We demonstrate that MSCs, labeled with a large (micron-scale) iron-fluorophore intracellular contrast particle, can readily be detected in vivo within beating hearts using MRI and that their incorporation into myocardial tissue can be confirmed ex vivo using confocal fluorescence microscopy.
An ideal agent for noninvasive tracking of therapeutic cells would have several essential characteristics. It should be nontoxic without altering cell viability, growth, differentiation, or other biological activity. It should not affect surrounding tissue if released by the carrier cell. It should be durable but ideally should have some elimination pathway. It should also permit repeated, nondestructive, noninvasive detection and should be detectable with satisfactory CNR and spatial and temporal resolution at realistic doses. It should accurately reflect the behavior of the cells it labels and should indicate the location, migration, and quantity of labeled cells. Finally, an ideal agent should indicate the true disposition of labeled cells after emigration, death, or phagocytosis; non-specific interstitial deposition should not misrepresent target cell bioactivity.
The larger IFP we used to label MSCs exhibits many of the above characteristics. Porcine MSCs can be labeled with preserved in vitro viability, proliferation, and differentiation capability as well as in vivo viability after allogeneic transplantation. IFP effects on surrounding cells and the characteristics of IFP elimination are not yet understood. We have demonstrated that IFP-labeled MSCs have useful contrast characteristics, because we can distinguish labeled MSCs from unlabeled MSCs in vitro and in fresh myocardial tissue. IFP-labeled MSCs can be detected in beating myocardium with millimeter-scale spatial resolution and 40-ms temporal resolution immediately after direct injection in pigs. The contrast is satisfactory within normal or infarcted myocardium, both of which may be targeted in future MSC therapies. We have identified a minimum detectable quantity (105
cells/injection) of cells using conventional cardiac MRI on a commercial scanner. This is at least one order of magnitude lower than projected injection doses of cellular agents14
and can accommodate tracer quantities of labeled MSCs admixed with unlabeled MSCs.
Moreover, we are able to detect labeled cells serially and noninvasively when animals are kept alive for up to 3 weeks. Animals in an ongoing study reproducibly have detectable signal voids after 12 weeks (data not shown). The label precisely colocalizes with recovered cells ex vivo when histologic sections are registered with high-resolution ex vivo MRI. Bare label injections seem to exit the myocardium soon after direct injection. When we recovered myocardium 3 weeks after IFP-labeled MSC injections, we found most IFP to remain incorporated within MSCs and not free in the interstitium. However, IFPs impart contrast based on their presence, not based on whether host cells are viable. In 2 animals, injected MSCs were labeled both with DAPI and IFP. Intracellular IFPs were found within DAPI-labeled cells, suggesting retention by administered MSCs rather than ingestion by host cells. However, we cannot exclude the possibility that resident or recruited phagocytes have reincorporated free IFP after MSC death or lysis.
The IFP generated significant T2
* contrast in vitro and in vivo, with T2
* values significantly shorter than neighboring normal and infarcted myocardium, enabling ready detection using conventional cardiac MR to assess both immediate and long-term localization. This is consistent with the known bulk magnetic susceptibility effects of iron oxide within IFP.15,16
We observed less T1
contrast effect of IFP. This is not surprising for magnetite particles embedded in polystyrene, which cannot associate as freely with nearby water protons. Cell injections were detected as signal voids (dark regions) on MRI using both FGRE and SSFP pulse sequences. Signal voids can result from a variety of causes and are thus less specific than signal enhancement methods in MRI and can be additionally obscured by signal averaging within voxels. Nevertheless, T2
* effects influence a larger volume than that occupied by the iron label itself (“blooming”), providing useful image amplification of small injections. However, whereas T2
* in vitro was linearly related to the concentration of labeled cells, in vivo cellular redistribution and aggregation will probably confound quantitation of retained or expanded stem cells. Alternative contrast agents using gadolinium chelates, although attractive for their T1
-shortening (signal enhancement) properties, require direct association with water protons (precluding sequestration within polymer beads) and are potentially toxic should free gadolinium be liberated during prolonged intracellular exposure. Polystyrene microspheres seem biocompatible but may be unattractive for implantation into patients, because they are not degraded and may not be excreted. Clinically approved ultrasmall iron oxide particles (Feridex, Berlex) generate significantly less MRI contrast than the IFP we describe here.9
Other biodegradable polymers such as dextran may provide a more suitable shell for magnetite labels should they be applied to human cellular therapeutics.
Although MSCs or marrow stromal cells, isolated based on density gradient centrifugation and plastic adherence, may contain both mature and progenitor cell population, there is evidence that these preparations contain many cells with multipotential capability in vitro as well as desirable effects when delivered to regions of myocardial injury.17–19
It is unlikely that our injection samples contain large numbers of other nonspecific phagocytes, such as macrophages. Most cells we inject into the myocardium have undergone multiple passages yet retain in vitro differentiation capacity. In this preliminary experience, we observed IFP-containing cells with preserved nuclear structure that have elongated and aligned with host myocardial fibers. Whether IFP-labeled MSCs indeed migrate, differentiate, and improve myocardial function after transplantation remains to be demonstrated in longer-term studies including careful controls.
This dual-fluorescence MRI contrast agent, incorporated into MSCs, imparts contrast characteristics favoring ready MRI detection and permits serial in vivo tracking of MSCs after endomyocardial delivery to the beating heart. In addition, this technology also allows accurate localization of injection sites and of retained cells within both normal and infracted myocardium. It may prove useful for real-time MRI-guided therapeutic endomyocardial injection of labeled MSCs.20
This new technique has potential to provide insight into stem cell retention, engraftment, and homing for cardiovascular cell therapy.