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Ashwini Ketkar-Atre,1 Tom Struys,1,2 Stefaan J Soenen,3 Ivo Lambrichts,2 Catherine M Verfaillie,4 Marcel De Cuyper,5 Uwe Himmelreich1 1Biomedical MRI/MoSAIC, Department of Imaging and Pathology, Biomedical Sciences Group, Katholieke Universiteit Leuven, Leuven, Belgium; 2Lab of Histology, Biomedical Research Institute, Hasselt University, Campus Diepenbeek, Agoralaan, Diepenbeek, Belgium; 3Lab for General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent, Belgium; 4Interdepartmental Stem Cell Institute, O&N IV, Katholieke Universiteit Leuven, Leuven, Belgium; 5Laboratory of BioNanoColloids, Interdisciplinary Research Centre, Katholieke Universiteit Leuven, Kortrijk, Belgium Abstract: The need to track and evaluate the fate of transplanted cells is an important issue in regenerative medicine. In order to accomplish this, pre-labelling cells with magnetic resonance imaging (MRI) contrast agents is a well-established method. Uptake of MRI contrast agents by non-phagocytic stem cells, and factors such as cell homeostasis or the adverse effects of contrast agents on cell biology have been extensively studied, but in the context of nanoparticle (NP)-specific parameters. Here, we have studied three different types of NPs (Endorem®, magnetoliposomes [MLs], and citrate coated C-200) to label relatively larger, mesenchymal stem cells (MSCs) and, much smaller yet faster proliferating, multipotent adult progenitor cells (MAPCs). Both cell types are similar, as they are isolated from bone marrow and have substantial regenerative potential, which make them interesting candidates for comparative experiments. Using NPs with different surface coatings and sizes, we found that differences in the proliferative and morphological characteristics of the cells used in the study are mainly responsible for the fate of endocytosed iron, intracellular iron concentration, and cytotoxic responses. The quantitative analysis, using high-resolution electron microscopy images, demonstrated a strong relationship between cell volume/surface, uptake, and cytotoxicity. Interestingly, uptake and toxicity trends are reversed if intracellular concentrations, and not amounts, are considered. This indicates that more attention should be paid to cellular parameters such as cell size and proliferation rate in comparative cell-labeling studies. Keywords: cell labeling, MR contrast agents, transmission electron microscopy, mesenchymal stem cells, multipotent adult progenitor cells, magnetic resonance imaging, nanoparticles, iron oxide

Abstract Introduction Auto-transplantation of third molars is frequently undertaken in order to restore a perfect occlusion and to improve mastication following a substantial loss of molars. However, little is known about the precise role of the periodontal membrane during this procedure. Therefore, we investigated if the epithelial rests of Malassez persist in the periodontal ligament of auto-transplanted teeth and, if so, whether these may show signs of a neuro-epithelial relationship. Case presentation We report a case of a 21-year-old Caucasian woman who underwent an auto-transplantation of two third molars. After two years, renewed progressive caries of the auto-transplanted teeth led to the removal of the auto-transplanted elements. The periodontal ligament was removed and studied with a light and transmission electron microscope. Conclusion In this report we examined the ultrastructure of the periodontal ligament after auto-transplantation in order to see if the periodontal ligament recovers completely from this intervention. We observed fully developed blood vessels and a re-innervation of the epithelial rests of Malassez which were proliferating following auto-transplantation. This proliferation might be critical in the remodelling of the alveolar socket in order to provide a perfect fit for the transplanted tooth. In order to minimalise the damage to the epithelial rests of Malassez, the extraction of the tooth should be as atraumatic as possible in order to provide an optimal conservation of the periodontal ligament which will be beneficial to the healing-process.

Aim: To assess the impact of the timing of soft-tissue augmentation (STA) on mean buccal bone changes following immediate implant placement (IPP) in the anterior maxilla., Materials and Methods: Patients with a failing tooth and intact buccal bone wall in the anterior maxilla (15-25) were enrolled in this randomized controlled trial. Following single IIP and socket grafting, they were randomly allocated to the control group (immediate STA performed during the same surgical procedure) or the test group (delayed STA performed 3 months later). Implants were placed with a surgical guide and immediately restored with an implant-supported provisional crown. Changes in bone dimensions were assessed using superimposed CBCT images taken prior to surgery and at 1-year follow-up. Clinical outcomes were registered at 1-year follow-up., Results: Twenty patients were randomized to each group (control: 16 females, 4 males, mean age 57.6; test: 9 females, 11 males, mean age 54.2). Ten patients in the control group and 13 patients in the test group had a thick bone wall phenotype. Estimated marginal mean horizontal buccal bone loss at 1 mm below the implant shoulder was -0.553 and -0.898 mm for the control and test group, respectively. The estimated mean difference of 0.344 mm in favour of the control group was not significant (95% CI: -0.415 to 1.104; p = 0.363). Also at all other horizontal and vertical levels, no significant differences could be observed between the groups. The combination of socket grafting and STA enabled counteraction of any buccal soft-tissue loss (≥ 0 mm) at 1 mm below the implant shoulder in 82% of the patients in the control group and in 75% of the patients in the test group (p = 1.000). The clinical outcome was favourable in both groups, yet implants in the control group demonstrated slightly less marginal bone loss (median difference 0.20 mm; 95% CI: 0.00-0.44; p = 0.028)., Conclusion: In patients with an intact and mainly thick buccal bone wall in the anterior maxilla, the timing of STA following IIP had no significant impact on mean buccal bone loss., Trial Registration: ClinicalTrials.gov identifier: NCT05537545., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The use of multimodal contrast agents can potentially overcome the intrinsic limitations of individual imaging methods. We have validated synthetic antiferromagnetic nanoparticles (SAF-NPs) as bimodal contrast agents for in vitro cell labeling and in vivo cell tracking using magnetic resonance imaging (MRI) and computed tomography (CT). SAF-NP-labeled cells showed high contrast in MRI phantom studies (r 2 * = 712 s -1 mM -1 ), while pelleted cells showed clear contrast enhancement in CT. After intravenous SAF-NP injection, nanoparticles accumulated in the liver and spleen, as visualized in vivo by significant MRI contrast enhancement. Intravenous injection of SAF-NP-labeled cells resulted in cell accumulation in the lungs, which was clearly detectable by using CT but not by using MRI. SAF-NPs proved to be very efficient cell labeling agents for complementary MRI- and CT-based cell tracking. Bimodal monitoring of SAF-NP labeled cells is in particular of interest for applications where the applied imaging methods are not able to visualize the particles and/or cells in all organs.

Gold nanoparticles offer the possibility to combine both imaging and therapy of otherwise difficult to treat tumors. To validate and further improve their potential, we describe the use of gold nanostars that were functionalized with a polyethyleneglycol-maleimide coating for in vitro and in vivo photoacoustic imaging (PAI), computed tomography (CT), as well as photothermal therapy (PTT) of cancer cells and tumor masses, respectively. Nanostar shaped particles show a high absorption coefficient in the near infrared region and have a hydrodynamic size in biological medium around 100 nm, which allows optimal intra-tumoral retention. Using these nanostars for in vitro labeling of tumor cells, high intracellular nanostar concentrations could be achieved, resulting in high PAI and CT contrast and effective PTT. By injecting the nanostars intratumorally, high contrast could be generated in vivo using PAI and CT, which allowed successful multi-modal tumor imaging. PTT was successfully induced, resulting in tumor cell death and subsequent inhibition of tumor growth. Therefore, gold nanostars are versatile theranostic agents for tumor therapy.

Alzheimer's disease is the most common neurodegenerative disease, and many patients also present with vascular dysfunction. In this study, we aimed to assess cerebral blood flow (CBF) and cerebrovascular response (CVR) as early, pre-symptomatic (3 months of age), imaging markers in a bigenic model of Alzheimer's disease (APP.V717IxTau.P301L, biAT) and in the monogenic parental strains. We further developed our previously published combination of pulsed arterial spin labeling perfusion MRI and hypo-ventilation paradigm, which allows weaning of the mice from the ventilator. Furthermore, the commonly used isoflurane anesthesia induces vasodilation and is thereby inherently a vascular challenge. We therefore assessed perfusion differences in the mouse models under free-breathing isoflurane conditions. We report (i) that we can determine CBF and hypoventilation-based CVR under ketamine/midazolam anesthesia and wean mice from the ventilator, making it a valuable tool for assessment of CBF and CVR in mice, (ii) that biAT mice exhibit lower cortical CBF than wild-type mice at age 3 months, (iii) that CVR was increased in both biAT and APP.V717I mice but not in Tau.P301L mice, identifying the APP genotype as a strong influencer of brain CVR and (iv) that perfusion differences at baseline are masked by the widely used isoflurane anesthesia., (© 2018 John Wiley & Sons, Ltd.)

Magnetoliposomes (MLs) were synthesized and tested for longitudinal monitoring of transplanted pancreatic islets using magnetic resonance imaging (MRI) in rat models. The rat insulinoma cell line INS-1E and isolated pancreatic islets from outbred and inbred rats were used to optimize labeling conditions in vitro. Strong MRI contrast was generated by islets exposed to 50 µg Fe/ml for 24 hours without any increased cell death, loss of function or other signs of toxicity. In vivo experiments showed that pancreatic islets (50-1000 units) labeled with MLs were detectable for up to 6 weeks post-transplantation in the kidney subcapsular space. Islets were also monitored for two weeks following transplantation through the portal vein of the liver. Hereby, islets labeled with MLs and transplanted under the left kidney capsule were able to correct hyperglycemia and had stable MRI signals until nephrectomy. Interestingly, in vivo MRI of streptozotocin induced diabetic rats transplanted with allogeneic islets demonstrated loss of MRI contrast between 7-16 days, indicative of loss of islet structure. MLs used in this study were not only beneficial for monitoring the location of transplanted islets in vivo with high sensitivity but also reported on islet integrity and hereby indirectly on islet function and rejection.

Pancreatic islets (PIs) transplantation is an alternative approach for the treatment of severe forms of type 1 diabetes (T1D). To monitor the success of transplantation, it is desirable to follow the location of engrafted PIs non-invasively. In vivo magnetic resonance imaging (MRI) of transplanted PIs is a feasible cell tracking method; however, this requires labeling with a suitable contrast agent prior to transplantation. We have tested the feasibility of cationic magnetoliposomes (MLs), compared to commercial contrast agents (Endorem and Resovist), by labeling insulinoma cells and freshly isolated rat PIs. It was possible to incorporate Magnetic Ressonance (MR)-detectable amounts of MLs in a shorter time (4 h) when compared to Endorem and Resovist. MLs did not show negative effects on the PIs' viability and functional parameters in vitro. Labeled islets were transplanted in the renal sub-capsular region of healthy mice. Hypointense contrast in MR images due to the labeled PIs was detected in vivo upon transplantation, while MR detection of PIs labeled with Endorem and Resovist was only possible after the addition of transfection agents. These findings indicate that MLs are suitable to image PIs, without affecting their function, which is promising for future longitudinal pre-clinical and clinical studies involving the assessment of PI transplantation., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

A major conceptual breakthrough in cell signaling has been the finding of EV as new biomarker shuttles in body fluids. Now, one of the major challenges in using these nanometer-sized biological entities as diagnostic marker is the development of translational methodologies to profile them. SPR offers a promising label-free and real time platform with a high potential for biomarker detection. Therefore, we aimed to develop a uniform SPR methodology to detect specific surface markers on EV derived from patient with CHD. EVs having an approximate size range between 30 and 100 nm (~48.5%) and 100-300 nm (~51.5%) were successfully isolated. The biomarker profile of EV was verified using immunogold labeling, ELISA and SPR. Using SPR, we demonstrated an increased binding of EV derived from patients with CHD to anti-ICAM-1 antibodies as compared to EV from healthy donors. Our current findings open up novel opportunities for in-depth and label-free investigation of EV., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background Aims: Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders affecting the normal hematopoietic differentiation process and leading to abnormal maturation and differentiation of all blood cell lineages. Treatment options are limited, and there is an unmet medical need for effective therapies for patients with severe cytopenias., Methods: We demonstrate that multipotent adult progenitor cells (MAPC) improve the function of hematopoietic progenitors derived from human MDS bone marrow (BM) by significantly increasing the frequency of primitive progenitors as well as the number of myeloid colonies., Results: This effect was more pronounced in a non-contact culture, indicating the importance of soluble factors produced by the MAPC cells. Moreover, the cells did not stimulate the growth of the abnormal MDS clone, as shown by fluorescent in situ hybridization analysis on BM cells from patients with a known genetic abnormality. We also demonstrate that MAPC cells can provide stromal support for patient-derived hematopoietic cells. When MAPC cells were intravenously injected into a mouse model of MDS, they migrated to the site of injury and increased the hematopoietic function in diseased mice., Discussion: The preclinical studies undertaken here indicate an initial proof of concept for the use of MAPC cell therapy in patients with MDS-related severe and symptomatic cytopenias and should pave the way for further investigation in clinical trials., (Copyright © 2017. Published by Elsevier Inc.)

We have characterized both acute and long-term vascular and metabolic effects of unilateral common carotid artery occlusion in mice by in vivo magnetic resonance imaging and positron emission tomography. This common carotid artery occlusion model induces chronic cerebral hypoperfusion and is therefore relevant to both preclinical stroke studies, where it serves as a control condition for a commonly used mouse model of ischemic stroke, and neurodegeneration, as chronic hypoperfusion is causative to cognitive decline. By using perfusion magnetic resonance imaging, we demonstrate that under isoflurane anesthesia, cerebral perfusion levels recover gradually over one month. This recovery is paralleled by an increase in lumen diameter and altered tortuosity of the contralateral internal carotid artery at one year post-ligation as derived from magnetic resonance angiography data. Under urethane/α-chloralose anesthesia, no acute perfusion differences are observed, but the vascular response capacity to hypercapnia is found to be compromised. These hemispheric perfusion alterations are confirmed by water [ 15 O]-H 2 O positron emission tomography. Glucose metabolism ([ 18 F]-FDG positron emission tomography) or white matter organization (diffusion-weighted magnetic resonance imaging) did not show any significant alterations. In conclusion, permanent unilateral common carotid artery occlusion results in acute and long-term vascular remodeling, which may have immediate consequences for animal models of stroke but also vascular dementia.

Stroke is the second most common cause of death and is a major cause of permanent disability. Given the current demographic trend of an ageing population and associated increased risk, the prevalence of and socioeconomic burden caused by stroke will continue to rise. Current therapies are unable to sufficiently ameliorate the disease outcome and are not applicable to all patients. Therefore, strategies such as cell-based therapies with mesenchymal stem cell (MSC) or induced pluripotent stem cell (iPSC) pave the way for new treatment options for stroke. These cells showed great preclinical promise despite the fact that the precise mechanism of action and the optimal administration route are unknown. To gain dynamic insights into the underlying repair processes after stem cell engraftment, noninvasive imaging modalities were developed to provide detailed spatial and functional information on the donor cell fate and host microenvironment. This review will focus on MSCs and iPSCs as types of widely used stem cell sources in current (bio)medical research and compare their efficacy and potential to ameliorate the disease outcome in animal stroke models. In addition, novel noninvasive imaging strategies allowing temporospatial in vivo tracking of transplanted cells and coinciding evaluation of neuronal repair following stroke will be discussed., (© 2016 Wiley Periodicals, Inc.)

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents.

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: In this study, we planned to assess if adult stem cell-based suicide gene therapy can efficiently eliminate glioblastoma cells in vivo. We investigated the therapeutic potential of mouse Oct4(-) bone marrow multipotent adult progenitor cells (mOct4(-) BM-MAPCs) in a mouse glioblastoma model, guided by multimodal in vivo imaging methods to identify therapeutic windows., Methods: Magnetic resonance imaging (MRI) of animals, wherein 5 × 10(5) syngeneic enhanced green fluorescent protein-firefly luciferase-herpes simplex virus thymidine kinase (eGFP-fLuc-HSV-TK) expressing and superparamagnetic iron oxide nanoparticle labeled (1 % or 10 %) mOct4(-) BM-MAPCs were grafted in glioblastoma (GL261)-bearing animals, showed that labeled mOct4(-) BM-MAPCs were located in and in close proximity to the tumor. Subsequently, ganciclovir (GCV) treatment was commenced and the fate of both the MAPCs and the tumor were followed by multimodal imaging (MRI and bioluminescence imaging)., Results: In the majority of GCV-treated, but not phosphate-buffered saline-treated animals, a significant difference was found in mOct4(-) BM-MAPC viability and tumor size at the end of treatment. Noteworthy, in some phosphate-buffered saline-treated animals (33 %), a significant decrease in tumor size was seen compared to sham-operated animals, which could potentially also be caused by a synergistic effect of the immune-modulatory stem cells., Conclusions: Suicide gene therapy using mOct4(-) BM-MAPCs as cellular carriers was effective in reducing the tumor size in the majority of the GCV-treated animals leading to a longer progression-free survival compared to sham-operated animals. This treatment could be followed and guided noninvasively in vivo by MRI and bioluminescence imaging. Noninvasive imaging is of particular interest for a rapid and efficient validation of stem cell-based therapeutic approaches for glioblastoma and hereby contributes to a better understanding and optimization of a promising therapeutic approach for glioblastoma patients.

Cell-based therapies are emerging as an alternative treatment option to promote functional recovery in patients suffering from neurological disorders, which are the major cause of death and permanent disability. The present study aimed to differentiate human dental pulp stem cells (hDPSCs) toward functionally active neuronal cells in vitro. hDPSCs were subjected to a two-step protocol. First, neuronal induction was acquired through the formation of neurospheres, followed by neuronal maturation, based on cAMP and neurotrophin-3 (NT-3) signaling. At the ultrastructural level, it was shown that the intra-spheral microenvironment promoted intercellular communication. hDPSCs grew out of the neurospheres in vitro and established a neurogenic differentiated hDPSC culture (d-hDPSCs) upon cAMP and NT-3 signaling. d-hDPSCs were characterized by the increased expression of neuronal markers such as neuronal nuclei, microtubule-associated protein 2, neural cell adhesion molecule, growth-associated protein 43, synapsin I, and synaptophysin compared with nondifferentiated hDPSCs. Enzyme-linked immunosorbent assay demonstrated that the secretion of brain-derived neurotrophic factor, vascular endothelial growth factor, and nerve growth factor differed between d-hDPSCs and hDPSCs. d-hDPSCs acquired neuronal features, including multiple intercommunicating cytoplasmic extensions and increased vesicular transport, as shown by the electron microscopic observation. Patch clamp analysis demonstrated the functional activity of d-hDPSCs by the presence of tetrodotoxin- and tetraethyl ammonium-sensitive voltage-gated sodium and potassium channels, respectively. A subset of d-hDPSCs was able to fire a single action potential. The results reported in this study demonstrate that hDPSCs are capable of neuronal commitment following neurosphere formation, characterized by distinct morphological and electrophysiological properties of functional neuronal cells.

Adult stem cells have been investigated increasingly over the past years for multiple applications. Although they have a more favorable safety profile compared to pluripotent stem cells, they are still capable of self-renewal and differentiate into several cell types. We investigated the behavior of Oct4-positive (Oct4(+)) and Oct4-negative (Oct4(-) ) murine or rat bone marrow (BM)-derived stem cells in the healthy brain of syngeneic mice and rats. Engraftment of mouse and rat Oct4-positive BM-derived hypoblast-like stem cells (m/rOct4(+) BM-HypoSCs) resulted in yolk-sac tumor formation in the healthy brain which was monitored longitudinally using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Contrast enhanced MRI confirmed the disruption of the blood brain barrier. In contrast, m/r Oct4-negative BM-derived multipotent adult progenitor cells (m/rOct4(-) BM-MAPCs) did not result in mass formation after engraftment into the brain. mOct4(+) BM-HypoSCs and mOct4(-) BM-MAPCs were transduced to express enhanced green fluorescent protein, firefly luciferase (fLuc), and herpes simplex virus-thymidine kinase to follow up suicide gene expression as a potential "safety switch" for tumor-forming stem cells by multimodal imaging. Both cell lines were eradicated efficiently in vivo by ganciclovir administration indicating successful suicide gene expression in vivo, as assessed by MRI, BLI, and histology. The use of suicide genes to prevent tumor formation is in particular of interest for therapeutic approaches where stem cells are used as vehicles to deliver therapeutic genes., (© 2014 AlphaMed Press.)

Purpose: The aim of the present study was to describe nerve fibres around osseointegrated implants in humans., Materials and Methods: Twelve mechanically failed implants, retrieved from 10 patients were collected from three dental centres over a period of 5 years. After implant removal, decalcified semi-thin sections (0.5 μm) were stained with thionic methylene blue for light microscopic analysis. In addition, an ultrastructural analysis was performed on serial ultra-thin sections (0.06 μm) using transmission electron microscopy., Results: Both myelinated and unmyelinated nerve fibres could be identified inside the Haversian canals of the osteonal bone near the implant threads. Myelinated fibres were also located at the woven bone around the implant. However, no differentiated nerve endings could be observed around the implants., Conclusions: This study shows the presence of nerve fibres in human peri-implant bone. Previous studies in animals showed that those fibres participate in the process of bone modelling and remodelling. Yet, the role of peri-implant bone innervation in the osseoperception phenomenon cannot be ruled out since the mechanism of mechanoreception in bone is not fully understood.

Blood brain barrier (BBB) disruption is used (pre)clinically as a measure for brain tumor malignancy and grading. During treatment it is one of the parameters followed rigorously to assess therapeutic efficacy. In animal models, both invasive and non-invasive methods are used to determine BBB disruption, among them Evans blue injection prior to sacrifice and T1-weighted magnetic resonance imaging (MRI) post contrast injection. In this study, we have assessed the BBB integrity with the methods mentioned above in two experimental high grade glioma models, namely the GL261 mouse glioblastoma model and the Hs683 human oligodendroglioma model. The GL261 model showed clear BBB integrity loss with both, contrast-enhanced (CE) MRI and Evans blue staining. In contrast, the Hs683 model only displayed BBB disruption with CE-MRI, which was not evident on Evans blue staining, indicating a limited BBB disruption. These results clearly indicate the importance of assessing the BBB integrity status using appropriate methods. Especially when using large therapeutic molecules that have difficulties crossing the BBB, care should be taken with the appropriate BBB disruption assessment studies.

Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific world because of their differentiation potential into osteoblasts, chondroblasts and adipocytes and possible transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothesized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angiogenic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence, MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogenesis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial cells will be evaluated critically., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters., Methods: First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, 124I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using 99mTcO4- radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cell's differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI., Results: The expression of both imaging reporter genes was functional and specific. An elution of 99mTcO4- from the cells was observed, with 31% retention after 3 h. After labeling cells with 124I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using 124I small-animal PET, CLI and BLI., Conclusions: This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury.

Magnetic nanoparticle (MNP) enabled cell visualization with magnetic resonance imaging (MRI) is currently an intensively studied area of research. In the present study, we have synthesized polyethylene glycolated (PEG) MNPs and validated their suitability as MR cell labeling agents in in vitro and in vivo experiments. The labeling of therapeutic potent mesenchymal stem cells (MSCs) with small core and large core MNPs was evaluated. Both MNPs were, in combination with a transfection agent, stably internalized into the MSCs and didn't show an effect on cell metabolism. The labeled cells showed high contrast in MRI phantom studies. For quantification purposes, the MRI contrast generating properties of cells labeled with small core MNPs were compared with large core MNPs and with the commercial contrast agent Endorem. MSCs labeled with the large core MNPs showed the highest contrast generating properties in in vitro phantom studies and in in vivo intracranial stereotactic injection experiments, confirming the size-relaxivity relationship in biological systems. Finally, the distribution of MSCs pre-labeled with large core PEGylated MNPs was visualized non-invasively with MRI in a glioma model., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.

Unlabelled: Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as (18)F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with (18)F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM)., Methods: Mouse MSCs and rat MAPCs were used for (18)F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with (18)F-FDG, and stem cell biodistribution was investigated using small-animal PET., Results: The optimal incubation period for (18)F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%-40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by (18)F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of (18)F-FDG from the cells., Conclusion: MSCs and MAPCs can be successfully labeled with (18)F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells' ultrastructural properties are not influenced by (18)F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with (18)F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.

Demyelination is one of the pathological hallmarks of multiple sclerosis (MS). To date, no therapy is available which directly potentiates endogenous remyelination. Interleukin-11 (IL-11), a member of the gp130 family of cytokines, is upregulated in MS lesions. Systemic IL-11 treatment was shown to ameliorate clinical symptoms in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. IL-11 modulates immune cells and protects oligodendrocytes in vitro. In this study, the cuprizone-induced demyelination mouse model was used to elucidate effects of IL-11 on de- and remyelination, independent of the immune response. Prophylactic-lentiviral- (LV-) mediated overexpression of IL-11 in mouse brain significantly limited acute demyelination, which was accompanied with the preservation of CC1(+) mature oligodendrocytes (OLs) and a decrease in microglial activation (Mac-2(+)). We further demonstrated that IL-11 directly reduces myelin phagocytosis in vitro. When IL-11 expressing LV was therapeutically applied in animals with extensive demyelination, a significant enhancement of remyelination was observed as demonstrated by Luxol Fast Blue staining and electron microscopy imaging. Our results indicate that IL-11 promotes maturation of NG2(+) OPCs into myelinating CC1(+) OLs and may thus explain the enhanced remyelination. Overall, we demonstrate that IL-11 is of therapeutic interest for MS and other demyelinating diseases by limiting demyelination and promoting remyelination.

Purpose: Recent ex vivo and pharmacological evidence suggests involvement of the endocannabinoid system in the pathophysiology of stroke, but conflicting roles for type 1 and 2 cannabinoid receptors (CB(1) and CB(2)) have been suggested. The purpose of this study was to evaluate CB(1) and CB(2) receptor binding over time in vivo in a rat photothrombotic stroke model using PET., Methods: CB(1) and CB(2) microPET imaging was performed at regular time-points up to 2 weeks after stroke using [(18)F]MK-9470 and [(11)C]NE40. Stroke size was measured using MRI at 9.4 T. Ex vivo validation was performed via immunostaining for CB(1) and CB(2). Immunofluorescent double stainings were also performed with markers for astrocytes (GFAP) and macrophages/microglia (CD68)., Results: [(18)F]MK-9470 PET showed a strong increase in CB(1) binding 24 h and 72 h after stroke in the cortex surrounding the lesion, extending to the insular cortex 24 h after surgery. These alterations were consistently confirmed by CB(1) immunohistochemical staining. [(11)C]NE40 did not show any significant differences between stroke and sham-operated animals, although staining for CB(2) revealed minor immunoreactivity at 1 and 2 weeks after stroke in this model. Both CB (1) (+) and CB (2) (+) cells showed minor immunoreactivity for CD68., Conclusion: Time-dependent and regionally strongly increased CB(1), but not CB(2), binding are early consequences of photothrombotic stroke. Pharmacological interventions should primarily aim at CB(1) signalling as the role of CB(2) seems minor in the acute and subacute phases of stroke.

Introduction: Auto-transplantation of third molars is frequently undertaken in order to restore a perfect occlusion and to improve mastication following a substantial loss of molars. However, little is known about the precise role of the periodontal membrane during this procedure. Therefore, we investigated if the epithelial rests of Malassez persist in the periodontal ligament of auto-transplanted teeth and, if so, whether these may show signs of a neuro-epithelial relationship., Case Presentation: We report a case of a 21-year-old Caucasian woman who underwent an auto-transplantation of two third molars. After two years, renewed progressive caries of the auto-transplanted teeth led to the removal of the auto-transplanted elements. The periodontal ligament was removed and studied with a light and transmission electron microscope., Conclusion: In this report we examined the ultrastructure of the periodontal ligament after auto-transplantation in order to see if the periodontal ligament recovers completely from this intervention. We observed fully developed blood vessels and a re-innervation of the epithelial rests of Malassez which were proliferating following auto-transplantation. This proliferation might be critical in the remodelling of the alveolar socket in order to provide a perfect fit for the transplanted tooth. In order to minimalise the damage to the epithelial rests of Malassez, the extraction of the tooth should be as atraumatic as possible in order to provide an optimal conservation of the periodontal ligament which will be beneficial to the healing-process.