Fig 1.
The structure of zebrafish embryo and experimental design.
(A) Sketch of zebrafish embryo at selected stage. (B) The image of cells-injected dechorionated zebrafish embryo at 4.3 hpf. (C) We drew schematic representation of experimental design.
Fig 2.
Expression of GFP in human ADSCs.
Human ADSCs were isolated from human adipose tissues and transduced with lentivirus vectors carrying GFP reporter gene. (A) spindle-like appearance of primary ADSCs at passage 3. (B) Phase-contrast microscopic image. (C) Fluorescence microscopic image. (D) merged image of (B) and (C). Original magnification of all images: 40 ×. (E) The effect of GFP expression on the proliferation of ADSCs.
Fig 3.
Detection of cell surface antigens.
Cell surface antigens of GFP-expressing ADSCs and control ADSCs were detected with flow cytometry. Nonspecific IgG was served as the control of background fluorescence. The positive percentage of cell surface was showed in the histograms.
Fig 4.
Survivals of human ADSCs transplanted into zebrafish embryos.
The GFP-expressing human ADSCs were transplanted into zebrafish embryos at 3.3–4.3 hpf after their chorions were removed, and followed by observation under fluorescence microscope at different time points. Each embryo was administered with about 10 cells, but the control did not receive cell transplantation and only their chorions were removed. (A1-D1) The representative control images of a same embryo standing for typical development stages at 12, 24, 48, and 96 hpf (bright). (A2-D2) The representative images of a same embryo captured under fluorescence microscope at indicated time points (bright + fluorescence). Original magnification: 40 ×. The white arrows indicate the location of GFP-expressing ADSCs (green) in the embryos and zebrafish. The insets in (C2) and (D2) show the enlarged images of transplanted GFP-expressing cells in the zebrafish. hpf: hour post-fertilization.
Fig 5.
GFP-expressing ADSCs in zebrafish were observed by using laser confocal fluorescence microscope.
Human ADSCs expressed GFP were xenotransplanted into zebrafish embryos at 3.3–4.3 hpf after their chorions were removed, and followed by observation under laser confocal fluorescence microscope at indicated time. Each embryo was injected with about 10 cells, and the control did not receive cell transplantation. (A) The control did not demonstrate any green fluorescence. (B) The representative image captured under a laser confocal fluorescence microscope at 15 dpf, displayed the GFP distributions (white arrows indicated) of transplanted cells in the zebrafish. Original magnification: 100 ×.
Table 1.
Effects of ADSCs transplantation on the development of zebrafish embryos.
Fig 6.
The proliferation of transplanted ADSCs detected by immunohistochemical staining.
The GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3–4.3hpf. The proliferation of transplanted cells were evaluated by immunohistochemical staining with rabbit anti-human Ki-67 monoclonal antibody at 6 days post fertilization. (A) Representative image of immunohistochemical staining demonstrated that the proliferating cells were Ki-67 positive staining (brownish, indicated by green arrow), suggesting that the transplanted human ADSCs could proliferate in the zebrafish. (B) The control that did not receive cell transplantation, was negative staining of Ki-67. Scale bar: 100 μm.
Fig 7.
The proliferation of transplanted ADSCs detected by immunofluorescence staining.
The GFP-expressing human ADSCs were transplanted into the fish embryos at 3.3–4.3 hpf and the immunofluorescence of whole-mount zebrafish larvae by using Ki-67 staining was performed as described in the materials and methods. The microphotos of the ADSCs or zebrafish larvae were captured by laser confocal scanning microscope. (A-C) The representative images of GFP-expressing human ADSCs seeded on the glass slides, and (A) indicates the cells expressed GFP (green), and (B) indicates the positive staining of Ki-67 (red). (C) is the merged image of (A) and (B). (D) The control zebrafish larva, which did not receive any transplantation, does not show green or red fluorescence. (E) and (I) Two representative fish larvae received the transplantation of GFP-expressing human ADSCs, show the different distributions of ADSCs in the fish. (F) and (J) are respectively the higher power images of the outlined areas in (E) and (I). (F) is the merged image of (G) and (H) under bright field; and (J) is the merged image of (K) and (L). (G) and (K) indicate the transplanted ADSCs expressed GFP (green). (H) and (L) demonstrate the positive staining of Ki-67 in the fish (red), indicating the proliferation of transplanted ADSCs in the fish.
Fig 8.
Detection of CD105 and CD31 expression of ADSCs.
Human ADSCs were xenotransplanted into the zebrafish embryos at 3.3–4.3hpf, and immunohistochemical staining and immunocytochemical staining were used to evaluate the expression of CD105 and CD31 of human ADSCs in the zebrafish at 48 hpf, and in culture dishes. (A-D) Representative images of immunohistochemical staining: the xenotransplanted ADSCs expressed positive CD105(brownish, indicated by green arrow) in the zebrafish embryos (A), and CD105 expression could not be detected in the control without ADSCs transplantation (B); the xenotransplanted ADSCs (indicated by red arrow) did not express CD31 antigen in the zebrafish embryos (C), and CD31could not be detected in the control without ADSCs transplantation (D). (E-H) Representative images of immunocytochemical staining: CD105 expression was detectable in the ADSCs cultured in the dishes before transplantation (E), and negative control for CD105 (without the addition of primary antibody) (F); CD31 expression was not detectable in the ADSCs cultured in the dishes before transplantation (G) and in the negative control (H). Scale bar: 100 μm. ab(+): added primary antibody; ab(-): no primary antibody.
Fig 9.
The transplanted ADSCs express CD105, but not CD31 in zebrafish.
The GFP-expressing human ADSCs were xenotransplanted into the zebrafish embryos at 3.3–4.3hpf, and immunofluorescence staining was performed to detect CD105 and CD31 expression of human ADSCs in the zebrafish at 48 hpf, and the images were captured with laser confocal scanning microscope. A-H: Representative images of immunofluorescence staining for zebrafish tissue sections. (A-D) Co-localization of CD105 and GFP in the transplanted ADSCs: (A) Bright field; (B) CD105 was positive; (C) GFP was positive; (D) Merged (A), (B) and (C). (E-H) The co-localization of CD31 and GFP in the transplanted ADSCs: (E) Bright field; (F) CD31 was negative; (G) GFP was positive; (H) Merged (E), (F) and (G). (I-N) Representative images of immunofluorescence staining for the ADSCs seeded on the glass slides. (I-K) The co-localization of CD105 and GFP in the human ADSCs before their transplantation: (I) CD105 expression was positive; (J) GFP was positive; (K) Merged (I) and (J). (L-N) The co-localization of CD31 and GFP in the human ADSCs before their transplantation: (L) CD31expression was negative; (M) GFP was positive; (N) Merged (L) and (M).