Background aims. Mesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the noninterference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI. Methods.We studied the effects of iron oxide-poly(L-lactide) nanoparticles in MSC with flow cytom-etry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant® proliferation testing, colony-forming unit-fibroblast (CFU-F) assays, flow chamber adhesion testing, immuno-logic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats. Results. It could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks. Conclusions. These particles qualify for studying MSC homing and trafficking via MRI.

Cell therapy using multipotent mesenchymal stromal cells (MSCs) is of high interest in various indications. As the pleiotropic effects mediated by MSCs rely mostly on their unique secretory profile, long-term persistence of ex-vivo-expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long-term persistence of nucleated cells and could therefore also be applied to MSCs. We have exposed human bone-marrow-derived MSCs to 30 or 60 Gy of g-irradiation and assessed cell proliferation, clonogenicity, differentiation, cytokine levels in media supernatants, surface receptor profile, as well as expression of proto-oncogenes/cell cycle markers, self-renewal/stemness markers, and DNA damage/ irradiation markers. Irradiated MSCs show a significant decrease in proliferation and colony-forming unitfibroblasts. However, a subpopulation of surviving cells is able to differentiate, but is unable to form colonies after irradiation. Irradiated MSCs showed stable expression of CD73 and CD90 and absence of CD3, CD34, and CD45 during a 16-week follow-up period. We found increased vascular endothelial growth factor (VEGF) levels and a decrease of platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB in culture media of nonirradiated cells. Irradiated MSCs showed an inverse pattern, that is, no increase of VEGF, and less consumption of PDGF-AA and PDGF-AB/BB. Interestingly, interleukin-6 (IL-6) levels increased during culture regardless of irradiation. Cells with lower sensitivity toward g-irradiation showed positive b-galactosidase activity 10 days after irradiation. Gene expression of both irradiated and nonirradiated MSCs 13-16 weeks after irradiation with 60 Gy predominantly followed the same pattern; cell cycle regulators CDKN1A (p21) and CDKN2A (p16) were upregulated, indicating cell cycle arrest, whereas classical proto-oncogenes, respectively, and self-renewal/stemness markers MYC, TP53 (p53), and KLF4 were downregulated. In addition, DNA damage/irradiation markers ATM, ATR, BRCA1, CHEK1, CHEK2, MDC1, and TP53BP1 also mostly showed the same pattern of gene expression as high-dose g-irradiation. In conclusion, we demonstrated the existence of an MSC subpopulation with remarkable resistance to high-dose g-irradiation. Cells surviving irradiation retained their trilineage differentiation capacity and surface marker profile but changed their cytokine secretion profile and became prematurely senescent.

(1) Background: Long COVID syndrome refers to long-term sequelae of the novel viral disease, which occur even in patients with initially mild disease courses. However, there is still little evidence of the actual organic consequences and their frequency, and there is no standardized workup to diagnose long COVID syndrome yet. In this study, we aim to determine the efficiency of a stepwise diagnostic approach for reconvalescent COVID-19 patients with cardiopulmonary symptoms. (2) Methods: The diagnostic workup for long COVID syndrome included three steps. In the first step, the focus was on broad applicability (e.g., blood tests and body plethysmography). In the second step, cardiopulmonary exercise testing (CPET) and cardiac MRI (CMR) were used. The third step was tailored to the individual needs of each patient. The observation period lasted from 22 February to 14 May 2021. (3) Results: We examined 231 patients in our long COVID unit (mean [SD] age, 47.8 [14.9], 132 [57.1%] women). Acute illness occurred a mean (SD) of 121 (77) days previously. Suspicious findings in the first visit were seen in 80 (34.6%) patients, prompting further diagnostics. Thirty-six patients were further examined with CPET and CMR. Of those, 16 (44.4%) had pathological findings. The rest had functional complaints without organ damage (“functional long COVID”). Cardiopulmonary sequelae were found in asymptomatic as well as severe courses of the initial COVID-19 disease. (4) Conclusions: A structured diagnostic pathway for the diagnosis of long COVID syndrome is practicable and rational in terms of resource allocation. With this approach, manifest organ damage can be accurately and comprehensively diagnosed and distinguished from functional complaints.

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