Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: Isolation of CD34 + VEGFR-3 + cells. (A) The mononuclear cells isolated from human umbilical cord blood. The cells are round and become fusiform after adhesion. (B) The mononuclear cells stained with 10% Giemsa solution. The nuclei of the cells are round-, ellipse-or horseshoe-shaped. (C) Features of CD34 + VEGFR-3 + cells. In the mononuclear cells, CD34 + VEGFR-3 + cells (arrows) are larger than CD34 + VEGFR-3 − cells. The right panel is the merged image of the left and middle panels and phase-contrast image. Bars (A–C) represent 10 μm. (D) Phenotype of the mononuclear cells analysed by dual-colour flow cytometry. CD34 + VEGFR-3 + cells and CD133 + VEGFR-3 + cells present in the mononuclear cells. Percentage of the positive cells was compared with isotype control. (E) CD133 is weekly expressed on the freshly sorted CD34 + VEGFR-3 + cells. Bar represents 10 μm.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Isolation, Staining, Flow Cytometry, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: Characterization of CD34 + VEGFR-3 + cells during differentiation. (A) Morphological changes of the cells during differentiation. At 2 hrs after induction with VEGF-C, the cells were round or ellipse. At day 7, the most cells became spindle-shaped. At day 14, the confluent monolayer of the cells demonstrated a typical cobblestone appearance. Bars represent 20 μm. (B) Microphotographs of a same colony. The colony enlarges gradually. Bars represent 20 μm (left panel) and 50 μm (middle and right panels) respectively. (C) Co-expression of CD34 and VEGFR-3 on the cells of the colony. Bar represents 10 μm. (D and E) Uptake of Dil-Ac-LDL and binding of UEA-1. The cells represent endothelial progenitor cell phenotype. Bars represent 10 μm.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Expressing, Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: Ultrastructural characterization of CD34 + VEGFR-3 + cells and the differentiated cells. The cells were treated with scanning (A, C and D) and transmission electron microscopies (B, E and F). (A) There are many microvilli on CD34 + VEGFR-3 + cell. Bar represents 2 μm. (B) In CD34 + VEGFR-3 + cell, the nucleus (N) is large, nuclear chromatin is rarefied. Mitochondrium (M), free ribosome (FR) and endoplasmic reticulum (ER) in the cytoplasm are rich. Bar represents 2 μm. (C and D) The cells induced with VEGF-C for 14 days. On the cell surface, there are numerous caveolae (arrows) and microvilli (arrowheads; C). Conjunctions (Conj) form between adjacent cells (D). Bars represent 1 μm (C) and 2 μm (D) respectively. (E and F) Organelles in the cells induced with VEGF-C for 14 days. There are more mitochondria (M), rough endoplasmic reticula (arrowheads) and phagocytic vesicles (white arrow) in the cytoplasm. Weibel-Palade body (black arrow) enwrapped by the membrane is observable. F is high-magnification of the selected area from E. N, nucleus. Bars represent 2 μm (E) and 0.5 μm (F) respectively.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Transmission Assay, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: Differentiation of CD34 + VEGFR-3 + cells into lymphatic endothelial cells. After induction with VEGF-C for 2 weeks, the cells express markers for lymphatic endothelial cells, 5′-Nase, LYVE-1 and Prox-1, and hyaluronan receptor CD44. (A) Expression of 5′-Nase and LYVE-1. Bar represents 20 μm. (B) Expression of Prox-1. The nucleus was counterstained with DAPI. Bar represents 10 μm. (C) Expression of CD44. Bar represents 20 μm.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: VEGFR-3 mRNA expression in CD34 + VEGFR-3 + cells. (A) Changes of VEGFR-3 mRNA expression after treatment with VEGF-C. Real-time PCR analysis shows that the sorted cells express VEGFR-3 mRNA. After treatment with VEGF-C, the level of VEGFR-3 mRNA expression increases and reaches the peak at 24 hrs. * P < 0.05 versus control and 2 hrs; ** P < 0.01 versus control and 2 hrs; # P < 0.01 versus 6 hrs; & P < 0.05 versus 12 hrs. (B) Screening of the effective VEGFR-3 siRNA. In silencing VEGFR-3 mRNA expression, VEGFR-3 siRNA no.3 is more effective than siRNA no.1 and siRNA no.2.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: Proliferation and transmigration of endothelial progenitor cells (EPC)-derived cells. EPCs were induced with VEGF-C for 14 days before accessing their capacity of proliferation and transmigration. (A) Effect of VEGF-C on proliferation of EPC-derived cells. EPC-derived cells were seeded onto the fibronectin-coated dishes. VEGF-C increases proliferation of the cells. After the cells are treated with VEGFR-3 siRNA, the effect of VEGF-C is inhibited. * P < 0.01 versus control; # P < 0.01 versus VEGF-C group and si-control + VEGF-C group. (B and C) Transmigration of EPC-derived cells. The cells were seeded on fibronectin-coated membrane of cell culture inserts. After stimulation of VEGF-C in the lower chamber, the cells in the upper side of the membrane (B) migrate to the lower side of the membrane (C) through pores (arrowheads). Arrows indicate the migrated cells. Giemsa staining. Bars represent 10 μm. (D and E) Scanning electron microphotographs of the cells on the membrane. There are more migrating cells (arrows) in VEGF-C group (D) compared with RNAi group (E). Arrowheads indicate pores. Bars represent 10 μm. (F) Effect of VEGF-C on transmigration of EPC-derived cells. VEGF-C promotes migration of the cells into lower chamber through pores of the membrane. After treatment with VEGFR-3 siRNA, effect of VEGF-C on cell transmigration decreases. * P < 0.01 versus the control group; # P < 0.01 versus VEGF-C group and si-control + VEGF-C group.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Transmigration Assay, Derivative Assay, Control, Membrane, Cell Culture, Staining, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: Effects of bFGF, VEGF and VEGF-C on migration of endothelial progenitor cell-derived cells. After wounding, the cells derived from CD34 + VEGFR-3 + cells migrate from the monolayer side to the scraped side. The cells were treated with bFGF, VEGF or VEGF-C (50 ng/ml) for 24 hrs. The number of cells migrated to the scraped side and maximal distance of cell migration in VEGF-C group are more than that in bFGF and VEGF groups. After pre-treatment with VEGFR-3 siRNA, the effect of VEGF-C on cell migration is inhibited. Arrowhead indicates the wound edge.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Migration, Derivative Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD34 + VEGFR-3 + progenitor cells have a potential to differentiate towards lymphatic endothelial cells

doi: 10.1111/jcmm.12233

Figure Lengend Snippet: Capillary-like structures formed by endothelial progenitor cells (EPC)-derived cells. When the cells on collagen gel were grown to submonolayer, the cells were treated with VEGF-C for 2 hrs. Then, upper layer of collagen gel on the cells was made. The cells were continued to be incubated for 24 hrs. (A and B) Microphotographs of capillary-like structures formed by EPC-derived cells. VEGF-C stimulates the cells to organize into capillary-like structures (A). After treatment with VEGFR-3 siRNA, the cells cannot form tubes well (B). Arrows indicate the tubes. Giemsa staining. Bars represent 20 μm. (C and D) Statistical analysis of length and area of the tubes. Length and area of the tubes in VEGF-C group are greater than those in the control and VEGFR-3 siRNA groups. * P < 0.01 versus the control group; # P < 0.01 versus VEGF-C group and si-control/VEGF-C group. (E) Microphotograph of capillary-like structures in semi-thin section. The tube displayed structures of capillaries with a lumen. The wall of the tube is very thin. Bar represents 5 μm. (F) Transmission electron microphotograph of capillary-like structures. The adjacent cells were extensively overlapping. There were tight junctions between the adjacent cells (arrows). The basal lamina is absent in the tube. The lumen (L) of the tube was irregular. CF, collagen fibres in the gel. Bar represents 1 μm. (G) LYVE-1 expression of capillary-like structures formed by EPC-derived cells in Matrigel. The cells were incubated in the medium containing 50 ng/ml VEGF-C for 12 hrs. The capillary-like structures in Matrigel were identified with LYVE-1 immunostaining. Bars represent 100 μm.

Article Snippet: For examining existence of CD34 + VEGFR-3 + cells in mononuclear cells, the cells were incubated with mouse anti-human CD34 antibody (1:100; Miltenyi Biotec, Auburn, CA, USA) and rabbit anti-human VEGFR-3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 50 min. at 4°C.

Techniques: Derivative Assay, Incubation, Staining, Control, Transmission Assay, Expressing, Immunostaining