Differentiation of Induced Pluripotent Stem Cells Decreases Immune Response Against Them

The goal of regenerative medicine is to replace dead or damaged cells, tissues and even organs with living, properly functioning cells tissues and organs. However, this goal has a few genuine barriers that include tumor formation in the case of pluripotent stem cells, poor cell survival, or even immunological rejection of the transplanted cells before they can render any long-term benefits. Induced pluripotent stem cells (iPSCs), which are made from adult cells by a combination of genetic engineering and cell culture techniques, can be made from a patient’s own mature cells and the differentiated into almost any tissue in the adult body. However, research with mouse iPSCs has shown that even stem cells produced from the subject’s own tissues can be rejected by the subject’s own immune system.

Immune rejection of iPSCs is a legitimate concern, but research from the Stanford University School of Medicine has shown that differentiation of iPSCs into more mature cells before transplantation into mice allows them to be tolerated by the immune system.

Joseph Wu, MD, PhD, director of the Stanford Cardiovascular Institute, said, “Induced pluripotent stem cells have tremendous potential as a source for personalized cellular therapeutics for organ repair. This study shows that undifferentiated iPS cells are rejected by the immune system upon transplantation in the same recipient, but that fully differentiating these cells allows for acceptance and tolerance by the immune system without the need for immunosuppression.”

Wu is the senior author of this publication, which appeared online on May 30th in Nature Communications. Lead authorship of this paper is shared by Patricia Almeida, PhD, and Nigel Kooreman, MD, and assistant professor of medicine Everett Meyer, MD, PhD.

Several other studies have suggested that differentiation of iPSCs can reduce their tendency to activate the immune system after transplantation. However, this study of Wu and others is the first to closely examine, at the molecular and cellular level, how this works.

“We’ve demonstrated definitively that, once the cells are differentiated, the immune response to iPS-derived cells is indistinguishable from its response to unmodified tissue derived from elsewhere in the body,” said lead author Nigel Kooreman.

Pluripotent stem cells have the capacity to differentiate into any cell in the adult body. Of the two types of pluripotent stem cells, embryonic stem cells are made from embryos and iPSCs are made in the laboratory from existing adult cells (e.g., skin or blood). Induced pluripotent stem cells are easier to come by than embryonic stem cells, they match the genetic background of the person from whom they were obtained, and they are not as ethically dubious as embryonic stem cells. Thus, in theory, iPSCs are a good option for any physician who wants to make patient-specific stem cells for potential therapies.

Previous studies in mice have shown, however, that even genetically identicaliPSCs can trigger an immune response after transplantation. Thus, Wu and his colleagues have, for the past six years, been investigating how to use immunosuppressive medications to dampen the body’s response to both embryonic andiPSCs and render them more amenable for clinical use (see AS Lee, et al., J Biol Chem 2011 286(37):32697-704; Durruthy-Durruthy L, et al.,PLoS One, 2014 9(4):e94231 and others).

In this recent study, Kooreman and his co-lead authors decided to examine the immune response against transplanted stem cells. They first transplanted undifferentiated iPS cells into the leg muscles of genetically identical recipient mice. These grafts were rejected and no iPSCs were detected six weeks after transplantation.

Next, Wu and his co-workers differentiated the iPSCs into blood vessel-making endothelial cells that line the interior of the heart and blood vessels and then transplanted them into genetically-identical mice. Kooreman, Almeida, and Meyer then compared the acceptance by the immune system of these iPSC-derived endothelial cells with that of naturally occurring endothelial cells derived from the aortic lining of genetically-identical donor mice. To emphasize once again, all the transplanted cells were genetically identical to the mice in which they were injected. Unlike the undifferentiated iPS cells, both the iPS-derived endothelial cells and the aortic endothelial cells survived for at least nine weeks after transplantation.

Next, Wu and his group repeated the experiment, but they removed the grafts 15 days after transplantation. They observed immune cells called lymphocytes in all grafts, but these immune cells were much more prevalent in the grafts of undifferentiated iPS cells. When the lymphocytes that infiltrated the grafts of undifferentiated iPSCs were compared with those in the differentiated iPSC-derived grafts and the endothelial grafts, their gene expression profiles differed significantly. Those lymphocytes in the undifferentiated iPSC grafts expressed high levels of genes known to be involved in robust immune responses, but lymphocytes in both types of endothelial cell grafts expressed higher levels of genes known to be involved in dampening the immune response and inducing self-tolerance.

Finally, Wu and others directly examined a specific type of lymphocyte called a T cell. Grafts of undifferentiated iPS cells harbored large numbers of T cells that were largely homogeneous, which is characteristic of a robust immune response. Conversely, T cell from grafts of the two types of endothelial cells were more diverse, which suggests a more limited immune response which is typically associated with a phenomenon known as self-tolerance.

“The immune response to the iPS-derived endothelial cells and the aortic endothelial cells, and the longevity of the grafts, was very similar,” said Kooreman. “If we specifically look at the T cells, we see they’re also very similar and that they look much different from grafts that are rejected.”

Wu, who is also a professor of cardiovascular medicine and of radiology, said, “This study certainly makes us optimistic that differentiation — into any nonpluripotent cell type — will render iPS cells less recognizable to the immune system. We have more confidence that we can move toward clinical use of these cells in humans with less concern than we’ve previously had.”

Preventing Rejection of Embryonic Stem Cell-Based Tissues

Embryonic stem cells (ESCs) are derived from human embryos. Because they are pluripotent, or have the capacity to make any adult cell type, ESCs are thought to hold great promise for cell therapy as a source of differentiated cell types.

One main drawback to the use of ESCs in regenerative medicine is the rejection of ESC-derived cells by the immune system of the patient. Transplantation of ESC-derived tissues would require the patient to take powerful anti-rejection drugs, which tend to have a boatload of severe side effects.

However, a paper reports a strategy to circumvent rejection of ESC-derived cells. If these strategies prove workable, then they might clear the way to the use of ESCs in regenerative medicine.

The first paper comes from the journal Cell Stem Cell, by Zhili Rong, and others (Volume 14, Issue 1, 121-130, 2 January 2014). In this paper, Rong and his colleagues from the laboratory of Yang Xu at UC San Diego and their Chinese collaborators used mice whose immune systems had been reconstituted with a functional human immune system. These humanized mice mount a robust immune response against ESCs and any cells derived from ESCs.

In their next few experiments, Xu and others genetically engineered human ESCs to routinely express two proteins called CTLA4-Ig and PD-L1. Now this gets a little complicated, but stay with me. The protein known as CTLA4-Ig monkeys with particular cells of the immune system called T cells, and prevents those T cells from mounting an immune response against the cells that display this protein on their surfaces. The second protein, PD-L1, also targets T cells and when T cells bind to cells that have this protein on their surfaces, they are completely prevented from acting.

CTLA-4 mechanism

Think of it this way: T cells are the “detectives” of the immune system. When they find something fishy in the body (immunologically speaking), they get on their “cell phones” and call in the cavalry. However, when these detectives come upon these cells, their cell phones are inactivated, and their memories are wiped. The detectives wander away and then do not remember that they ever came across these cells.

Further experiments showed that any derivatives of these engineered ESCs, (teratomas, fibroblasts, and heart muscle cells) were completely tolerated by the immune system of these humanized mice.

This is a remarkable paper. However, I have a few questions. Genetic engineering of these cells might be potentially dangerous, depending upon how it was done, where in the genome the introduced genes insert, and how they are expressed. Secondly, if cells experience any mutations during the expansion of these cells, these mutations might cause the cells to be detected by the immune system. Third, do these types of immune repression last long-term? Clearly more work will need to be done, but these questions are potentially addressable.

My final concern is that if this procedure is used widespread, it might lead to the wholesale destruction of human embryos. Human embryos, however, are the youngest, weakest, and most vulnerable among us. What does that say about us if we do not value the weakest among us and dismember them for their cells? Would we allow this with toddlers?

Thus my interest and admiration for this paper is tempered by my concerns for human embryos.

Engineered Tissues for Transplantation

Xenotransplantation refers to the transplantation of organs from non-human animals into human patients. Such a procedure can increase the availability of organs for transplantation, but proteins and sugars on the surfaces of animal cells that are not found in human bodies can elicit an immune response against these xenotransplanted organs and tissues. For example, the human immune system recognizes a sugar molecule that coats the surface of pig blood vessels but is absent from human tissues called alpha-1,3-galactose (α-gal). In 2003, David Cooper, who runs the transplantation program at the University of Cape Town Medical School, engineered pigs without the α-1,3-galactosyltransferase gene that produces the α-gal residues. However, there were other problems with pig organs as well.

Tissue engineered organs are grown from a patient’s own cells. Such organs should help increase the availability of organs and avoid the problems of immune rejection that plague the field of xenotransplantation. “Cartilage, skin, and bone are already on the market. Blood vessels are in clinical trials. The progress has been really gratifying,” says Laura Niklason of Yale University.

Such engineered tissues consist of either flat planes or hollow tubes and are relatively simple to produce. Also, they consist of a small number of cell types. However, solid organs, such as the lungs, heart, liver, and kidneys, pose a greater challenge, since they are bigger and contain dozens of cell types. In addition, they have a complex architecture and an extensive network of the most essential component, which are the blood vessels. “Every cell needs to eat and breathe, and each one needs to be close to a source of nutrition and oxygen,” says Joseph Vacanti, who is in charge of the liver transplantation program at Boston Children’s Hospital in Massachusetts. Still, Vacanti is optimistic that it should be possible to produce even these complex organs through tissue engineering. “People differ about whether it’ll be achieved in 5 or 100 years, but most people in the field believe that it’s a realistic goal,” he says.

In 2008, Harald Ott of Massachusetts General Hospital and Doris Taylor of the University of Minnesota dramatically demonstrated the potential of organ engineering by growing a beating heart in the laboratory. These know first-hand, the need for organs for transplantation, since as physician-scientists, they often see patients who badly need transplants, but have no available organs for transplantation. To make engineered hearts, they began by using detergents to strip the cells from the hearts of dead rats. This left behind an extracellular matrix (a white, ghostly, heart-shaped frame of connective proteins such as collagen and laminin). Ott and Taylor used this matrix as a scaffold, and they seeded it with cells from newborn rats and incubated it in a bioreactor, which is a vat that provides cells with the right nutrients, and simulates blood flow. Four days later, the muscles of the newly formed heart began contracting, and after eight days, it started to beat.

This technique is extremely labor-intensive and is known as whole organ decellularization. Think of it as knocking down a house’s walls to reveal its frame, and then replastering it anew with different materials. Because the frame is of the same structure as the original organ and retains the complicated three-dimensional architecture of the organ which includes the branching network of blood vessels. Additionally, it also preserves the armamentarium of complex sugars and growth factors that covers the matrix and provides signaling signposts for growing cells. These signals will nudge the cells into the proper shapes and structures. “The matrix really is smart,” says Taylor. “If we put human cells on human heart matrix, they organize in remarkable ways. We can spend the next 20 years trying to understand what’s in a natural matrix and recreate that, or we can take advantage of the fact that nature’s put it together perfectly.”

Ott and Taylor’s groundbreaking feat of tissue engineering has since been duplicated for several other organs, including livers, lungs, and kidneys. Rodent versions of all have been grown in labs, and some have been successfully transplanted into animals. Recellularized organs have even found their way into human patients. Between 2008 and 2011, Paolo Macchiarini from the Karolinska Institute in Sweden fitted nine people with new tracheas. These tracheas were built from their own cells grown on decellularized scaffolds. Most of these operations were successful (although three of the scaffolds partially collapsed for unknown reasons after implantation). Decellularization has one big drawback: it still depends on having an existing organ, either from a donor or an animal. These disadvantages led Macchiarini to devise a different approach. In March 2011, he transplanted the first trachea built on an artificial, synthetic polymer scaffold. His patient was an Eritrean man named Andemariam Teklesenbet Beyene, who had advanced tracheal cancer and had been given 6 months to live. “He’s now doing well. He’s employed, and his family have [sic] come over from Eritrea. He has no need for immunosuppression and doesn’t take any drugs at all,” says Macchiarini. A few months later, he treated a second patient—an American named Christopher Lyles—in the same way, although Lyles later died for reasons unrelated to the transplantation.

Macchiarini now has gained approval from the US Food and Drug Administration to perform these transplants in the United States on a compassionate basis, for those patients who have no other options. “The final organ will never ever be as beautifully perfect as a natural organ,” says Macchiarini, “but the difference is that you don’t need a donation. It can be offered to a patient in need within days or weeks.” By contrast, even if a donor is found, a simple trachea can take a few months to regrow using a decellularized scaffold.

Other scientists have enjoyed similar success with other organs. In 1999, Anthony Atala of the Wake Forest Institute for Regenerative Medicine successfully grew bladders using artificial scaffolds. He subsequently transplanted them into seven children afflicted with spina bifida. By 2006, all the children had gained better urinary control. Atala has just completed Phase II trials of his artificial bladders.

Vacanti thinks that artificial scaffolds are the future of organ engineering, and the only way in which organs for transplantation could be mass-produced. “You should be able to make them on demand, with low-cost materials and manufacturing technologies,” he says. Such mass production is relatively simple for organs such as tracheas or bladders, since these are simply hollow tubes or sacs. Such tissue engineering is much more difficult for the lung or liver, which have much more complicated structures. However, Vacanti thinks it will be possible to simulate their architecture with computer models, and then fabricate them with modern printing technology, which uses inkjet technology to squirt stem cells unto three-dimension scaffolds that fit the size of the organ of interest. “They obey very ordered rules, so you can reduce it down to a series of algorithms, which can help you design them,” says Vacanti. However, Taylor says that even if the architecture is correct, the scaffold would still need to contain the right surface molecules to guide the growth of any added cells. “It seems a bit of an overkill when nature has already done the work for us,” she says.

Whether the scaffold used by tissue engineers are natural or artificial, clinicians need to seed it with patient’s cells. For bladders or tracheas, enough cells can be collected from the patient by means of a small biopsy. Unfortunately, this will not work if the organ is diseased, or if it is a complex structure composed of multiple tissue types, or, as in the heart, if its cells do not normally divide normally. In such cases, clinicians will need either stem cells, which can divide and differentiate into any cell type, or progenitor cells that are restricted to specific organs. Since 2006, one source of stem cells has been adult tissues, which scientists can now reprogram back into a stem-cell like state using just a handful of genes. Induced pluripotent stem cells or iPSCs, could then be coaxed to develop into a tissue of choice. “For me, the cells have always been the most difficult part,” says Vacanti, “and I’d say the iPSCs are the ideal solution.”