Non-invasive imaging reveals conditions that impact distribution and persistence of cells after in vivo administration

In recent years, biomedical and translational research has focussed on exploring the potential of regenerative medicine therapies (RMTs) to treat a vast number of diseases [1]. A primary safety concern of RMTs, especially if based on stem cells, is their potential to form tumours, due to their proliferative and multi-potential differentiation characteristics [2].

Mesenchymal stem/stromal cells (MSCs) isolated from bone marrow, adipose tissue or umbilical cord are being tested in clinical trials for a range of conditions, but in many cases, preclinical safety data are not available, and the authors fail to report whether the cells cause any adverse effects. Bone marrow-derived (hBM)-MSCs have been used for many years and appear safe [3], but a review of adipose-derived MSCs concluded that while adverse events are rare, they nevertheless do occur and are likely to be related to underlying health conditions of the patients or administration route [4]. Human umbilical cord-derived (hUC)-MSCs have only recently been introduced in clinical trials, with more than 50% of these initiated within the last 3 years (a summary of registered trials is presented in Additional file 1). hUC-MSCs are less immunogenic than other types of MSCs, which contributes to their attraction as clinical RMTs. However, because of their low immunogenicity in combination with higher proliferative behaviour, these cells may also pose a greater potential risk [5]. Yet until now, their safety profile has not been robustly assessed. The importance of preclinical safety testing is highlighted by a recent report where a tumour developed in a patient’s spinal cord following intrathecal administration of stem cells [6].

Assessing the safety of cell therapies by tracking their distribution and fate over time after administration can be achieved in preclinical models. Many animal studies use lipophilic membrane dyes, such as PKH26 or CM-Dil, to label the cells, which requires culling of animals at various time points and histological analysis [7–11]. The key flaws of this approach are (1) the detection of false-positive cells because lipophilic dyes have the potential to transfer to host cells [12]; (2) very large animal numbers, infringing on the principles of the 3Rs (Replacement, Reduction, Refinement); and (3) the failure to longitudinally monitor the cell fate in each individual animal over time. By contrast, non-invasive imaging technologies have opened up exciting new possibilities for preclinical assessment of the safety of cell therapies by allowing longitudinal in vivo cell tracking to monitor cell biodistribution and persistence. Preclinical imaging technologies for cell tracking, some of which have clinical relevance, include magnetic resonance imaging (MRI) to detect cells labelled with superparamagnetic iron oxide nanoparticles (SPIONs), multispectral optoacoustic tomography (MSOT) to detect cells labelled with gold nanorods (GNRs) or near-infrared red fluorescent protein [13–17] and bioluminescence imaging (BLI) for the detection of cells expressing the genetic reporter, firefly luciferase [18–20]. Genetic reporters are particularly advantageous because signals are only generated from living cells, thus allowing the monitoring of cell proliferation and tumour growth and avoiding problems based on nanoparticle dissociation from cells, which can lead to false-positive signals. However, the spatial resolution of BLI is poor, making it difficult to precisely locate the cells [18]. By contrast, both preclinical MSOT and MRI have much higher spatial resolution (150 μm and 50 μm, respectively), providing details of the inter- and intra-organ distribution of administered cells. Moreover, as MRI is routinely used in the clinic, it provides a bridge for preclinical and clinical studies.

An advanced approach to longitudinal in vivo cell tracking is the use of multi-modal imaging strategies that combine cell labels and reporters, including dual-labelling with both the luciferase reporter gene for BLI, and either gold nanorods (GNRs) for photoacoustic imaging [21–24] or superparamagnetic iron oxide nanoparticles (SPIONs) for MRI [25–28]. Such multi-modal imaging approaches benefit from the sensitivity of the luciferase-based signal conferred by living cells, in combination with the high resolution of MRI and photoacoustic imaging systems to detect the nanoparticles inside organs, allowing a comprehensive longitudinal analysis of cell fate and safety risks.

The most common way to administer cells systemically in small animals is via the intravenous (IV) route through the tail vein [29], delivering cells directly to the lungs where they are sequestered as a consequence of the pulmonary first-pass effect [30–35]. Previous reports have suggested that IV-administered cells labelled with lipophilic dyes bypass the lungs, but this is likely due to false-positive staining. For example, in renal regenerative studies, PKH26 dye-labelled IV-administered cells have been reported to engraft in injured kidneys and replace damaged renal cells [9–11, 36], but a more recent study using this lipophilic dye in combination with GFP expression shows that while the dye can sometimes be detected in the kidneys, the cells remain trapped in the lungs [32]. These recent findings are corroborated by in vivo cell tracking studies which show that after IV injection, transplanted cells predominantly accumulate in the lungs [19, 33, 34], fail to integrate or differentiate into tissue-specific cell types and disappear within 7 days [19, 20, 37].

Although the IV route is also frequently used in clinical trials, administration via the arterial circulation is not uncommon. For instance, clinical trials testing the potential of cell therapies to treat myocardial infarction administer cells into the coronary arteries or left cardiac ventricle [24, 38], while in patients with peripheral artery disease or stroke, intra-arterial injection via the femoral or carotid artery, respectively, is frequently employed [39]. Intra-arterial administration will also lead to systemic distribution to other organs, including the brain, and cells passing through the blood-brain barrier could pose an important safety concern. However, a detailed analysis of cell fate after intra-arterial cell administration has so far not been reported [4].

Here, we have implemented a multi-modal imaging approach comprising BLI, MSOT and MRI, to assess biodistribution and fate of different cell types following venous and arterial administration in healthy mice. Some of these cell types are currently being used in clinical trials, including hUC-MSCs (Additional file 1), hBM-MSCs [40], kidney-derived cells [41] and macrophages [42]. We show that our multi-modal imaging approach allows us to determine the immediate distribution of the cells with respect to the route of administration, and to assess the long-term fate of mouse and human MSCs, and their propensity to form tumours. Our findings demonstrate that the multi-modal imaging platform allowing longitudinal cell tracking is an important tool to identify safety concerns of cells used in clinical trials.

Cells were trypsinised, pelleted and resuspended in ice-cold phosphate-buffered saline (PBS) and kept on ice until injection. One hundred microlitres of cell suspension was administered to mice via intravenous (IV) or ultrasound-guided intracardiac (IC) injection. A description and comments on this method of administration are provided in Additional file 3. Tolerance of dosing of cells was adjusted in separate experiments.

Here, we have employed a novel platform approach of non-invasive preclinical imaging encompassing BLI, MRI and MSOT to assess the biodistribution and persistence of a range of mouse and human cell types following IV and IC administration in healthy mice. These cells included mouse MSCs, kidney stem cells and macrophages, as well as human kidney-derived cells and two types of human MSCs, which are already being tested as cell therapies in clinical trials.

As expected, immediate analysis after IV administration revealed that apart from macrophages, all other cell types were mostly sequestered in the lungs, although small numbers of hUC-MSCs could be detected in other organs following ex vivo analysis. After IC administration, all cell types showed a widespread distribution. However, irrespective of the administration route, analysis using BLI and MRI determined that cells disappeared from major organs within 24–48 h, which based on the loss of BLI signals, was likely due to cell death.

A number of preclinical cell therapy studies have reported that IV-administered cells reached the relevant injured organs, where they incorporated and contributed to the regeneration of healthy and functional tissue [7, 9, 10, 51, 52]. It had been postulated that the mechanisms by which the cells, especially MSCs, reached the injured organs was based on the release of trophic factors which attracted the cells to the relevant injury site [33, 53]. An important problem with these studies was that cells had mostly been labelled with membrane dyes (DiI or PKH26) which were detected in the injured organ. However, as we have recently demonstrated, membrane dyes easily dissociate from the cells, leading to misinterpretation of the presence of administered cells [32]. Therefore, more sophisticated approaches to cell labelling and tracking have been established, including bioluminescence imaging of luciferase-transduced cells [17, 54]. This method is specific to living cells because the signal is dependent on production of the enzyme luciferase. Here, we have demonstrated, as others before [32, 34, 55, 56], that IV-administered cells are generally sequestered in the first vascular bed they encounter, which is the lung microvasculature, where they die and then disappear within 2 days.

Since a number of preclinical studies have shown that administered cells fail to incorporate and directly contribute to the regeneration of the damaged tissue in injured organs despite leading to beneficial effects in organ function and histology, it is highly relevant to understand the mechanisms by which efficacy is provided. Increasing evidence points towards paracrine factors that are released by the dying cells. This probably occurs through activation of immunomodulatory pathways that initiate regeneration or, possibly, also via other protective functions [31, 32, 56]. It is currently not understood whether these effects are based on direct and short-term interactions between cytokines and immune cells, stimulating an anti-inflammatory switch, or if epigenetic changes are involved, which could lead to long-term effects.

We explored an alternative systemic administration route by injecting cells directly into the left ventricle of the heart and demonstrated that this route delivered the cells directly to organs, instead of sequestration in the lungs. Our observation that cells are cleared very quickly from the major organs following IC administration indicates that the arterial route poses no significant advantage for cell therapy administration. However, it is possible that small numbers of cells reaching the organs after IC administration may still be able to locally exert beneficial roles through paracrine effects before they are cleared. Therefore, further research is required to explore the local role of IC-delivered cells.

Using our platform of imaging techniques, we were able to provide some mechanistic insight into the fate of macrophages after administration, which have been demonstrated to confer beneficial effects in organ injury models [57, 58]. Macrophages have been previously shown to home to the liver and spleen after passage through the lungs [59]. However, the dynamics of this homing process had not been described. Using multi-modal BLI and MSOT [24], we could monitor macrophage accumulation in the liver and spleen for 4.5 h continuously at high temporal resolution. We found that labelled macrophages immediately started to accumulate in the liver and spleen, particularly in the first ~ 90 min, which indicated that some of the macrophages instantly passed through the pulmonary circulation.

While BLI has the advantage of highly sensitive body-wide detection of luciferase-expressing cells, its spatial resolution is poor, which prevents organ-focussed imaging. To visualise cells within major organs such as the kidney and brain and monitor their fate over time, we implemented a bimodal approach comprising BLI and MRI, taking advantage of the high spatial resolution of MRI in addition to the high sensitivity of BLI and the fact that luciferase activity is dependent on cell viability [15–18].

It should be noted that labelling of cells for preclinical imaging approaches raises a number of questions regarding the interpretation of the findings. While lentiviral labelling is typically a process that results in stable expression of the reporter protein of interest, and as in our case of luciferase, with high sensitivity, exposure to lentiviral particles and the resulting expression of exogenous proteins could possibly affect the immunogenicity of the cells [60], which may have downstream effects on recognition by the endogenous immune system. Because we have used SCID mice for most of our studies, especially those with human MSCs, possible immunogenicity will unlikely have affected the results. In addition, luciferase signal will disappear when cells are phagocytosed, but may remain if cells undergo cell fusion. Therefore, loss of BLI signal is a clear sign of loss of cells through cell death, while continued presence of signal could also indicate a change in cell phenotype by cell fusion. By contrast, SPION or GNR labels may still be detectable after cells have been phagocytosed or fused, potentially leading to misinterpretation. To our knowledge, SPIONs and GNRs have not shown to modify the immunogenicity of labelled cells. To avoid false signals, dual or triple labelling of cells using a combination of genetic direct reporters and nanoparticles could address this problem. We have recently shown that a combination of labels can indeed provide detailed information on the dynamics of cell fate, as GNR-labelled mouse MSCs which also expressed a near-infrared reporter protein disappeared within 2 days after IC injection from the various organs in which they could be detected initially, and instead GNRs accumulated in the liver. By contrast, the reporter protein visualised the increase in cells in tumours forming in sites similar to the ones reported here [24]. A genetic reporter is highly suitable for the detection of multiplying cells, for example in tumours, where the GNRs or other nanoparticles would get diluted out with increasing numbers of cell divisions.

Our detailed analysis of the biodistribution of mMSCs after IC injection using in vivo and subsequently ex vivo MR imaging techniques revealed that SPION-labelled cells were scattered throughout the brain, while in the kidneys, they were restricted to the cortical regions. Ex vivo histological staining and fluorescence microscopy demonstrated that cells in the kidneys were found only within the glomeruli, bounded by endothelial cells within the microvasculature, where they appeared to be trapped. Similarly, cells in the brain were only localised within the microvasculature, indicating that they lack the capacity to pass through the blood-brain barrier. These results demonstrate that the mMSCs cannot extravasate into the brain and kidneys.

During long-term cell tracking of the BALB/c-derived mMSCs, we observed tumour formation in skeletal muscle following IC administration to a similar degree in immunocompetent BALB/c mice as in BALB/c SCIDs. mMSCs also gave rise to tumours in an unrelated inbred strain, albeit at a slower rate, while in an unrelated outbred strain, small foci of mMSCs expanded at early time points and later regressed. Taken together, these data suggest that the adaptive immune system might not be able to recognise tumours derived from syngeneic MSCs and that the genetic background of the host appears to have an effect on the propensity of MSCs to form tumours. This could be a concern for human trials using autologous MSCs where the ability of the cells to form tumours may not be detected by the recipient’s immune system. Furthermore, the results suggest that the risk of tumour formation might depend on undefined genetic factors that would vary from patient to patient. However, it is important to note that karyotype testing of the mMSC line used here revealed a range of chromosomal abnormalities, which could contribute to their propensity to form tumours. In a recent study, a clonal mouse MSC line derived from the immortomouse was found to persist for up to 33 weeks after intra-arterial injection, in the distal femur of SCID mice in which the respective leg had been injured by irradiation [55]. However, while the BLI signal of firefly luciferase expressed by the mMSCs increased over time, tumours were not reported.

Our observation that mMSCs distributed to most organs following IC injection, but tumours were predominantly localised in the skeletal muscles and not within the organs they originally appeared in, raises the question of how tumour formation is regulated in different organs and tissues. Our data indicate that the cells had a ‘survival advantage’ in muscular tissue, but not in the brain and the kidneys, from which they failed to extravasate. We hypothesise that following IC administration, a small number of MSCs were able to extravasate from the capillaries in the skeletal muscle where they started to proliferate. The mechanisms that regulate the ability of the mMSCs to extravasate and form tumours in the skeletal muscle but not in other organs are not known, and further analysis is required to determine the molecular and cellular factors controlling this process.

Our results also show that the cells failed to home to and populate the bone marrow, which is surprising given the cells had been originally isolated from this tissue [61]. The D1 mMSC line used here has not previously been reported to generate invasive tumours, since subcutaneously injected cells provided no evidence of metastasising, even if they proliferated at the injection site [23, 62]. Our observation that the mMSCs did not form tumours outside the lung following IV administration is therefore consistent with this finding. However, the formation of osteosarcomas in the skeletal muscle after IC administration of mMSCs in this study is in line with the previously described formation of osteosarcomas after adoptive transfer of primary MSCs, particularly for cells expanded in vitro, and is a major safety concern in therapies using MSCs [63, 64].

Since these observations suggested that arterial administration of MSC-based cell therapies could have important safety implications, we followed the fate of MSC derived from the bone marrow or the umbilical cord of healthy human donors. We confirmed that neither of these cells presented any major chromosomal aberrations, even after transduction with the luciferase reporter. While in most animals the cells became undetectable within a few days after IV administration, in a few mice, the hUC-MSCs persisted longer, albeit transiently, in other body regions where their presence was not expected. Of note, a transient persistence of hUC-MSCs was not observed after IC administration, nor for hBM-MSCs using IV or IC administration routes. We suggest that this unusual behaviour is not linked to cell size, because the hUC-MSCs are not smaller than mKSCs or mMSCs, but could possibly be due to their surface proteins, allowing some of the cells to escape the lungs [33, 65]. The observation that hUC-MSC foci appeared in a small number of mice, grew in size, but later disappeared, was difficult to explain, especially given that the mice were SCIDs and thus lacked an adaptive immune system. It is possible that the cells eventually elicited a xenogeneic response involving macrophages and natural killer cells [66], after initially suppressing the native immune system, which is one of their central properties [67, 68]. Alternatively, the hUC-MSCs may have expanded in the animal but then become senescent and died, irrespective of the host’s ability to mount an immune response. Thus, after an 8-week period of cell tracking, we could not observe any tumour growth by in vivo or ex vivo BLI in all SCID mice to which hUC-MSCs had been administered by either IV or IC injection. However, the observation that the IV-injected hUC-MSCs persisted for a time period in 25% of the animals indicates that these cells carry greater safety risks and suggests that clinical therapies with these cells should proceed with caution with an appropriate risk management plan. Further preclinical studies are needed to determine the mechanisms by which hUC-MSCs were able to persist as well as eventually disappear in order to better define the potential for tumourigenicity. The imaging platform presented here provides the necessary biotechnology for preclinical evaluation of the potential tumourigenicity of cell products used for cell transplantation, for which there is presently no internationally recognised guideline.

Cell-based therapies are currently being considered for a range of diseases, some of which are already undergoing clinical trials. A robust biodistribution and safety assessment is essential for understanding cell fate and ensuring patient welfare. Here, we demonstrate a safety assessment platform that can expose not only the general organ distribution of potential cell therapies, but also a detailed view of their presence within different organs. Importantly, by using this imaging platform, we show that the route of administration affects the range of organs that the cells can reach and, particularly, their propensity to form tumours. Our assessment suggests that cells are short-lived irrespective of whether they are administered via the venous or arterial circulation and that the risk of cell persistence or tumour formation is dependent on the cell type, route of administration and immune status of the host. Crucially, we show that clinically used, human umbilical cord-derived mesenchymal stem/stromal cells form transient unexpected self-limited proliferations in various anatomical regions when administrated intravenously. The implications of this observation require further investigations and should be taken into account when clinical trials are considered.