Gaining Mechanistic Insights into Cell Therapy Using Magnetic Resonance Imaging
Cell therapy remains a promising approach to treat neurological disorders with evidence of clinical efficacy emerging. However, the mechanisms underlying recovery and the conditions to achieve therapeutic success are poorly understood. Uncovering putative mechanisms and their interaction is essential to progress cell therapy from an interesting and promising possibility to a robust and consistent treatment that can be used to treat large cohorts of patients. Although there is a heavy focus on stem cell biology, relatively little effort has been dedicated to understanding the in vivo mechanisms that underlie efficacy. In vivo imaging and the development of appropriate biomarkers will be essential to gain a better mechanistic understanding of cell therapy in the treatment of neurological disease. The availability of magnetic resonance imaging in a clinical setting and its use in animal models allow for its use as a unique platform for mechanistic studies of cell therapy in a translational context.
KeywordsMRI Cell therapy Mechanism Neural stem cells Bone marrow stem cell Imaging
The promise of cell therapy as an efficacious treatment for neurological conditions not only is contingent upon a thorough understanding of stem cell biology, but also requires optimization of the preparation and delivery of these cells and a deeper understanding of their interaction with the disease-damaged brain [1, 2]. Although a variety of cell sources have been identified from developing, as well as adult tissues , three physiological processes have been proposed as key mechanisms for cell therapy to promote improvements in behavioral impairments in neurological conditions: (1) integration of implanted cells to replace lost or damaged cells, (2) supply of paracrine or juxtacrine factors leading to improvements in host tissue function, and/or (3) induction of host (e.g., inflammatory, neurogenesis) responses that lead to changes in the host tissue facilitating recovery. Considering the varied and synergistic effects of these processes that can be induced by implanted cells, it is extremely challenging to pinpoint precise processes as being the pivotal mechanisms leading to recovery . Indeed, no one factor may be sufficient for recovery; rather, a cascade of interactions may be necessary to induce physiological changes that yield functional improvements . As the brain consists of highly specialized regions, a further key component is the spatial sphere of action of these physiological processes. This can be very localized, as in the case of cell integration into existing functional or dysfunctional circuitry , or very diffuse, as in the case of a systemic factor release that affects the entire brain .
Considering that the various physiological processes require a spatial and temporal monitoring, it becomes evident that non-invasive in vivo serial monitoring can provide unique insights into the mechanisms involved in recovery. Indeed, cell therapy in a clinical setting is likely to require the use of diagnostic imaging to define patients who will benefit from the intervention and also to guide and monitor the effects of cell therapy. Clinical imaging techniques, such as computer tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), have all been incorporated in early translational trials . For instance, PET imaging of dopaminergic receptor binding has been crucial to demonstrate the functional activity of fetal tissue transplants in animal models , as well as patients with Parkinson’s disease . Although the ultimate goal is to use imaging to gain a mechanistic understanding of cell therapy in patients, this needs to be rooted in a thorough validation of imaging biomarkers that requires preclinical animal studies. Moreover, these animal studies provide the testing grounds to develop new approaches that could yield mechanistic insights.
Location, Location, Location—Monitoring Cell Delivery and Distribution
For cells to exert their physiological action, their location is pivotal. For instance, neural stem cells implanted into the peri-infarct parenchyma of a stroke promote recovery, whereas cells injected into the lateral ventricle did not . Considering the varied extent and topology of lesions caused by neurological conditions, such as stroke and traumatic brain injury, guidance of injection to the parenchyma is, hence, a crucial step to ensure that the transplanted cells can be efficacious . Nevertheless, in other cases, direct targeting of the lesion cavity is important and MRI can provide the required guidance in terms of volume and location to perform a safe and efficient injection [13, 14]. Although most preclinical studies in rodents use a predefined implantation site irrespective of lesion size and location, neurosurgical planning is an essential component for other species, such as non-human primates , pig , and, of course, humans . MR imaging can also be used to assess potential iatrogenic complications, such as injection tract damage  or the formation of cerebral microemboli after intra-arterial (i.a.) infusion of cells . Real-time monitoring of the delivery of cells has been described by tagging cells with MRI contrast agents prior to injection . This approach can also afford a determination of the accuracy of cell placement using cellular MRI , although consideration has to be given to the potential biological effects of the contrast agents that could potentially interfere with the cells’ efficacy . Co-injection of contrast agent in the infusate could, however, serve as a short-lived substitute to monitor delivery . As the brain is a highly specialized organ and the efficacy of cells is likely due to their interaction with local tissue environments, defining the anatomical site, as well as the tissue characteristics of the environment, will likely be important to gain a mechanistic understanding of the cells’ physiological action and will likely form the basis for future studies.
Assessing Survival and Differentiation of Implanted Cells
In organ transplantation, the survival of the “replacement organ” is essential for the survival of the host. However, in cell therapy, the host organ remains and, hence, the survival or rejection of injected cells might not be crucial for survival of the host or even for clinical efficacy of the cells administered. Although one would expect cell survival to be essential for recovery, there is evidence that this is not the case. Indeed, for some applications, the rejection response or short-term immunomodulation of the local inflammatory environment may be sufficient to promote improvement . Non-invasive monitoring of the survival or rejection of cells therefore provides a unique means to assess the presence of implanted cells in a given tissue. Survival of cells, however, is not merely a function of “seeing” implanted cells tagged with a MRI contrast agent, as these can persist after cell death . It has been suggested that surviving cells will yield a dilution and gradual attenuation of the signal due to proliferation and migration within the tissue, whereas contrast agent from dead cells will be more clustered around the injection site . An alternative approach is to use MR-based nanosensors that, for instance, report on local pH changes surrounding transplanted cells to report on their viability . However, interpretation of these signals is complicated and reliance on an additional imaging modality, such as bioluminescence, which only produces a signal for live cells is advocated to further develop cell vitality-based MR imaging . Indeed, bioluminescence currently provides the gold standard for the non-invasive in vivo assessment of cell survival .
Beyond cell survival, the phenotypic differentiation of cells is key for some stem cells to exert their physiological functions within a tissue (e.g., neural stem cells function to produce differentiated neuronal cells). Evidence from fetal tissue transplantation in Parkinson’s disease shows that dyskinetic side effects can be due to the ratio of serotonergic neurons within the dopaminergic transplant , highlighting the importance of achieving appropriate differentiation of transplanted cells. The in vivo assessment of cell phenotypes remains, nevertheless, a major challenge for MR imaging and is also reliant on using specific markers. At a more general level, the ability to distinguish between undifferentiated and differentiated cells can be considered. Undifferentiated cells typically proliferate, which leads to a higher metabolic activity, but also certain specific metabolites being present. To this end, MR spectroscopy can be used to distinguish between undifferentiated and differentiated cells based on elevated levels of phosphocholine + glycerophosphocholine, as well as myo-inositol . However, MR spectroscopy does not readily lend itself to imaging, i.e., the ability to localize signals to a small area of tissue with a small number of cells, although it is suitable to characterize large tissue grafts in patients . Thymidine analogs for PET imaging have been described to measure cell proliferation , and there is some effort to achieve a similar visualization using MRI , but so far, no study has applied these approaches to the imaging of cell therapy. Contrast agent-based approaches have been described to assess the functional status of transplanted cells , as well as their differentiation into inhibitory GABAergic neurons . Although reliable cell surface imaging (e.g., receptors) for specific cell types is available using PET imaging , the poor spatial resolution and the inability to distinguish transplanted from host cells still limit its use to monitor differentiation of transplanted cells. However, in the case of Parkinson’s disease, where dopaminergic cells are placed ectopically in the striatum, it is well suited to provide a macroscopic assessment of dopamine replacement in this location through the differentiation of cells into dopaminergic neurons . At present, therefore, there is a dramatic lack of in vivo tools to directly visualize the differentiation of transplanted cells.
Determining Functional Effects and Circuit Integration
A major advance to probe for circuit integration of transplanted cells has been achieved through the combination of optogenetics and fMRI (ofMRI). This approach takes advantage of being able to specifically activate cells containing optical actuators that are genetically encoded (e.g., channelrhodopsin). If only transplanted cells contain this gene, light can selectively stimulate transplanted cells to produce neuronal activity. Using fMRI, in contrast, can measure the response to this stimulus across the whole brain to indicate which areas are being activated by the optogenetic stimulus. Unlike the fMRI of activity in the fetal transplants, ofMRI affords probing of the downstream connectivity of implanted cells [54••], hence, the effect of transplanted cells on host signal processing. An improvement of this approach over electrophysiology is that localization of implanted cells for recordings through an electrode is very challenging, and it is difficult to determine where the downstream connections are formed, whereas optogenetics can stimulate all relevant transplanted cells within a region of interest and record the global downstream effects using fMRI. Nevertheless, one has to be cautious and question as to how many cells will need to form connections within a given region to yield of a sufficient fMRI signal.
Uncovering putative mechanisms and their interaction is essential to progress cell therapy from an interesting and promising innovation to a robust and consistent treatment. Nevertheless, considering the varied modes of action of cell therapy, as well as its interaction and responsiveness to the tissue environment over time, histological studies are insufficient to investigate putative mechanisms. Non-invasive in vivo imaging techniques constitute an essential complementary line of analysis. Specifically, the biodistribution of cells within a treated organ or the whole organism, their migration, and survival within regions of damage, in addition to their impact on local and distant tissue function, are all pivotal aspects of their mode of action that will be crucial to guarantee the success of cell therapy in the clinic. A greater emphasis on in vivo imaging is, hence, required with a specific focus on uncovering the sufficient and necessary conditions for cell therapy to be efficacious.
MM gratefully acknowledges funding by the National Institute for Neurological Disease and Stroke (R01NS08226; R21NS088167) and National Institute for Biomedical Imaging and Bioengineering (R01EB016629).
Compliance with Ethical Standards
Conflict of Interest
Michel Modo declares that he has no conflict of interest.
Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.
Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance
- 5.Chou CH, Modo M. Human neural stem cell-induced endothelial morphogenesis requires autocrine/paracrine and juxtacrine signaling. Sci Rep. 2016;6:29029. doi:10.1038/srep29029.
- 18.Cui LL, Kerkela E, Bakreen A, Nitzsche F, Andrzejewska A, Nowakowski A, et al. The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and infusion velocity. Stem Cell Res Ther. 2015;6:11. doi:10.1186/scrt544.CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Modo M, Hitchens TK, Liu JR, Richardson RM. Detection of aberrant hippocampal mossy fiber connections: ex vivo mesoscale diffusion MRI and microtractography with histological validation in a patient with uncontrolled temporal lobe epilepsy. Hum Brain Mapp. 2016;37(2):780–95. doi:10.1002/hbm.23066.CrossRefPubMedGoogle Scholar
- 27.Janowski M, Walczak P, Kropiwnicki T, Jurkiewicz E, Domanska-Janik K, Bulte JW, et al. Long-term MRI cell tracking after intraventricular delivery in a patient with global cerebral ischemia and prospects for magnetic navigation of stem cells within the CSF. PLoS One. 2014;9(2), e97631. doi:10.1371/journal.pone.0097631.CrossRefPubMedPubMedCentralGoogle Scholar
- 31.Bible E, Dell’Acqua F, Solanky B, Balducci A, Crapo PM, Badylak SF, et al. Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodeling in the stroke-damaged rat brain by (19)F- and diffusion-MRI. Biomaterials. 2012;33(10):2858–71. doi:10.1016/j.biomaterials.2011.12.033.CrossRefPubMedPubMedCentralGoogle Scholar
- 33.•Nicholls FJ, Ling W, Ferrauto G, Aime S, Modo M. Simultaneous MR imaging for tissue engineering in a rat model of stroke. Sci Rep. 2015;5:14597. doi:10.1038/srep14597. First example of non-invasive imaging of neural stem cells and endothelial cells implanted into a stroke cavity to visualize their relative distribution in the same subject. This study demonstrated that with ever more advanced tissue engineering approaches, significant imaging developments are required to meet the need for in vivo monitoring in translational paradigms.
- 34.Lappalainen RS, Narkilahti S, Huhtala T, Liimatainen T, Suuronen T, Narvanen A, et al. The SPECT imaging shows the accumulation of neural progenitor cells into internal organs after systemic administration in middle cerebral artery occlusion rats. Neurosci Lett. 2008;440(3):246–50. doi:10.1016/j.neulet.2008.05.090.CrossRefPubMedGoogle Scholar
- 35.Khabbal J, Kerkela E, Mitkari B, Raki M, Nystedt J, Mikkonen V, et al. Differential clearance of rat and human bone marrow-derived mesenchymal stem cells from the brain after intra-arterial infusion in rats. Cell Transplant. 2015;24(5):819–28. doi:10.3727/096368914X679336.CrossRefPubMedGoogle Scholar
- 37.Cianciaruso C, Pagani A, Martelli C, Bacigaluppi M, Squadrito ML, Lo Dico A, et al. Cellular magnetic resonance with iron oxide nanoparticles: long-term persistence of SPIO signal in the CNS after transplanted cell death. Nanomedicine (Lond). 2014;9(10):1457–74. doi:10.2217/nnm.14.84.CrossRefGoogle Scholar
- 38.Berman SC, Galpoththawela C, Gilad AA, Bulte JW, Walczak P. Long-term MR cell tracking of neural stem cells grafted in immunocompetent versus immunodeficient mice reveals distinct differences in contrast between live and dead cells. Magn Reson Med. 2011;65(2):564–74. doi:10.1002/mrm.22613.CrossRefPubMedGoogle Scholar
- 43.Chung YL, El Akabawy G, So PW, Solanky BS, Leach MO, Modo M. Profiling metabolite changes in the neuronal differentiation of human striatal neural stem cells using 1H-magnetic resonance spectroscopy. Neuroreport. 2013;24(18):1035–40. doi:10.1097/WNR.0000000000000056.CrossRefPubMedPubMedCentralGoogle Scholar
- 54.••Byers B, Lee HJ, Liu J, Weitz AJ, Lin P, Zhang P, et al. Direct in vivo assessment of human stem cell graft-host neural circuits. Neuroimage. 2015;114:328–37. doi:10.1016/j.neuroimage.2015.03.079. Mechanistic insights into neuronal connectivity are very challenging using conventional approaches. The combination of optogenetic and functional magnetic resonance imaging to interrogate the functional impact of transplanted cells provides a major step forward to assess cell replacement in neurological conditions.
- 56.•Englander ZA, Sun J, Laura C, Mikati MA, Kurtzberg J, Song AW. Brain structural connectivity increases concurrent with functional improvement: evidence from diffusion tensor MRI in children with cerebral palsy during therapy. Neuroimage Clin. 2015;7:315–24. doi:10.1016/j.nicl.2015.01.002. Evidence that non-invasive imaging, such as diffusion tensor imaging, are key outcome measures to evaluate the efficacy of cell therapy in patients and that more preclinical studies are needed to validate these biomarkers.CrossRefPubMedPubMedCentralGoogle Scholar