Journal: Molecular Genetics & Genomic Medicine

Article Title: Precise CCM1 gene correction and inactivation in patient‐derived endothelial cells: Modeling Knudson's two‐hit hypothesis in vitro

doi: 10.1002/mgg3.755

Figure Lengend Snippet: Generation and characterization of patient‐derived BOECs. (a) Pedigree of the CCM index case (III:2; arrow). (b) Sequence of the heterozygous frameshift variant c.2012delA in the CCM1 gene of III:2. (c) Schematic domain structure for CCM1 and localization of the pathogenic c.2012delA; p.(Asn671Thrfs*36) mutation in its FERM domain. ANK = ankyrin repeat domain; FERM = band Four.1 Ezrin Radixin Moesin; NPxY/F = Asn‐Pro‐X‐Tyr/Phe motif. (d) Brightfield and (e) spheroid sprouting of patient‐derived BOECs. Strong expression of CD31/PECAM‐1 (green) and very few cells expressing SM22α (red, *)(f, g) as well as immunopositivity for CD146 (green, h, i) and CD34 (green, j, k) in BOECs established from a healthy donor (f, h, j) and the index patient III:2 (g, i, k) confirmed their endothelial phenotype. Scale bars indicate 100 µm (f, g) or 400 µm (d, h‐k)

Article Snippet: The following antibodies were used: monoclonal mouse anti‐human CD31 (BBA7, R&D Systems, Minneapolis, MN), polyclonal rabbit anti‐SM22α (ab14106, Abcam, Cambridge, UK), monoclonal mouse anti‐human CD146 (MAB932, R&D Systems), polyclonal rabbit anti‐human CD34 (HPA036723, Sigma‐Aldrich, St. Louis, MO), polyclonal rabbit anti‐KLF4 (PA5‐27441, Thermo Fisher Scientific), secondary goat anti‐mouse IgG antibody, Alexa Fluor 488 (A‐11029, Thermo Fisher Scientific), and goat anti‐rabbit IgG antibody, Alexa Fluor 555 (A‐21429, Thermo Fisher Scientific).

Techniques: Derivative Assay, Sequencing, Variant Assay, Mutagenesis, Expressing

Journal: Biomaterials

Article Title: Cell-based approach for 3D reconstruction of lymphatic capillaries in vitro reveals distinct functions of HGF and VEGF-C in lymphangiogenesis.

doi: 10.1016/j.biomaterials.2015.11.027

Figure Lengend Snippet: Fig. 1. Morphological and molecular characteristics of a human 3D reconstructed lymphatic microvascular network in vitro. (A) Schematic outline of the method for in vitro reconstruction of the lymphatic microvascular network within a connective tissue substitute. Image of the construct at the end of experiment is shown on the right. (B, C) Lymphatic vascular network visualized by immunofluorescent staining for CD31 (red), imaged by confocal microscopy (B), or computationally reconstructed using Imaris software (C). (D) Side view of the 3D network shown in C, at higher magnification. Note lymphatic capillary lumens (white arrows), blind-ends (yellow arrows), and sprouts (green arrows). Inter- connected networks can be seen in two planes, as indicated with the dotted line. (E) Immunostaining for CD31 and Imaris reconstruction showing numerous filopodia protruding from a lymphatic capillary in vitro. (F, G) Double-immunofluorescent staining for podoplanin (F) and CD31 (G) on the whole reconstructed tissue sample. (H) Immunohistochemical staining for Ki67 (brown) in a tissue cross-section. Cell nuclei are shown in blue. Note Ki67þ LECs in the two vessels shown. (I, J) Immunohistochemistry on serial tissue sections for CD31 (I) and Prox-1 (J). (KeN) TEM images showing typical ultra-structural features of lymphatic capillaries: overlapping endothelial cells (K, M, N), button-like adherens junctions (green arrows, K, M, N), discontinuous basement membrane (red arrow in L), anchoring filaments (red arrow in N), single layer of LECs, and lack of mural cell coverage (L, N). Scale bars, 5 mm (A), 500 mm (B, C), 100 mm (D, F, G), 50 mm (H, I, J), 10 mm (E), and 500 nm (KeN). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Whole-mounts and frozen sections were stained with sheep anti-human CD31 (1:100, R&D systems AF806), anti-human podoplanin (1:50, Angiobio 11-003), and corresponding secondary antibodies labeled with Alexa-555 or Alexa-633 (Molecular Probes, Eugene, Oregon, USA).

Techniques: In Vitro, Construct, Staining, Confocal Microscopy, Software, Immunostaining, Immunohistochemical staining, Immunohistochemistry, Membrane

Journal: Biomaterials

Article Title: Cell-based approach for 3D reconstruction of lymphatic capillaries in vitro reveals distinct functions of HGF and VEGF-C in lymphangiogenesis.

doi: 10.1016/j.biomaterials.2015.11.027

Figure Lengend Snippet: Fig. 3. Effects of HGF/c-Met and VEGF-C/VEGFR-3 inhibition on 3D in vitro lymphangiogenesis. (A, B) Formation of lymphatic microvascular network in the presence of an anti- VEGFR-3 blocking antibody (2.5 mg/ml), c-Met inhibitor SU11274 (1 mM), or both. Lymphatic vasculature was visualized by immunostaining of the whole construct with a CD31 antibody and reconstructed in 3D with the Imaris software. B is a higher magnification of an area shown in A. (C) Cross-sections of the LEC-fibroblast constructs treated with an anti-VEGFR-3 antibody, c-Met inhibitor or both as indicated, and immunostained for the lymphatic marker podoplanin. (DeG) Quantitative analyses of the lymphatic network volume (D), number of vessels (E), vessel size distribution (F) and connectivity (G) upon treatments as indicated. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 500 mm (A), 50 mm (B), 100 mm (C).

Article Snippet: Whole-mounts and frozen sections were stained with sheep anti-human CD31 (1:100, R&D systems AF806), anti-human podoplanin (1:50, Angiobio 11-003), and corresponding secondary antibodies labeled with Alexa-555 or Alexa-633 (Molecular Probes, Eugene, Oregon, USA).

Techniques: Inhibition, In Vitro, Blocking Assay, Immunostaining, Construct, Software, Marker

Journal: Scientific Reports

Article Title: A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment

doi: 10.1038/s41598-019-44113-6

Figure Lengend Snippet: Interconnected vascular network formation in 3D skin substitutes 7 days after epidermal differentiation. ( a – c ) HUVEC were seeded at 0.1 to 1 × 10 5 cells per culture insert; FN-G-coated NHDF content (1 × 10 7 cells) remained constant. FN-G-coated NHDF and HUVEC were cocultured at 1,000:1, 500:1, or 100:1, and then with HEKn (1 × 10 6 cells). ( a ) Whole-mount CD31 immunofluorescence analysis revealed that HUVEC developed branching vessels within the dermal layer. The data are representative of 2 independent experiments (n = 6/condition). ( b , c ) Quantification of vascular structures to determine the vessel density ( b ) and branching index (branching points/unit area) ( c ). The data are the mean ± SD (n = 6). * p < 0.05 and ** p < 0.01 determined by one-way ANOVA with Tukey’s multiple comparison post hoc test.

Article Snippet: The following primary antibodies were used for immunostaining of: a human–specific CD31 (1:200, NBP2-15202, clone C31.3; Novus Biologicals, Centennial, CO, USA), a mouse–specific CD31 (1:100, 14-0311-82, clone 390; Invitrogen), HLA-Class I ABC (1:2,500, ab70328, clone: EMR8-5; Abcam), and laminin 5 (1:200; ab14509, Abcam).

Techniques: Immunofluorescence, Comparison

Journal: Scientific Reports

Article Title: A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment

doi: 10.1038/s41598-019-44113-6

Figure Lengend Snippet: In vitro evaluation of the therapeutic potential of pre-vascularized 3D skin substitutes. ( a – e ) HUVEC (0.2 × 10 5 cells/insert) mixed with FN-G-coated NHDF were cocultured, subsequently covered with HEKn, then cultured for up to an additional 7 days. ( a ) Macroscopic view of the construct in the culture insert. Scale bar: 10 mm. ( b – e ) Histological and immunohistochemical staining with hematoxylin and eosin ( b ), Masson’s trichrome ( c ), anti-laminin 5 ( d , arrows = basement membrane), and anti-CD31 (e, arrows = CD31 + blood vessel). Scale bars: 500 μm ( b ), 100 μm ( c – e ).

Article Snippet: The following primary antibodies were used for immunostaining of: a human–specific CD31 (1:200, NBP2-15202, clone C31.3; Novus Biologicals, Centennial, CO, USA), a mouse–specific CD31 (1:100, 14-0311-82, clone 390; Invitrogen), HLA-Class I ABC (1:2,500, ab70328, clone: EMR8-5; Abcam), and laminin 5 (1:200; ab14509, Abcam).

Techniques: In Vitro, Cell Culture, Construct, Immunohistochemical staining, Staining, Membrane

Journal: Scientific Reports

Article Title: A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment

doi: 10.1038/s41598-019-44113-6

Figure Lengend Snippet: Quantification of wound vasculature in vivo . ( a ) Immunohistochemical detection of mouse-specific CD31 (mCD31) and human-specific CD31 (hCD31) at 7 and 14 days after grafting. Dashed lines indicate the skin substitute–host interface. Scale bars: 100 μm. ( b ) Quantification of CD31 + blood vessels in the dermal areas of grafts. The data are the mean ± SD (n = 5). ** p < 0.01 compared with the non-vascularized control (unpaired Student’s t -test). ( c ) Immunofluorescence image stained for mouse-CD31 (red) and human-CD31 (green) of pre-vascularized substitute-treated wounds at day 14 after grafting. Nuclei were stained with DAPI (blue). White arrowheads indicate human–mouse chimeric vessel. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining of: a human–specific CD31 (1:200, NBP2-15202, clone C31.3; Novus Biologicals, Centennial, CO, USA), a mouse–specific CD31 (1:100, 14-0311-82, clone 390; Invitrogen), HLA-Class I ABC (1:2,500, ab70328, clone: EMR8-5; Abcam), and laminin 5 (1:200; ab14509, Abcam).

Techniques: In Vivo, Immunohistochemical staining, Control, Immunofluorescence, Staining