using iPSCs over embryonic stem cells is that the immune system of the patient should not reject tissues and cells made from iPSCs. This would eliminate the need for immune suppression drugs, which can be rather toxic.

Cynthis Dunbar from the National Heart, Lung, and Blood Institute said of her experiments, “We have been able to design an animal model for testing of pluripotent stem cell therapies using the rhesus macaque, a small monkey that is readily available and has been validated as being closely related physiologically to humans.

Dr. Dunbar continued: “We have used this model to demonstrate that tumor formation of a type called a ‘teratoma’ from undifferentiated autologous iPSCs does occur; however, tumor formation is very slow and requires large numbers of iPSCs given under very hospitable conditions. We have also shown that new bone can be produced from autologous iPSCs as a model for their possible clinical application.”

Dunbar and her team used a excisable polycistronic lentiviral vector called STEMCCA (Sommer et al., 2010) that expressed four genes: human OCT4, SOX2, MYC, and KLF4 to make iPSCs from skin cells. After they had derived culturable iPSCs from rhesus monkeys (made under feeder-free conditions), Dunbar and her group seeded them on ceramic scaffolds that are used by reconstructive surgeons to fill in or rebuild bone. Interestingly, these cells regrew bone in the monkeys.

The differentiated iPSCs formed no teratomas, but monkeys that had received implantations of undifferentiated iPSCs formed teratomas in a dose-specific manner.

Dunbar and her colleagues note that this approach might be beneficial for people with large congenital bone defects or other types of traumatic injuries. Having said that, it is doubtful that bone replacement therapies will be the first human iPSC-based treatment, since bone defects are not life-threatening, even though they can seriously compromise the quality of a patient’s life.

“A large animal preclinical model for the development of pluripotent or other high-risk/high-reward generative cell therapies is absolutely issues of tissue integration of homing, risk of tumor formation, and immunogenicity,” said Dunbar. “The testing of human-derived cells in vitro or in profoundly immunodeficient mice simply cannot model these crucial preclinical safety and efficiency issues.”

This NIH team is now collaborating with other labs to differentiate macaque iPSCs into liver, heart, and white blood cells for to test them for eventual pre-clinical trials in hepatitis C, heart failure, and chronic granulomatous disease, respectively.

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.