Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue

AbstractSorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34−, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1−/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34− cells. Permeabilized cs-DLK1−/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31−). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34− and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34− perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1−/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45−/CD31− cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity

Palisade Endings Are a Constant Feature in the Extraocular Muscles of Frontal-Eyed, But Not Lateral-Eyed, Animals

Purpose To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs). Methods Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done. Results In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exhibited an EOM-specific distribution. In particular, the number of palisade endings in the medial recti was significantly higher than in the other rectus muscles. In the lateral-eyed animals, palisade endings were infrequent and, when present, their total number was rather low. They were only found in ungulates (sheep, calf, pig, and horse) and in rabbit. In rodents (rat, guinea pig, mouse, and gerbil) palisade endings were found infrequently (e.g., rat) or were completely absent. Palisade endings in frontal-eyed species and in some lateral-eyed species (pig, sheep, calf, and horse) had a uniform morphology. They generally lacked α-bungarotoxin staining, with a few exceptions in primates. Palisade endings in other lateral-eyed species (rabbit and rat) exhibited a simplified morphology and bound α-bungarotoxin. Conclusions Palisade endings are not a universal feature of mammalian EOMs. So, if they are proprioceptors, not all species require them. Because in frontal-eyed species, the medial rectus muscle has the highest number of palisade endings, they likely play a special role in convergence

Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging

Design and implantation of bionic implants for restoring impaired hair cell function relies on accurate knowledge about the microanatomy and nerve fiber pathways of the human inner ear and its variation. Non-destructive isotropic imaging of soft tissues of the inner ear with lab-based microscopic X-ray computed tomography (microCT) offers high resolution but requires contrast enhancement using compounds with high X-ray attenuation. We evaluated different contrast enhancement techniques in mice, cat, and human temporal bones to differentially visualize the membranous labyrinth, sensory epithelia, and their innervating nerves together with the facial nerve and middle ear. Lugol’s iodine potassium iodine (I2KI) gave high soft tissue contrast in ossified specimens but failed to provide unambiguous identification of smaller nerve fiber bundles inside small bony canals. Fixation or post-fixation with osmium tetroxide followed by decalcification in EDTA provided superior contrast for nerve fibers and membranous structures. We processed 50 human temporal bones and acquired microCT scans with 15 μm voxel size. Subsequently we segmented sensorineural structures and the endolymphatic compartment for 3D representations to serve for morphometric variation analysis. We tested higher resolution image acquisition down to 3.0 μm voxel size in human and 0.5 μm in mice, which provided a unique level of detail and enabled us to visualize single neurons and hair cells in the mouse inner ear, which could offer an alternative quantitative analysis of cell numbers in smaller animals. Bigger ossified human temporal bones comprising the middle ear and mastoid bone can be contrasted with I2KI and imaged in toto at 25 μm voxel size. These data are suitable for surgical planning for electrode prototype placements. A preliminary assessment of geometric changes through tissue processing resulted in 1.6% volume increase caused during decalcification by EDTA and 0.5% volume increase caused by partial dehydration to 70% ethanol, which proved to be the best mounting medium for microCT image acquisition

Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells.

INTRODUCTION: We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. METHODS: Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. RESULTS: Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. CONCLUSION: We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers

Data_Sheet_2_Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging.pdf

<p>Design and implantation of bionic implants for restoring impaired hair cell function relies on accurate knowledge about the microanatomy and nerve fiber pathways of the human inner ear and its variation. Non-destructive isotropic imaging of soft tissues of the inner ear with lab-based microscopic X-ray computed tomography (microCT) offers high resolution but requires contrast enhancement using compounds with high X-ray attenuation. We evaluated different contrast enhancement techniques in mice, cat, and human temporal bones to differentially visualize the membranous labyrinth, sensory epithelia, and their innervating nerves together with the facial nerve and middle ear. Lugol’s iodine potassium iodine (I₂KI) gave high soft tissue contrast in ossified specimens but failed to provide unambiguous identification of smaller nerve fiber bundles inside small bony canals. Fixation or post-fixation with osmium tetroxide followed by decalcification in EDTA provided superior contrast for nerve fibers and membranous structures. We processed 50 human temporal bones and acquired microCT scans with 15 μm voxel size. Subsequently we segmented sensorineural structures and the endolymphatic compartment for 3D representations to serve for morphometric variation analysis. We tested higher resolution image acquisition down to 3.0 μm voxel size in human and 0.5 μm in mice, which provided a unique level of detail and enabled us to visualize single neurons and hair cells in the mouse inner ear, which could offer an alternative quantitative analysis of cell numbers in smaller animals. Bigger ossified human temporal bones comprising the middle ear and mastoid bone can be contrasted with I₂KI and imaged in toto at 25 μm voxel size. These data are suitable for surgical planning for electrode prototype placements. A preliminary assessment of geometric changes through tissue processing resulted in 1.6% volume increase caused during decalcification by EDTA and 0.5% volume increase caused by partial dehydration to 70% ethanol, which proved to be the best mounting medium for microCT image acquisition.</p

Data_Sheet_1_Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging.pdf

<p>Design and implantation of bionic implants for restoring impaired hair cell function relies on accurate knowledge about the microanatomy and nerve fiber pathways of the human inner ear and its variation. Non-destructive isotropic imaging of soft tissues of the inner ear with lab-based microscopic X-ray computed tomography (microCT) offers high resolution but requires contrast enhancement using compounds with high X-ray attenuation. We evaluated different contrast enhancement techniques in mice, cat, and human temporal bones to differentially visualize the membranous labyrinth, sensory epithelia, and their innervating nerves together with the facial nerve and middle ear. Lugol’s iodine potassium iodine (I₂KI) gave high soft tissue contrast in ossified specimens but failed to provide unambiguous identification of smaller nerve fiber bundles inside small bony canals. Fixation or post-fixation with osmium tetroxide followed by decalcification in EDTA provided superior contrast for nerve fibers and membranous structures. We processed 50 human temporal bones and acquired microCT scans with 15 μm voxel size. Subsequently we segmented sensorineural structures and the endolymphatic compartment for 3D representations to serve for morphometric variation analysis. We tested higher resolution image acquisition down to 3.0 μm voxel size in human and 0.5 μm in mice, which provided a unique level of detail and enabled us to visualize single neurons and hair cells in the mouse inner ear, which could offer an alternative quantitative analysis of cell numbers in smaller animals. Bigger ossified human temporal bones comprising the middle ear and mastoid bone can be contrasted with I₂KI and imaged in toto at 25 μm voxel size. These data are suitable for surgical planning for electrode prototype placements. A preliminary assessment of geometric changes through tissue processing resulted in 1.6% volume increase caused during decalcification by EDTA and 0.5% volume increase caused by partial dehydration to 70% ethanol, which proved to be the best mounting medium for microCT image acquisition.</p

Primary antibodies used for immunohistochemistry.

<p>Primary antibodies used for immunohistochemistry.</p

Tumour microtissue formation.

<p>SEM pictures were taken after ten days: Monocultures of A549 and SV80 were seeded in a ratio of 2500 cells/40 μl, whereas Colo699 monocultures were cultured in form of 1250 cells/40 μl. All co-cultures were seeded in a carcinoma cell: fibroblast ratio of 1∶2/40 μl (A549 co-cultures: 2500 cancer cells +5000 fibroblasts / Colo699 co-cultures: 1250 cancer cells +2500 fibroblasts). (A/B) A549 monocultures, (C/D) A549 co-cultures; (E/F) Colo699 monocultures, (G/H) Colo699 co-cultures; (I/J) SV80 monocultures. All co-cultures displayed a more homogenous and rounder microtissue surface compared to monocultures.</p

Cytokeratin 7 expression pattern.

<p>IHC staining of A549, SV80 monocultures and A549 co-cultures after ten days (Bar in 1–3: 100 μm). Cytokeratin 7 staining could only be observed in cancer cells but not in SV80 cells.</p