In vivo Stem Cell Imaging-Regulatory Challenges and Advances


In vivo Stem Cell Imaging-Regulatory Challenges and Advances

On April 19, Geron Corp. presented safety data on the first patient in its groundbreaking clinical trial of GRNOPC1.

GRNOPC1 is Geron's spinal cord injury therapeutic, comprising human embryonic stem cell-derived oligodendrocyte progenitor cells; it is the first therapeutic based on embryonic stem cells to reach the clinic.

The statement says, in effect, that nothing happened – which basically is good news for a Phase 1 safety study. [1] The researchers noted no serious adverse events, nor any adverse events associated with the cells or the injection procedure themselves (though there were two adverse events associated with the immunosuppresents used in the protocol). The researchers also found no sign of immune reaction to the transplanted cells, "no adverse changes or evidence of cavitation," and "no significant change in neurological status."

To collect these data, the researchers used neurological tests, immune monitoring, and of course, MRI scans. Yet these are all indirect probes of Geron's therapeutic. The company has no direct way to measure the activity, location, or abundance of the implanted cells themselves, because those cells are not labeled.

"The short answer is that there is no way for us in the clinical trial to know for certain what the fate of our cells is," says Ed Wirth, Medical Director of the GRNOPC1 program at Geron. "However, we tested hundreds of laboratory animals, where human cells can readily be detected histologically, and found human cells at the injury site, and no cells outside the spinal cord."

Researchers doing pre-clinical work in laboratory animals have a variety of reagents and imaging modalities they can use to tag their cells and follow their progress, but few if any of these approaches are approved for human use, and many can be used only in animals. In lieu of these options, Geron uses a battery of tests, including some nine MR scans in the first year, to closely monitor patient progress in its Phase 1 clinical trial.

Charles Lin, Associate Professor of Dermatology at Harvard Medical School, says being blind to the fate of infused cells in the body is a dangerous way to conduct a clinical trial. "It's like a black box, putting the cells in a black box and then seeing what comes out at the end," he says.

Lin uses fluorescence-based in vivo imaging in his studies of the bone marrow microenvironment in mice. But that strategy isn't very translatable, he says – besides the challenge in getting regulatory approval for fluorescent dyes, there's also the issue of signal intensity: it's difficult to detect light emitting from deep within the human body. Similar issues plague another animal-friendly imaging strategy, bioluminescence.

Other imaging strategies are more readily adaptable to the clinic, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). MRI, says Jeff Bulte, Professor of Radiology and Director of Cellular Imaging in the Institute for Cell Engineering at the Johns Hopkins University School of Medicine, offers excellent anatomic detail and cellular resolution; PET and SPECT, which require radioactive tracers, are highly specific, but offer no anatomic detail and thus must be combined with other imaging modalities.

By Bulte's reckoning seven clinical MRI cell-tracking studies have been published using magnetically labeled cells, four of which involved stem cells. [2] None of these studies was performed in the United States, Bulte says, a reflection perhaps of the difficult regulatory landscape cellular therapeutics developers must navigate to move their therapies into the clinic.

Bulte's own study, performed in Israel, was a Phase 1 clinical trial involving 15 patients with multiple sclerosis and 19 with amyotrophic lateral sclerosis. [3] The patients were injected with autologous mesenchymal stem cells intravenously. Nine patients received stem cells labeled with iron oxide nanoparticles in the form of an FDA-approved imaging agent called Feridex, which can be detected in so-called T2-weighed MRI images. Analysis of the resulting data, Bulte and his coauthors note, suggests the injected cells migrated into the spinal cord and brain. But they cannot be certain that the cells were still viable, they concede, as the cells may have died with the label persisting for some time. (To make such a determination, either PET or MRI reporter genes are required, he says, the development of which is now the focus of ongoing research.)

This kind of ambiguity is one reason Wirth cites in the company's decision not to label its therapeutic cells; other reasons he suggests are both regulatory and technical.

First, Wirth explains, there is the concern from regulatory agencies that labeling the cells could alter their behavior or biology. As a result, if labeled cell are to be used in human clinical trials, they must first be shown to be safe in preclinical trials. And since those labeled cells are what was tested in clinical trials, it would be they – and not their unlabeled counterparts – that would then be approved as a therapy by FDA.

That, says Wirth, means that, though it is technically possible to conduct a clinical trial using labeled cells, it is problematic on a practical level.

"That requires an industry sponsor or other entity wanting to take a cell into clinical trials to make a commitment to a particular labeling strategy very, very early in the preclinical process and basically committing to that labeling strategy all the way through to the clinic and essentially all the way to market," Wirth explains.

Complicating matters, he continues, some strategies work well in preclinical studies but fail in larger animals. Spinal cord repair, for instance, can easily be imaged in rats using bioluminescence, but not in humans, because the tissue depth is too great for the emitted light to reach the detector. But successful strategies can also fail. Feridex, the imaging reagent used in Bulte's 2010 study, was a popular and FDA-approved compound that was readily taken up by cells. Many groups spent considerable time and resources developing and optimizing labeling and imaging strategies based on this agent, only to have it pulled from the market by its developer for financial reasons.

"A lot of work is essentially up in smoke there," Wirth says.

Heike Daldrup-Link, Associate Professor of Radiology in the Molecular Imaging Program at Stanford University, was one researcher who got burned by that financial decision. Feridex, she says, worked well in her hands, easily labeling the human bone marrow-derived mesenchymal stem cells she studies in a rat model of arthritis. Since that compound was pulled, she has switched to Ferumoxytol, another iron oxide nanoparticle that has been FDA-approved for use as an iron supplement.

But though both materials contain iron oxide, they are not identical. Whereas Feridex can enter cells on its own, Ferumoxytol has to be actively delivered, for instance using protamine sulfate. "We have to start over again, like optimizing our imaging protocols, labeling and so on," Daldrup-Link says.

Another issue, says Wirth, is spatial resolution. MRI offers excellent anatomic detail, but cannot easily see fewer than tens or even hundreds of thousands of cells, says Wirth. That "may or may not be sufficient, depending on the application," he says, but it certainly makes it difficult using MRI to detect the migration of a small number of cells to some other (unintended) location. MRI also cannot easily distinguish different cell types, meaning it cannot tell the difference between (in Geron's case) oligodendrocyte progenitor cells and macrophages that might take up the label once the injected cells die. That's the same problem Bulte and his team alluded to in their study, and it's a significant issue with stem cell therapies, notes Wirth; "Many of the cells that are injected die either immediately or shortly after injection," a fact that is true of many cell-based therapies, he says.

Wirth says the company, with help from Hans Keirstead at the University of California, Irvine, did do some preliminary work using labeled cells, but ultimately abandoned the idea.

As a result, he says, both preclinical and clinical studies were more complicated than they might otherwise have been. For instance, because the team could not image an animal repeatedly (or longitudinally) to track cells in vivo over time, they had to use more animals and sacrifice them at specific time points to collect the biological and histological data they required. On the clinical front, he says, the inability to track the cells in vivo means it will be harder for the team to interpret its MRI data. They can use MRI imaging to, for instance, detect the formation or lack thereof of fluid-filled "cavities" at the injury site, but they cannot tell whether those injured sites are filled with injected cells or the patient's own cells (that could only be determined histologically or by biopsy).

The company's use of unlabeled cells will also make it harder to calculate optimum dosages in future studies, Wirth says. "Although labeled cells might partially address the issue, we have found that long-term filling of the spinal-cord lesion cavity to be a useful surrogate outcome for cell survival in animal studies."

Of course, a lack of cell labeling is not always so problematic, says Wirth. Chondrocytes, for instance, produce cartilage, which is easily visualized by MRI. And islet cells can be monitored with a simple blood test, precluding the need for cell labeling.

Which is fortunate, because Wirth says future Geron projects also are unlikely to involve cell labeling, at least in the near term. "Barring the availability of a commercially approved labeling agent for cells …, I don't envision us having a straightforward way to go with a labeled cell product at this time," he says.

[1] "Geron Corporation Reports 2011 First Quarter Financial Results and Highlights," April 27, 2011.

[2] J. Bulte, "In vivo MRI cell tracking: clinical studies," AJR Am J Roentgenol, 193[2]:314-25, 2009.

[3] D. Karussis et al., "Safety and Immunological Effects of Mesenchymal Stem Cell Transplantation in Patients With Multiple Sclerosis and Amyotrophic Lateral Sclerosis," Arch Neurol, 67[10]:1187–94, 2010.