Inducing Translation (Nature Biotechnology)

June, 2013

The field of induced pluripotent stem cells is barely seven years old, but already three groups are close to launching clinical trials. Ken Garber looks into the prospects and perils.

Last December, Advanced Cell Technology (ACT) of Marlborough, Massachusetts, made its initial filing with the US Food and Drug Administration (FDA) for a proposed clinical trial using platelets derived from induced pluripotent stem (iPS) cells. If approved, it could be the first clinical trial involving iPS cells, which are reprogrammed adult cells, typically fibroblasts or hematopoietic precursors. But other groups are not far behind. Researchers at Stanford University in Palo Alto, California, have also met with the FDA about a proposed trial of genetically corrected keratinocytes to treat epidermolysis bullosa, a rare skin disease. And ophthalmologist and stem cell biologist Masayo Takahashi, of the RIKEN Center for Developmental Biology in Kobe, Japan, is awaiting final government approval for a trial of iPS cell–derived retinal pigment epithelium (RPE) to treat the wet form of age-related macular degeneration (AMD).

Each of these efforts faces obstacles and dangers that are particular to the pathology of the chosen disease and the nature of the replacement cells used to treat it. And beyond those perils loom the unknowns that accompany any venture into completely new medical territory. Nevertheless, these groups and others (Table 1) are already seriously bidding to make the leap into humans.


Clinical success would be much more than a technological stunt for the field because iPS cells offer vital and unique capabilities. Compared with human embryonic stem (ES) cells, “iPS cells just make such tremendous sense as a therapy,” says Sue O’Shea, a stem cell biologist at the University of Michigan in Ann Arbor. Sourcing the cells is one big reason. “You’re not going to be able to get ES cells from me or you,” she notes.

Similarly, iPS cells have theoretical advantages over adult stem cells as therapies. They are a potentially unlimited source of proliferating cells, in contrast to the limited donor cell availability and the impaired proliferation capacity of many types of adult stem cells.  And giving back to patients their own cells, thus avoiding immune responses, could solve immune rejection—the most vexing problem in transplantation medicine. “You can imagine personalized medicine, where you take a pluripotent cell but you make it from that same individual,” says Mahendra Rao, director of the Center for Regenerative Medicine at the US National Institutes of Health. “You solve both the source issue and you likely have solved the immune issue.”

…and minuses

But stem cell therapy faces several technical limitations. Replacing neurons in the brain, for example, would require that they connect, often over long distances, to form proper neural circuits and networks, something very unlikely to happen spontaneously. Glial cell transplantation avoids this problem, but most common brain diseases involve neurons. Another problem has been the differentiation of mature cell subtypes from iPS cells. For example, heart researchers are still struggling to derive homogeneous populations of atrial, ventricular and nodal cardiomyocyte subtypes. And getting sufficient cell quantities remains a hurdle for blood cell replacement therapy. “I don’t see how it could be cost effective or scalable without some new major breakthrough,” says Rao.

In terms of the safety of the cells themselves, iPS cell–derived cells appear to look and act remarkably like the natural cell types they are meant to replace, but there are a growing number of documented changes caused by reprogramming. For example, some studies show a high mutation rate in iPS cells, and they may also be more likely to acquire chromosomal aberrations, as well as gene duplications and deletions.

There are other concerns. “The process of iPS cell generation is still sort of a brute force process,” says Rao. “We use four different genes, we expose [cells] to high levels of these things, and then somehow we think that the cells will figure out how to adapt.” To a remarkable extent, they do, but iPS cells are prone to “epigenetic memory”—the failure to reprogram cell methylation patterns—and to other epigenetic aberrations1. The extent to which epigenetic memory is an issue remains controversial. For example, Alex Meissner, a stem cell biologist at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts , writes in an e-mail that epigenetic memory at a given locus affects some iPS cell lines but not others, tends to diminish over multiple passages and “is not a big issue or concern.”

Another worry is that female iPS cells undergo a change in X chromosome inactivation. Normally, one of the two X chromosomes is randomly inactivated in each cell in the body. There is evidence that iPS cells suffer an erosion of such X chromosome inactivation over time, thus disrupting dosage compensation, the alignment of X chromosome products between male and female2. And iPS cells appear to favor activation of one X chromosome over the other during reprogramming3. Such skewing is a feature of several X-linked diseases.

The fact that reprogrammed iPS cells do not go through the fertilization process like ES cells could also be a problem.   The resulting differences in telomere maintenance, mitochondrial content, and imprinting, says Rao, may or may not cause problems for iPSC-derived cells.

Then there’s cancer. The reported genetic mutations caused by reprogramming are enriched in genes that play a role in cancer and the copy number variants seen tend to be duplications of oncogenes and deletions of tumor suppressor genes—neither of which is a good thing. In addition, no one knows whether cells, once transplanted into the target organ, will fully adapt to their new environment. “The worst scenario is they drift…into something that’s more phenotypically immature and more susceptible to forming a tumor,” says Stanford cardiologist and stem cell biologist Joseph Wu.

Besides the theoretical cancer risk accompanying reprogramming, some undifferentiated cells may contaminate the cell preparations used for transplantation and give rise to teratomas or teratocarcinomas. Teratomas are disorganized tumors containing differentiated cells from all three somatic lineages. Teratocarcinomas contain persisting undifferentiated stem cells and are highly malignant. As differentiation protocols improve, such tumors are becoming less of a concern, says Wu. Nevertheless, the combined malignancy risk from mutation events and residual undifferentiated cells is influencing the translational choices that researchers are making.

Second skin

One example is the Stanford effort to use iPS cell–derived keratinocytes to treat recessive dystrophic epidermolysis bullosa (RDEB). There are perhaps 500 people in the US with this devastating blistering disease of the skin. Many don’t survive past their teen years, and those who do must cope with widespread chronic wounds that lead to disfigurement, notably of the hands, and difficulty eating due to mouth and esophageal blistering and scarring. In late 2009, Stanford dermatologists Alfred Lane and Anthony Oro, with stem cell biologist Marius Wernig, received a four-year, $11.7-million grant from the California Institute for Regenerative Medicine in San Francisco to develop an FDA-approved clinical trial by mid-2014.

It’s an ambitious goal, since it requires overcoming   loss-of-function mutations in the type VII collagen gene, COL7A1, that cause RDEB. Because type VII collagen helps form the anchoring fibrils that connect the epidermis to the underlying dermis, its loss leads to the separation of these layers at the slightest trauma, causing blisters and, eventually, open wounds and massive scars. The Stanford group proposes to biopsy the patient’s own fibroblasts, reprogram them into iPS cells,  genetically correct the type VII collagen defect, then differentiate these cells into keratinocytes, the main collagen-producing cells. Then they’ll fashion these keratinocytes into skin grafts, similar to Genzyme’s Epicell grafts now used to treat burns, and graft them onto affected areas on patients’ bodies.

Because the protocol involves ex vivo gene therapy as well as cell transplantation, the regulatory hurdles seem high, but Lane is optimistic that the group can reach the clinic by the mid-2014 deadline. “People are critical of the FDA, but we’ve found that they recognize the severity of this disease,” he says.

Although the group had not published preclinical data as of early May, Lane says graft experiments in a severe combined immunodeficient (SCID) mouse model have shown that the genetically corrected cells can grow and develop skin that has normal type VII collagen, and produce normal anchoring fibrils. The FDA, he adds, has accepted this model.

But regulators are, obviously, concerned about undifferentiated cells contaminating the keratinocyte grafts and causing cancer. “That’s a big question the FDA has for us,” says Lane, who hopes to eventually put the concern to rest. “We think that if you have cultured keratinocytes from iPS cells, and you had iPS cells mixed in with them, that the iPS cells in the graft are just going to fall right off [and die], because they won’t have the natural keratinocyte adhesion characteristics,” he says. This remains to be proven, however. “That’s something we’re working on,” says Lane.

Cancer concerns also influenced the choice of fibroblasts as the starting cells to be reprogrammed. Keratinocytes are a more obvious cell type from which to make more keratinocytes. But because they form the outermost layers of the skin, and are directly exposed to ultraviolet radiation from the sun, keratinocytes appear to undergo a higher rate of spontaneous mutation than fibroblasts. So starting with fibroblasts somewhat dampens the cancer risk.

RDEB patients are already at an extremely high risk of developing a very aggressive form of squamous cell carcinoma. Stem cell therapy could heighten that risk. The Stanford group will be using a method based on homologous recombination to correct the COL7A1 mutations, thus minimizing aberrant gene regulatory events arising from random genomic integration. But they’ll be using a retroviral (integrating) vector to deliver the reprogramming factors to fibroblasts because, Lane explains, the group generated much of its safety data using such vectors, before nonintegrating reprogramming methods came into wide use. Risks aside, “the advantage for us is that when we graft their skin we can then watch the skin,” says Lane. “If they would develop what looks like a squamous cell carcinoma, we should be able to remove it very early.”

Lane considers keratinocyte grafts for RDEB a good test case for iPS cell therapy. “We feel that this disease is so severe, that we understand the immunological and the physical disease fairly well, and that the things that we’re doing on the skin may be less risky than if we were going internally,” he says.

Stem cells for the eye—again

Cancer also enters into the risk equation for Takahashi’s proposed trial of RPE transplants in wet AMD. Besides the general concerns about reprogramming-related mutations and residual undifferentiated cells, RPE may have other vulnerabilities. Although primary human tumors of the eye are rare, it’s possible that iPS cell–derived RPE still maintains the ability to divide and proliferate. And in AMD the eye is flooded with cytokines that could potentially maintain such transplanted cells in a proliferative state. Takahashi and others are working on obtaining pure, homogeneous populations of RPE cells and are developing methods to detect proliferating cells before implantation.

Despite the risks, RPE has become the cell type of choice for transplantation in retinal disease, owing partly to its biological importance and partly to expedience. In fact, two human trials of ES cell–derived cells, now underway, involve RPE replacement. The RPE forms a monolayer of retinal cells sandwiched between photoreceptors on the inside and Bruch’s membrane on the outside. The choroid, a vascular covering that supplies blood to the outer layers of the retina, lies just outside Bruch’s membrane. The RPE, Bruch’s membrane and choroidal endothelial cells together form the blood-retina barrier.

RPE is critically important. It removes the damaged outer segments of photoreceptor rods and cones, which are constantly turning over; it preserves and replenishes the supply of vitamin A, which forms part of the light-absorbing molecule rhodopsin; and it controls the transport of nutrients and the removal of waste products produced by the highly metabolically active photoreceptors. In addition, the RPE “in a polarized fashion is continually secreting cytokines and neurotrophic factors either to the photoreceptor side or to the choroidal side,” says Sheldon Miller, scientific director of the division of intramural research at the National Eye Institute (NEI).

RPE is gradually destroyed and damaged in the dry and wet forms of AMD, respectively, so restoring an intact RPE should at least slow vision loss, and could even restore photoreceptor function, assuming photoreceptors are intact. But photoreceptor and choroid defects also contribute to AMD, so RPE transplants alone aren’t likely to reverse the disease process. “To really solve the disease issue, one would like to transplant…at least two of the three cell types,” says NEI molecular developmental biologist Kapil Bharti.

Nevertheless Miller and Bharti at the NEI, like Takahashi in Japan, are working on iPS cell–derived RPE alone for transplantation in AMD. RPE is really the only choice for now. “The outer segment of photoreceptors—which is the place where critical visual function happens—people have not been able to differentiate in vitro from human iPS cells,” explains Bharti. And transplanted photoreceptors would need to be connected synaptically, he adds, a daunting prospect. Differentiation and connection problems also make choroidal endothelial cell transplants impractical. “RPE seems like a low-hanging fruit,” says Bharti.

But in AMD the Bruch’s membrane suffers damage from inflammation, which could affect the ability of RPE to properly attach. Implanting the RPE monolayer on a biodegradable scaffold, Miller and Bharti’s approach, should help. Takahashi has found that  mounting RPE on a collagen scaffold ex vivo enables implantation (with scaffold removed) of the RPE in its proper polarized orientation—a key requirement, says Miller. But clinical trials of transplanted normal human adult RPE, both autologous and allogeneic, in AMD have been disappointing. The whole issue of proper attachment remains unresolved.

Takahashi, at the May 2012 annual meeting of the Association for Research in Vision and Ophthalmology in Fort Lauderdale, Florida reported survival of autologous grafts in monkeys forup to six months.  Both Takahashi’s group and the NEI group have compared RPE, derived from iPS cells, to primary human RPE, and found them similar in terms of morphology, secretion of growth factors, gene expression patterns and other measures.

Autologous RPE grafts aren’t completely free of rejection risk. Although tissue in the normal eye is normally protected from immune rejection inflammation is a major component of AMD, and breaching of the blood-retina barrier in the disease results in an influx of immune cells.  There could be graft-harming bystander effects from microglial activation or T-cell stimulation; an infection leading to an autoimmune reaction; or an immune reaction against antigens on the RPE itself. What’s more, autologous RPE might still express the same genetic risk factors that made it susceptible to disease in the first place. Takahashi, in an e-mail, writes that the primary etiology of AMD is senescence, not a genetic defect, so this shouldn’t be an issue.

Elsewhere, ACT’s preliminary report on the first two patients to receive ES cell–derived RPE is encouraging4. There was no sign of inflammation in either eye for four months after transplantation. In one patient, there was evidence of RPE survival and engraftment, which, says ACT chief scientific officer Bob Lanza, has continued at least through early May, 20 months out, with no signs of rejection.  The other patient initially didn’t follow the immunosuppressive drug regimen.) “On at least one of the patients there was no… immunological adverse event,” says Miller. “That’s an important piece of information for the entire field.”

Takahashi has received institutional review board approval for her trial and it’s now under consideration by a committee at the Japanese Ministry of Health, Labor and Welfare. f approved, Takahashi believes that the trial could begin around the middle of next year. But her group had not yet published animal model data as Nature Biotechnology went to press. That worries Lanza. “We had years and years of data in multiple models, so we knew what to expect,” Lanza says of his company’s ES cell–RPE work. “You have to do lifetime animal studies, at least in the United States.” Lanza considers the Takahashi trial premature. “To go in with a nucleated cell just now…I think it would be a little early to be trying something like that,” he says.

Miller and Bharti, for their part, say Takahashi should proceed with the trial, based on data they’ve seen her present at meetings. But a lot is at stake. “If something goes wrong, it doesn’t just ruin the company or the individual’s reputation, it hurts the entire field,” Lanza argues.

Platelets: a numbers game

Lanza considers his company’s platelet trial a much better initial test for iPS cells. “Since platelets don’t have nuclei, they cannot form tumors, which makes them ideal for the first iPS cell clinical trial,” he writes in an e-mail. And platelets can be safely irradiated to kill off any nucleated cells that may be contaminating the infusion. Even without irradiation, says Lanza, the company has methods to ensure that no nucleated cells contaminate the platelet dose. “Platelets make a very appealing kind of test case, because you can really ratchet down the oncogenic worry factor,” agrees hematologist Andrew Leavitt, director of the blood and marrow transplant laboratory at the University of California, San Francisco.

ACT’s trial, says Lanza, will be a phase 1 / 2 dose escalation study of patient-specific iPS cell-derived platelets in refractory thrombocytopenia.  Because the FDA is requiring GMP (good manufacturing practice) cell lines, “We are working now to find a provider… or a partnership to give us IPS lines that we could use int his trial,” said ACT CEO Gary Rubin in a May 9 conference call.  The company hopes to demonstrate that such platelets behave in vivo like normal platelets.

According to Lanza, ACT’s iPS cell–derived platelets “appear to be completely comparable” to normal platelets in terms of size, structure, morphology, organelles and platelet-specific markers. And, he says, they’re functional both in vitro and in vivo, and respond to vascular injury in mice. (As of early May, ACT had not yet published any of these data.)

The body makes platelets out of megakaryocytes, bone marrow cells that grow to an enormous size and then release fragments in a highly orchestrated fashion. These cytoplasmic fragments become circulating platelets. Unfortunately, the useful shelf life of donated platelets is only five days. “And the first two to three days, they’re not even available because they’re undergoing extensive infectious disease testing, like all donated blood products,” says Leavitt. Platelets cannot be frozen, because freezing renders them nonfunctional. And donation is a two-hour process that discourages some prospective donors. “There is a huge need for platelets, in part because people aren’t donating them,” says Leavitt. “Supply and demand in platelets runs so close all the time. There’s room for a better mousetrap.”

Fortunately, platelets usually don’t have to be matched to patients.  Immunogenicity is much less an issue than for nucleated cells. “Platelets are non-immunogenic in most instances, so you could put platelets from the same original iPS cells into virtually anybody,” says Lanza.  Lanza’s vision is to generate and freeze many lines of iPS cell–derived megakaryocyte progenitors, thawing them as needed to generate platelets. “In theory your bank could treat every patient on the planet, unlimited. They just grow forever,” he enthuses. ACT’s ten-year goal, says Lanza, is to produce a million standard therapeutic doses (units) of iPS cell– and/or ES cell–derived platelets.

But there’s one big obstacle to achieving this vision, and that’s getting individual iPS cell–derived megakaryocytes to produce platelets in quantity. “The bottleneck in our protocol right now isn’t getting the megakaryocytes, it’s getting [them] to make enough platelet,” says Lanza. For a variety of reasons, only partly understood, megakaryocytes in culture don’t shed platelets at anywhere near the rate in vivo. “There’s something about the body that you’re not replicating in the dish,” says Leavitt. Lanza says the company is working on the problem.

Massive numbers of platelets are needed. one unit of platelets, the standard dose, is between 3 × 1011 and 6 × 1011platelets. Leavitt points out that a single platelet donor can now supply between one and two units per sitting. Even if ACT could generate therapeutically meaningful platelet quantities, Leavitt asks, “could we do it for billions of dollars less by just having a campaign for donors?”

ACT just needs more time, says Lanza. “We do not expect to put the Red Cross out of business anytime soon,” he admits. “But just because it may take a few years to improve the efficiency…to be competitive with the current approach of recruiting donors, it doesn’t mean you should abandon a new technology that could potentially ‘revolutionize’ this aspect of medicine.”

No easy path

None of the likely inaugural iPS cell–based clinical trials is without major issues. But Harvard University’s George Daley, for his part, doesn’t want the problems and risks to obscure the great potential of the young field. “I think it’s miraculous that reprogramming works at all,” he says.  And the field is advancing quickly, with reports of new reprogramming methods appearing almost monthly.  (See Box.)  But technical limitations are inevitable when it comes to practical applications, Daley adds, and will take decades to solve.  “We all have to move forward with prudence,” Daley says. “It’s not going to be easy. It’s going to take us a long time to figure out the principles to make cells deliverable as medicines.”

Ken Garber, Ann Arbor, Michigan


  1. Lister, R. et al, Nature 471, 68-73 (2011).
  2. Mekhoubad, S. et al, Cell Stem Cell 10, 595-609 (2012)
  3. Pomp, O. et al, Cell Stem Cell 9, 156-65 (2011).
  4. Schwartz, S.D. et al., Lancet 379, 713-720 (2012).
  5. Vierbuchen, T. et al, Nature 463, 1035-1041 (2010).
  6. Ieda, M. et al., Cell 142, 375-386 (2010).



BOX: Skipping pluripotency

If the ability to reprogram cells to pluripotency still strikes many biologists as magical, then the direct conversion, or ‘transdifferentiation,’ of one differentiated cell type to another seems positively miraculous. But evidence for transdifferentiation existed as far back as 1987, and it can now be done to create a variety of cell types.

For example, in 2010 Marius Wernig of Stanford University directly converted fibroblasts to functional neurons by introducing just three transcription factors5, and later that year Deepak Srivastava of the University of California, San Francisco, accomplished the transdifferentiation of fibroblasts into cardiomyocytes, also using three factors6. Since then reports of transdifferentiation have proliferated.

In theory, direct conversion of one differentiated cell type to another, avoiding the pluripotency stage altogether, could solve many of the problems of iPS cell–derived cells. The process takes weeks, not months—enabling more timely therapy—and the cancer risk from residual undifferentiated cells should be greatly reduced or eliminated. This will probably result in an easier pathway to regulatory approval.

But transdifferentiation has a long way to go. “It’s very exciting, but there are a lot of things that need to be worked out,” says Stanford’s Joseph Wu. “The efficiency is very low, 1%, some people can’t even duplicate that 1%.” And the cells that do convert, says Wu, make up a heterogeneous mixture, with some expressing the right genes yet not assuming the correct phenotype. “And keep in mind that this is not a renewable resource,” he adds. iPS cell–derived cells can be replenished by going back to the same iPS cell line, but to make more transdifferentiated cells one would have to start over, again transducing the necessary transcription factors. That makes replicability an issue. “I’m worried about it because you never have the same starting population,” says Sue O’Shea of the University of Michigan.

For now, iPS cells, for all their own problems of efficiency, uniformity and replicability, are superior to transdifferentiated cells. But that may not be true forever. “The direct differentiation methods will evolve, and they will absolutely offer alternatives to the iPS intermediate steps,” says Nancy Stagliano, CEO of iPierian in S. San Francisco, California, which is developing disease models for drug discovery using iPS cells.

Table 1 Selected iPS cell–based therapy initiatives



Cell type


Disease indications


Current stage


Advanced Cell Technology Megakaryocytes (for platelets) Refractory thrombocytopenia, leukemia, aplastic anemia Pre-IND


Masayo Takahashi


Retinal pigment epithelium Age-related macular degeneration (wet type) IRB approval, awaiting Health Ministry approval; clinical trial could begin mid-2014
Alfred Lane, Anthony Oro, Marius Wernig Stanford University Keratinocytes Recessive dystrophic epidermolysis bullosa (RDEB) Pre-pre-IND stage; clinical trial could begin in mid-2014
Koji Eto, Kyoto University, with Megakaryon Corporation (Tokyo) Megakaryocytes (for platelets) Thrombocytopenia with leukemia, or requiring bone marrow or cord blood transplantation; cancer Phase 1/2 planned for 2014 or 2015
Steve Goldman, (University of Rochester, New York) Oligodendrocyte precursor (OP) cells Multiple sclerosis Planning 2015 trial using tissue-derived cells; later will test human iPS cell-derived OP cells
Mahendra Rao, NIH Dopaminergic neurons Parkinson’s disease Preclinical; IND filing in late 2014 possible
Jun Takahashi, (Kyoto University) Dopaminergic neurons Parkinson’s disease Clinical trial could begin at end of 2015
Jeffrey Goldberg, Bascom Palmer Eye Institute, University of Miami (Florida) Retinal ganglion cells Glaucoma and other optic neuropathies Moving into GMP production, preclinical toxicology studies
Jakub Tolar & John Wagner (University of Minnesota, Minneapolis) Keratinocytes and hematopoietic grafts RDEB Preclinical animal models
Fernando Ptossi (Instituto Leloir, Buenos Aires, Argentina) Dopaminergic neurons Parkinson’s disease Proof of concept
Angela Christiano,Columbia University; withNew York Medical College and Stony Brook Medicine Three-dimensional skin equivalents from fibroblasts and keratinocytes, RDEB Proof of concept


NIH, National Institutes of Health; IND, investigational new drug; IRB, institutional review board; GMP, good manufacturing practice; OPCs, Oligodendrocyte precursor cells