Optimizing stem cell transplantation
Researchers use labeling technique and MRI to track cells in vivo
Studies have shown that stem cell transplantation could aid in the restoration of brain function in patients with medical conditions such as Parkinson’s and Huntington’s diseases, and stroke. The timing and site of transplantation can affect the stem cells’ migration and differentiation, however.
Therefore, to optimize clinical transplantation paradigms, researchers will need to monitor the cells’ migration over time to determine the optimal parameters for transplantation.
Because it is noninvasive and offers high spatial resolution, MRI provides an excellent means to track stem cells in vivo — in contrast to invasive modalities, which require the sacrifice of animals at multiple time points. Investigators have shown that they can achieve sufficient MR contrast by tagging stem cells with superparamagnetic iron oxide nanocomposites; they also have demonstrated the use of nanocomposites for tracking the cells after stroke. These studies were performed with nonhuman stem cells, though, and did not characterize fully the effect of the nanocomposites on stem cell biology. Such characterization is necessary if the method is to be used to label human stem cells for clinical applications.
Researchers have demonstrated MR imaging of stem cells labeled with superparamagnetic iron oxide nanocomposites to track the cells after transplantation to aid in the restoration of brain function. The coronal sections in panels (A) and (C) above show migration of the cells in an ischemic brain after stroke induced in a rat model, one week (A) and five weeks (C) after transplantation. Migration toward the lesion is apparent after five weeks. Panels (B) and (D) are three-dimensional reconstructions of the posterior view of the brain, again one and five weeks after transplantation, respectively. Here, pink represents the graft and green, the stroked area. Images reprinted with permission of PNAS.
In the June 12 issue of PNAS, researchers at Stanford University School of Medicine in California reported a study in which they explored the effects of superparamagnetic iron oxide labeling on human central nervous system stem cells grown as neurospheres both in vitro and in vivo. Using MRI, they monitored the migration of the labeled cells transplanted into the rodent brain. “The questions we asked were: How does tagging of the human clinical grade stem cells affect their biology? [and] Can we detect and track them using MRI in the naïve and stroke brain?” said lead author Raphael Guzman.
The researchers performed in-depth analysis of the effects of labeling on the stem cells, including its effects on cell proliferation, differentiation, survival and electrophysiology. First, though, they had to develop the superparamagnetic iron oxide labeling technique to use with their specific cells, taking into account the fact that they wanted to use FDA-approved substances. To this end, they looked to a 2004 study authored by A.S. Arbab et al in the journal Blood, in which the researchers described a method using protamine sulfate instead of transfection agents that can be used only in the laboratory, Guzman said. “We had to find an optimal ratio of the nanocomposites to protamine sulfate to yield maximal transfection rates with minimal toxic effect.”
Guzman’s team tested the possible effects of the labeling technique on stem cell biology. Using a combination of in vitro immunohistochemistry, electrophysiology and in vivo experiments, the scientists confirmed that it had no adverse effects. They observed no statistically significant difference in the differentiation potential between labeled and unlabeled cells, for example, or in the electrophysiological characteristics.
They then turned to testing of stem cell behavior and migration in the neonatal and adult rodent brain, performing MRI with a 4.7-T, 40-cm animal scanner system from Varian Inc. of Palo Alto, Calif. They used an imaging protocol consisting of scout imaging in two planes followed by a spin-echo sequence and a 3-D gradient echo scan.
First, they looked at the MRI detection threshold of the labeled cells by using phantom studies. A number of factors contribute to this, including the superparamagnetic iron oxide concentration per cell and the cell density after integration into the host, as well as various MRI parameters (field strength and signal-to-noise ratio, for example).
They examined the iron oxide concentration per cell over time to determine how dilution might affect the detection threshold and noted that, in vitro, the relative iron oxide concentration decreased by 50 percent every three days. Still, they showed they could detect clusters of cells up to 18 weeks after transplantation, assuming that cells retain their iron oxide content once cells differentiate.
With respect to cell density, they observed a detection limit of 1000 cells in vitro. However, concentrations of cells are high in vitro. The density of cells drops considerably when the cells begin to migrate and integrate in a site-specific manner.
Using MRI, they tracked the stem cells in vivo after transplantation into the rodent brain. In the uninjured neonatal brain, in contrast to the uninjured adult brain, migration of the cells occurred spontaneously. In the adult brain, targeted migration toward stroke occurred only after injury. They noted a correlation between transcortical migration and the distance between the graft site and the lesion; specifically, targeted migration did not occur when the distance was greater than 1 mm. Previous reports indicated that longer migrations are possible, but these used rodent-derived rather than the human-derived neural stem cells used in the present study.
The investigators also noted that the labeling method could be used to monitor cell death during a graft. Shown at left is a graft undergoing necrosis two (A), seven (B), and 35 (C) days after transplantation. Cell death is indicated by loss of MRI signal and graft volume on the right side of the images.
They reported other findings: for example, that the superparamagnetic iron oxide-labeled human neural stem cells are not impaired in their differentiation into neuronal and glial lineages, that they exhibit electrophysiological characteristics similar to those of mature neurons, and that they respond to microenvironmental cues as they should after transplantation in the rodent brain. Finally, the researchers observed that they could monitor the death of transplanted cells using in vivo MRI.
Neurotransplantation can play a significant role in the treatment of acute and chronic central nervous system disease; in vivo imaging can contribute in a variety of ways in addition to tracking of cell migration and integration — including postoperative visualization of the graft location and monitoring of graft survival. The Stanford researchers are working to develop its potential further.
“We are studying alternative routes of cell delivery and developing new ways for noninvasive tracking of transplanted stem cells using combined MRI and PET imaging,” Guzman said.
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