Abstract
The development of objective and quantitative imaging approaches for tracking stem cell fate, including stem cell location, survival, engraftment, and differentiation, may lead to better patient stratification and eventual successful translation of cardiac stem cell therapy into the clinic.
Summary:
Transplantation of human mesenchymal stem cells (MSCs) is an exciting area of medical research, and cell-based tissue regeneration seems only a matter of time before becoming a reality. This development could dramatically alter our perspective about disease. Much hype has accompanied the potential of stem cell therapy to restore organs to their original state; however, we remain a long way from that goal. The development of objective and quantitative imaging approaches for tracking stem cell fate, including stem cell location, survival, engraftment, and differentiation, may lead to better patient stratification and eventual successful translation of cardiac stem cell therapy into the clinic. The contributions of Willmann et al (1) represent an important step toward this goal.
The Setting
Encoded into the DNA of every cell is the complete blueprint for regenerating any type of tissue, yet only a tiny percentage of cells, the stem cells, retain the ability to differentiate. The promise of stem cell therapy is to unlock this code and harness it for the treatment of disease. The embryonic stem cell, because of its potential for differentiation into any other type of cell, offers the most flexibility for tissue regeneration. In January 2009, the U.S. Food and Drug Administration approved the first human trial of embryonic stem cell therapy for spinal cord repair (2). However, because of the controversial nature of embryonic stem cells, more research has been conducted on adult stem cells, such as human MSCs, which retain the potential to develop into a more limited menu of tissue types: muscle, tendons, and cartilage, among other connective tissues. The opportunity to regenerate diseased tissues has led to high expectations.
However, human adult stem cell therapy has had a mixed track record (3). Hematopoetic stem cells have been successfully applied in bone marrow therapy, but other adult stem cell therapies have not proved as successful. Determining the fate of the stem cells with imaging could improve the monitoring of stem cells, thus enabling a better understanding of stem cell therapy.
But how will we image stem cells? In this issue of Radiology, Willmann et al (1) describe the use of reporter genes combined with a PET radiotracer to monitor the viability of human MSCs after intramyocardial injection into rats and swine and demonstrate the challenges facing this technology.
The Science
There are a variety of ways to tag human MSCs so that they can be monitored in vivo. If the human MSCs are induced to phagocytize iron oxide particles, they can be detected with magnetic resonance (MR) imaging. This method is capable of very high sensitivity and resolution but does not provide information on cell differentiation or viability (4). The amount of iron gradually diminishes as cells divide. Optical imaging is also possible by using transfected genes, which produce endogenous fluorescent proteins (eg, green fluorescent protein) or enzymes (luciferase for bioluminescence imaging). However, the penetration of light in tissue does not allow noninvasive imaging in larger animals. Positron emission tomography (PET) is appealing because it is highly sensitive and quantitative and can be performed in humans; when combined with computed tomography (CT), it can also accurately localize radiotracer uptake.
In order to label a stem cell, it must be stably transfected with a reporter gene. Adenovirus (altered so it cannot replicate) containing the cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV-sr39tk) was genetically engineered into human MSCs, which were then injected into the heart muscle of rats and swine during open thoracotomy. The PET radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG), a thymidine analog, was administered to the animal, and the transfected human MSCs phosphorylated the FHBG, thus entrapping it within myocardial cells. The beauty of this method is that subsequent generations of the human MSCs (when stably transduced with the reporter gene) will, in theory, retain the reporter gene so that the human MSCs can be monitored over many generations of cell division subject to the limits of DNA repair mechanisms, which may eventually excise the introduced gene.
In their experiment, Willman et al (1) performed intramyocardial injections of human MSCs as a model to induce muscle regeneration in heart disease. First, the Ad-CMV-HSV-sr39tk was expressed in cells in proportion to the dose of the gene and radioactivity. In rats, radiotracer uptake was noted on micro-PET images of the heart. However, when the same experiment was performed in swine (approximately 40 kg vs 0.3 kg), no radiotracer uptake was initially seen. It was only after co-injection of human MSCs with matrigel, a proteinaceous mixture that simulates the extracellular environment, that the cells appeared to “take” and produce transduced cells expressing the mutant thymidine kinase reporter gene. It is unclear whether this would be feasible in humans given the potential for immunogenicity. Even so, imaging in the swine with a human PET scanner yielded relatively subtle radiotracer uptake compared with imaging in the rat. This work demonstrates that it is feasible to image human MSCs with a reporter gene strategy by using clinical PET units; however, there are still many technical challenges in achieving adequate radiotracer uptake in larger animals.
The Practice
Clinical use: Simply knowing that the injected stem cells are still viable is an important step in stem cell therapy, but it is just the first step. Localization, viability, differentiation, and therapeutic effect will be potential roles for imaging. Localization may require the development of image-guided interventional techniques to place catheters directly into the damaged tissue. Transient expression of the gene will be a problem that will need to be overcome if human MSCs are to be monitored over long time. Viability may be demonstrated by means of the reporter gene construct, provided it remains stably transfected. The next clinical question will be whether the stem cells differentiate into the desired tissue type. By using other constructs, it may be possible to design reporter genes that only activate during differentiation of MSCs into heart muscle. Finally, traditional imaging tests (eg, nuclear cardiology and cardiac MR imaging) will be used to monitor actual improvements in heart function. Deconstructing stem cell therapy failures will be a major role for imaging.
Future opportunities and challenges: Reporter gene paradigms could be used widely for stem cell therapies to monitor localization, engraftment, differentiation, and therapy for a wide range of organs. Moreover, this work points out an important limitation of current PET imaging. Although PET is acknowledged to be highly sensitive, the current human PET equipment results in relatively large voxels in which the signal from a small number of labeled human MSCs will be averaged with many host cells that are unlabeled. Partial volume averaging is a technical challenge for imaging human MSCs in humans, which places higher demands on spatial resolution than is needed for conventional PET scans. Higher-resolution PET cameras, with 1–2-mm resolution, are possible but only at great expense using existing technology (5). As PET moves into other frontiers, such as human MSC imaging, partial volume averaging looms as a challenge for PET camera design and awaits technology developments that will enable smaller, more numerous, and affordable detectors.
This trial also points out the need to develop new adjuvants to improve the retention of newly injected human MSCs. While matrigel was successful in this experiment, it is unclear it will be feasible in humans, and research in biopolymer scaffolds is urgently needed to prolong the effectiveness of human MSCs.
Footnotes
See also the article by Willmann et al in this issue.
Funding: The author is an employee of the National Institutes of Health.
References
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