Journal: Neurology Research International

Article Title: Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury

doi: 10.1155/2011/891605

Figure Lengend Snippet: Neural and human markers with differentiation of the hNT2.19 cell line in vitro. The hNT2.19 cell line was treated for two weeks with retinoic acid and mitotic inhibitors and lifted to substrate-coated 8-well plastic TC slides for differentiation and immunohistochemistry for neuron-specific markers. As soon as 4 days in vitro, a variety of neural markers appeared, which remained strong until at least 6 wks of differentiation: TuJ1 (a), hNSE (b), NFL (c), NFM (d), and NFH (e). For comparison, the negative control hNT2.6 cell line was cultured similarly as the hNT2.19 cells and is here stained for TuJ1 (f). Magnification bar = 20 nm, (a–f).

Article Snippet: For immunohistochemistry of sectioned spinal cord tissues, the polyclonal antibody anti-5HT (ab10385; dilution 1/100 (in vivo)) was purchased from Abcam Inc, Cambridge, MA, and the antihuman TuJ1 antibody (Neuron-specific class III beta-tubulin) was purchased from Neuromics, Edina, MN (MO15013; dilution 1/100 (in vivo).

Techniques: In Vitro, Immunohistochemistry, Comparison, Negative Control, Cell Culture, Staining

Journal: Neurology Research International

Article Title: Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury

doi: 10.1155/2011/891605

Figure Lengend Snippet: Transplant of hNT2.19 and hNT2.6 cell lines in the severe contusive SCI model: TuJ1 and 5HT immunohistochemistry. Rats were injured with severe contusive SCI followed at two weeks by hNT2.6 (a, b) or hNT2.19 (c, d) cell grafts. Sagittal spinal cord sections were examined at 8 wks after SCI for evidence of surviving lumbar subarachnoid hNT2.6 (a, b) or hNT2.19 (c, d) cell line grafts, utilizing TuJ1 (a, c) or 5HT (b, d) immunohistochemistry. The hNT2.19 and control hNT2.6 (10 6 cells/injection), which had been differentiated for two weeks in vitro, were injected into the subarachnoid space two weeks after the SCI. Cell graft sites were colocalized with 5HT (b, d) and the human-specific marker TUJ1 (neuron-specific class III β -tubulin; (a, c)). There are many surviving hNT2.19 (c) and hNT2.6 (a) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI and about 6 weeks after cell transplant. Adjacent sections with the same grafted hNT2.19 (d) and hNT2.6 cells (b) are stained for 5HT, but only the hNT2.19 cells (d) are labeled for 5HT (arrows).

Article Snippet: For immunohistochemistry of sectioned spinal cord tissues, the polyclonal antibody anti-5HT (ab10385; dilution 1/100 (in vivo)) was purchased from Abcam Inc, Cambridge, MA, and the antihuman TuJ1 antibody (Neuron-specific class III beta-tubulin) was purchased from Neuromics, Edina, MN (MO15013; dilution 1/100 (in vivo).

Techniques: Immunohistochemistry, Control, Injection, In Vitro, Marker, Staining, Labeling

Journal: Scientific Reports

Article Title: Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

doi: 10.1038/s41598-019-52204-7

Figure Lengend Snippet: Characterization of vascularized neural organoids. Neural spheres consisting of Sox1 + neuroepithelial cells ( A ) and Brachyury + mesodermal spheres. ( B ) Spheres were brought in co-culture. The formation of chimeric neuro-mesodermal aggregates was observed ( C ). The aggregates were cultured for up to 280 days ( D ). ( E ) HE-staining of sections showing the interface between neuroepithelial (left side) and mesenchymal part (right side). ( F – G ) Immunofluorescence analyses showing the Pax6 + neuroepithelium and CD31 + endothelial cells. ( H – D ) The neural part consists of Sox1 + stem cells and TUJ1 + neurons. ( J – K ) At the interface between neuroepithelium and mesenchyme, CD31 + vessels form a perineural plexus. ( L ) A capillary at the perineural plexus in an ED 5 chicken embryo is depicted. Blood cells show green autofluorescence. ( M ) Within the mesodermal part an extensive network of blood vessels can be observed. M shows a maximum intensity projection of a whole mount stained organoid. ( N ) HE staining of paraffin sections from the mesodermal part of an organoid. The picture shows a vessel structure with clear lumen (asterisk) and a small capillary (arrowhead). ( O – S ) Transmission electron microscopic pictures showing the endothelium of vessel structures within the organoids. *Periendothelial cells, EC: endothelial cells, BM: basement membrane, IJ: intercellular junction, CV: caveolae, MV: microvesicle.

Article Snippet: Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used.

Techniques: Co-Culture Assay, Cell Culture, Staining, Immunofluorescence, Transmission Assay, Membrane

Journal: Scientific Reports

Article Title: Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

doi: 10.1038/s41598-019-52204-7

Figure Lengend Snippet: Characterization of the neural part of the organoid. Pictures show organoids at culture day 210. ( A ) HE-stained sections reveal ventricle-like cavities inside the neural tissue. ( B – C ) Neuroepithelial cells lining the ventricle-like structures stain positive for Sox1 and Nestin, while the cells surrounding the stem cell zone express the neuronal marker protein MAP2. ( C ) Shows a higher magnification of B. ( D ) The apical side of the neuroepithelium is marked by N-Cadherin + cell-cell junctions. TUJ1 + cells can be found towards the basal side indicating neuronal differentiation. ( E ) Besides TUJ1 + neurons, GFAP + radial glia cells or astrocytes can be found located towards the basal side of the epithelium.

Article Snippet: Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used.

Techniques: Staining, Marker

Journal: Scientific Reports

Article Title: Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

doi: 10.1038/s41598-019-52204-7

Figure Lengend Snippet: Vascularization of the neural part of the organoid and invasion of microglia-like cells. ( A ) At day 180 a CD31 + endothelial network infiltrating the Pax6 + neural part of the organoid was observed. ( B ) shows a higher magnification of A. ( C ) Infiltrating vessels were found within the neural tissue surrounding Sox1 + neuroepithelial structures. ( D – E ) CD31 + capillaries within the neural part of the organoid closely associated with GFAP + astrocytes/radial glia and TUJ1 + neurons. ( F ) GFAP + cells form close contacts with CD31 + endothelial cells (see inset). ( G ) Iba1 + macrophage-like cells were observed in close association with CD31 + capillaries within the mesodermal part of the organoid. ( H ) Iba1 + cells were also found within the neural part of the organoid being tightly associated with TUJ1 + neurons suggesting a microglia-like identity. ( I ) Microglia-like cells show a cellular morphology similar to microglial cells at the transition from an amoeboid to a ramified appearance. ( A , B , F , G , H ) Show maximum intensity projections of whole-mount stained cleared organoids. ( C , D , E , I) show stained paraffin sections.

Article Snippet: Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration

doi: 10.3390/ijms241713642

Figure Lengend Snippet: Spinal cord cells (SCC) cultivated in direct biomaterial contact are labeled with anti-βIII-tubulin (TUJ1, green, neurons) and glial fibrillary acidic protein (GFAP, red, astrocytes) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue, cell nucleus). Immunofluorescence of SCC in direct contact with ( A ) control (CTL), ( B ) chitosan (CHI), ( C ) poly (ε-caprolactone) (PCL), and ( D ) poly (L-lactic acid) (PLLA). ( E – H ) Zoom boxes with high magnification of the selected area in A–E evidencing TUJ1 labeling. ( I – L ) Zoom boxes with high magnification of the selected area in A–E evidencing GFAP labeling. ( M ) Graph representing the percentage of cells labeling TUJ1 and GFAP in each biomaterial. Bars represent standard errors of the mean. ** and ## represent p < 0.01 for TUJ1 and GFAP, respectively. Scale bar: 50 μm; scale bar for zoom boxes: 5 μm.

Article Snippet: Marker antibodies, TUJ1 (60052, Stemcell Technologies, Vancouver, BC, Canada) and GFAP (60048, Stemcell Technologies, Vancouver, BC, Canada), and isotype controls, mouse IgG2A PerCP-conjugated Isotype control (IC003C, R&D Systems, Minneapolis, MN, USA), were added per manufacturer’s instructions and incubated for 1 h at 4 °C in the dark.

Techniques: Labeling, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration

doi: 10.3390/ijms241713642

Figure Lengend Snippet: Analysis of the neural progenitor cells derived from neurospheres (NPCdn) spread and core area in the biomaterial scaffolds. NPCdn of green fluorescent protein (GFP)-mouse cultivated in direct contact with the biomaterial scaffolds and labeled with anti-βIII-tubulin (TUJ1, red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunocytochemistry of NPCdn-GFP labeling TUJ1 and DAPI: ( A ) control (CTL), ( B ) chitosan (CHI), ( C ) poly(ε-caprolactone) (PCL), ( D ) poly(L-lactic acid) (PLLA). NPCdn-GFP images showing neurosphere and outgrowth area boundaries: ( E ) CTL, ( F ) CHI, ( G ) PCL, and ( H ) PLLA. ( I ) Graph comparing the neurosphere spread ratio. ( J ) Graph comparing neurosphere core area. Bars represent standard errors of the mean. Black dots represent the value of each sample. * p < 0.05. Scale bar: 50 μm.

Article Snippet: Marker antibodies, TUJ1 (60052, Stemcell Technologies, Vancouver, BC, Canada) and GFAP (60048, Stemcell Technologies, Vancouver, BC, Canada), and isotype controls, mouse IgG2A PerCP-conjugated Isotype control (IC003C, R&D Systems, Minneapolis, MN, USA), were added per manufacturer’s instructions and incubated for 1 h at 4 °C in the dark.

Techniques: Derivative Assay, Labeling, Immunocytochemistry

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration

doi: 10.3390/ijms241713642

Figure Lengend Snippet: Emergence of βIII-tubulin (TUJ1, neuron) and glial fibrillary acidic protein (GFAP, astrocyte) in neural progenitor cells derived from human induced pluripotent stem cells (hiPSCs-NPC) cultivated over the scaffolds after DIV 15 in free-specific growth factor conditions. ( A ) Schematic illustration of hiPSCs-NPC cultures. (B – D ) TUJ1 marker plotted in flow cytometry graphs of hiPSC-NPC seeded on ( B ) poly (ε-caprolactone) scaffold (PCL), ( C ) poly (L-lactic acid) scaffold (PLLA), and ( D ) chitosan scaffold (CHI). ( E – G ) GFAP marker plotted in flow cytometry graphs of hiPSC-NPC seeded on ( E ) PCL, ( F ) PLLA, and ( G ) CHI. ( H ) Percentage of TUJ1 and GFAP in hiPSC-NPC on DIV 15. Bars represent standard errors of the mean. Illustration made on Biorender.com. * p < 0.05.

Article Snippet: Marker antibodies, TUJ1 (60052, Stemcell Technologies, Vancouver, BC, Canada) and GFAP (60048, Stemcell Technologies, Vancouver, BC, Canada), and isotype controls, mouse IgG2A PerCP-conjugated Isotype control (IC003C, R&D Systems, Minneapolis, MN, USA), were added per manufacturer’s instructions and incubated for 1 h at 4 °C in the dark.

Techniques: Derivative Assay, Marker, Flow Cytometry