A novel zebrafish embryo xenotransplantation model to study primary human fibroblast motility in health and disease

Abstract

Fibroblasts have a central role in the maintenance of tissue homeostasis and repair after injury. Currently, there are no tractable, cost-effective model systems for studying the biology of human fibroblasts in vivo. Here we demonstrate that primary human fibroblasts survive transplantation into zebrafish embryos. Transplanted cells migrate and proliferate, but do not integrate into host tissues. We used this system to study the intrinsic motility of lung fibroblasts from a prototype fibrotic lung disease, idiopathic pulmonary fibrosis (IPF). IPF fibroblasts displayed a significantly higher level of motility than did fibroblasts from nonfibrotic lungs. This is the first in vivo examination of primary human lung fibroblast motility in health and disease using zebrafish models.

FIG. 1.

Representative images of grafts at mid-gastrula (3 h post transplantation). A solitary graft positioned close to the geometric center of the blastoderm. (A) Phase contrast; (B) fluorescence; (C) merged image. Shown is a lateral view with the animal pole upwards. Arrows indicate graft location. Scale bar: 100 μm.

FIG. 2.

Formation of the fibrotic reticulum. Representative images of the IPF and HLF grafts localized to the head and trunk regions of the 1 and 2 dpf host embryos. Left panel: Fluorescence: High cell motility is characteristic of the IPF grafts in 2 dpf embryos. Arrows pointing at the processes, wedge indicating the graft margin, * marking the foci. Yolk autofluorescence (background) and no signal in head and trunk regions in embryos without grafts (CON). Right panel: Brightfield images showing graft-related malformations in the head (unilateral anophthalmia and microphthalmia) and trunk (lump, axial deformities) regions in the graft-bearing embryo, and no deformities in embryos without grafts (CON); e, eye; ov, otic vesicle; p, pericardium; Y, yolk sack. Lateral view. Scale bar: 250 μm.

FIG. 3.

Proliferation of the engrafted cells in the 2 dpf host embryos. (A) Representative image of the embryo cross-section immunostained with anti-human cd59/Cy-3 antibodies. (1) FITC: CFSE-labeled graft cells; (2) TRITC: CD59/Cy-3-positive cells; (3) DAPI; (4) Merged image. Arrows point to the areas with CFSE-/CD59+ cells. Scale bars: 50 μm. (B) Representative image of the embryo sagittal section immunostained with human anti-Ki67 antibody and HRP. Arrow points to the graft area in the head region of the embryo. Inset: Ki67+ cell nuclei in the graft body. Arrows point to the labeled nuclei of the engrafted cells. Scale bars: 100 μm in B and 30 μm in B inset.

FIG. 4.

Graft morphology and measurements.

FIG. 5.

Motility of engrafted fibroblasts measured by aggregate length of graft's processes. (A) Box-plot showing all data for each of the 8 IPF and 8 HLF cell lines; (B) Boxplot showing aggregated data for IPF versus HLF.

FIG. 5.

Motility of engrafted fibroblasts measured by aggregate length of graft's processes. (A) Box-plot showing all data for each of the 8 IPF and 8 HLF cell lines; (B) Boxplot showing aggregated data for IPF versus HLF.