Immunohistochemical Localization and Characterization of Putative Mesenchymal Stem Cell Markers in the Retinal Capillary Network of Rodents

Perivascular cells of microvascular niches are the prime candidates for being a reservoire of mesenchymal stem cell (MSC)-like cells in many tissues and organs that could serve as a potential source of cells and a target of novel cell-based therapeutic approaches. In the present study, by utilising typical markers of pericytes (neuronal-glial antigen 2, NG2, a chondroitin sulphate proteoglycan) and those of MSCs (CD146 and CD105) and primitive pluripotent cells (sex-determining region Y-box 2, Sox2), the phenotypic traits and the distribution of murine and rat retinal perivascular cells were investigated in situ. Our findings indicate that retinal microvessels of juvenile rodents are highly covered by NG2-positive branching processes of pericytic (perivascular) cells that are less prominent in mature capillary networks of the adult retina. In the adult rodent retinal vascular bed, NG2 labeling is mainly confined to membranes of the cell body resulting in a pearl-chain-like distribution along the vessels. Retinal pericytes, which were identified by their morphology and NG2 expression, simultaneously express CD146. Furthermore, CD146-positive cells located at small arteriole-tocapillary branching points appear more intensely stained than elsewhere. Evidence for a differential expression of the two markers around capillaries that would hint at a clonal heterogeneity among pericytic cells, however, is lacking. In contrast, the expression of CD105 is exclusively restricted to vascular endothelial cells and Sox2 is detected neither in perivascular nor in endothelial cells. In dissociated retinal cultures, however, simultaneous expression of NG2 and CD105 was observed. Collectively, our data indicate that vascular wall resident retinal pericytes share some phenotypic features (i.e. CD146 expression) with archetypal MSCs, which is even more striking in dissociated retinal cultures (i.e. CD105 expression). These findings might have implications for the treatment of retinal pathologies

First Experience With The GoBack-Catheter For Successful Crossing of Complex Chronic Total Occlusions in Lower Limb Arteries

Purpose: To evaluate the use of the GoBack-catheter (Upstream Peripheral Technologies) in complex revascularizations in lower limb arteries. Materials and Methods: In this retrospective single-center study, the results of the first 100 consecutive patients including 101 limb-revascularizations, performed between May 2018 and July 2020 with the study device, were analyzed. In all cases, guidewire-crossing failed, and all lesions were chronic total occlusions (CTO), either de novo, reocclusions, or in-stent reocclusions. Successful crossing was defined as passing the CTO using the study device. Patency at discharge and after 30 days was defined as less than 50% restenosis on duplex sonography, without target lesion revascularization. Results: Median lesion length was 24 cm and 38 patients (37.6%) had a calcium grading according to the peripheral arterial calcium scoring system (PACSS) of 4 or 5. In 20.8% of patients, an occluded stent was treated. CTOs involved the femoropopliteal segment in 91.1%, iliac arteries in 5.9%, and tibial arteries in 7.9%. The GoBackcatheter was employed for entering into or crossing through parts or the full length of a CTO or an occluded stent as well as for re-entering into the true lumen after subintimal crossing. The device was used via contralateral and ipsilateral antegrade as well as retrograde access with an overall technical success rate of 92.1%. In 3 patients minor bleeding occurred at the crossing or re-entry site, which were managed conservatively. Thirty-day adverse limb events comprised minor amputations in 4 patients (4.0%), 1 major amputation (1.0%), and reocclusions in 7 limbs (6.9%). Conclusion: The new GoBack-catheter offers versatile endovascular applicability for complex CTO recanalization in a broad range of peripheral vascular interventions with a high technical success and low complication rate

Multipotent glia-like stem cells mediate stress adaptation

The neural crest-derived adrenal medulla is closely related to the sympathetic nervous system; however, unlike neural tissue, it is characterized by high plasticity which suggests the involvement of stem cells. Here, we show that a defined pool of glia-like nestin–expressing progenitor cells in the adult adrenal medulla contributes to this plasticity. These glia-like cells have features of adrenomedullary sustentacular cells, are multipotent, and are able to differentiate into chromaffin cells and neurons. The adrenal is central to the body’s response to stress making its proper adaptation critical to maintaining homeostasis. Our results from stress experiments in vivo show the activation and differentiation of these progenitors into new chromaffin cells. In summary, we demonstrate the involvement of a new glia-like multipotent stem cell population in adrenal tissue adaptation. Our data also suggest the contribution of stem and progenitor cells in the adaptation of neuroendocrine tissue function in general

Imaging of nanoparticle-labeled stem cells using magnetomotive optical coherence tomography, laser speckle reflectometry, and light microscopy

Cell transplantation and stem cell therapy are promising approaches for regenerative medicine and are of interest to researchers and clinicians worldwide. However, currently, no imaging technique that allows three-dimensional in vivo inspection of therapeutically administered cells in host tissues is available. Therefore, we investigate magnetomotive optical coherence tomography (MM-OCT) of cells labeled with magnetic particles as a potential noninvasive cell tracking method. We develop magnetomotive imaging of mesenchymal stem cells for future cell therapy monitoring. Cells were labeled with fluorescent iron oxide nanoparticles, embedded in tissue-mimicking agar scaffolds, and imaged using a microscope setup with an integrated MM-OCT probe. Magnetic particle-induced motion in response to a pulsed magnetic field of 0.2 T was successfully detected by OCT speckle variance analysis, and cross-sectional and volumetric OCT scans with highlighted labeled cells were obtained. In parallel, fluorescence microscopy and laser speckle reflectometry were applied as two-dimensional reference modalities to image particle distribution and magnetically induced motion inside the sample, respectively. All three optical imaging modalities were in good agreement with each other. Thus, magnetomotive imaging using iron oxide nanoparticles as cellular contrast agents is a potential technique for enhanced visualization of selected cells in OCT

Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes

Background: Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. Methodology and Principal Findings: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca2+ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. Conclusions and Significance: Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic

First Experience With The GoBack-Catheter For Successful Crossing of Complex Chronic Total Occlusions in Lower Limb Arteries

Purpose: To evaluate the use of the GoBack-catheter (Upstream Peripheral Technologies) in complex revascularizations in lower limb arteries. Materials and Methods: In this retrospective single-center study, the results of the first 100 consecutive patients including 101 limb-revascularizations, performed between May 2018 and July 2020 with the study device, were analyzed. In all cases, guidewire-crossing failed, and all lesions were chronic total occlusions (CTO), either de novo, reocclusions, or in-stent reocclusions. Successful crossing was defined as passing the CTO using the study device. Patency at discharge and after 30 days was defined as less than 50% restenosis on duplex sonography, without target lesion revascularization. Results: Median lesion length was 24 cm and 38 patients (37.6%) had a calcium grading according to the peripheral arterial calcium scoring system (PACSS) of 4 or 5. In 20.8% of patients, an occluded stent was treated. CTOs involved the femoropopliteal segment in 91.1%, iliac arteries in 5.9%, and tibial arteries in 7.9%. The GoBackcatheter was employed for entering into or crossing through parts or the full length of a CTO or an occluded stent as well as for re-entering into the true lumen after subintimal crossing. The device was used via contralateral and ipsilateral antegrade as well as retrograde access with an overall technical success rate of 92.1%. In 3 patients minor bleeding occurred at the crossing or re-entry site, which were managed conservatively. Thirty-day adverse limb events comprised minor amputations in 4 patients (4.0%), 1 major amputation (1.0%), and reocclusions in 7 limbs (6.9%). Conclusion: The new GoBack-catheter offers versatile endovascular applicability for complex CTO recanalization in a broad range of peripheral vascular interventions with a high technical success and low complication rate

First Experience With The GoBack-Catheter For Successful Crossing of Complex Chronic Total Occlusions in Lower Limb Arteries

Purpose: To evaluate the use of the GoBack-catheter (Upstream Peripheral Technologies) in complex revascularizations in lower limb arteries. Materials and Methods: In this retrospective single-center study, the results of the first 100 consecutive patients including 101 limb-revascularizations, performed between May 2018 and July 2020 with the study device, were analyzed. In all cases, guidewire-crossing failed, and all lesions were chronic total occlusions (CTO), either de novo, reocclusions, or in-stent reocclusions. Successful crossing was defined as passing the CTO using the study device. Patency at discharge and after 30 days was defined as less than 50% restenosis on duplex sonography, without target lesion revascularization. Results: Median lesion length was 24 cm and 38 patients (37.6%) had a calcium grading according to the peripheral arterial calcium scoring system (PACSS) of 4 or 5. In 20.8% of patients, an occluded stent was treated. CTOs involved the femoropopliteal segment in 91.1%, iliac arteries in 5.9%, and tibial arteries in 7.9%. The GoBackcatheter was employed for entering into or crossing through parts or the full length of a CTO or an occluded stent as well as for re-entering into the true lumen after subintimal crossing. The device was used via contralateral and ipsilateral antegrade as well as retrograde access with an overall technical success rate of 92.1%. In 3 patients minor bleeding occurred at the crossing or re-entry site, which were managed conservatively. Thirty-day adverse limb events comprised minor amputations in 4 patients (4.0%), 1 major amputation (1.0%), and reocclusions in 7 limbs (6.9%). Conclusion: The new GoBack-catheter offers versatile endovascular applicability for complex CTO recanalization in a broad range of peripheral vascular interventions with a high technical success and low complication rate

Transfer of endocytic organelles between ARPE-19 cells.

<p>(A-F) Mixed populations of CTG (green) and DID (red) labelled ARPE-19 cells were cocultured for up to 24 hours and analyzed by fluorescence microscopy. As acceptor population, cells were labelled with CTG (A-B). As an organelle donor population, cells were labelled with DiD leading to red fluorescently labelled endocytic organelles (C-D). After populations were mixed, images were obtained 1 hour (A, C, E) and 24 hours (B, D, F) after seeding. The arrows show identical cells in panels B, D, F, respectively. Double-positive cells are indicative of organelle transfer (arrow in F). Scale bar, 20 μm. (G-H) FACS analyses revealed a significant (p < 0.001, t-test) transfer of 11.04% labelled endocytic organelles after 24 hours cultivation in comparison to 1 hour. A representative dot plot from the flow cytometry analysis is shown in (H).</p

Membrane nanotubes of ARPE -19 cells contained F-actin but not microtubules.

<p>Fluorescence image of ARPE-19 cells stained with phalloidin-TRITC for actin (A, B) anti-ß-tubulin (C, D) and nucleus (DAPI). Images represent the cytoskeleton in control (no treatment A, C, E) and after treatment with 15 µM nocodazole for 24 h (B, D, F). (A, B) Fluorescence image of F-actin. Actin fibres were visible in the cells and clearly seen is a straight connection, representing the TNT structure, between the cells (arrows). (C, D) Fluorescence image of ARPE-19 cells stained with mAb against β-tubulin revealed the lack of β-tubulin in the TNTs (arrows). (E, F) Merged pictures with actin (red), ß-tubulin (green) and nucleus (blue). Arrows mark TNTs. Scale bar, 20 µm.</p