The initial fetal human retinal vasculature develops by vasculogenesis

Abstract

There is increasing evidence that the hemangioblast, a common progenitor for hematopoietic cells and endothelial cells, participates in embryonic and extra-embryonic vasculogenesis in some organs. Whether resident angioblasts or endothelial progenitor cells (EPCs) contribute to human retinal vasculogenesis is still a matter of controversy. To address this controversy, fetal human retinas of 6-23 weeks gestation (WG) were examined using immunohistochemistry and a panel of antibodies against endothelial cell markers (CD34, CD31), a marker for retinal angioblasts and endothelium (CD39/ecto-ADPase), and a marker for precursors and hemangioblasts (CXCR4). Confocal microscopic spectral analysis and double labeling with Ki67 was used to identify the proliferating cell types. In the inner neuroblastic layer of the 6-8 WG retina and in the putative ganglion cell layer in avascular regions of older eyes (14 WG-20 WG), scattered CD39+ angioblasts were well in advance of forming vasculature. There was a layer of CXCR4+ cells in the inner retina that was reduced in size with development. As blood vessels formed, CD39+ cells were always well in advance of the vascular front and they expressed CXCR4. This demonstrates that a pool of resident angioblasts express CD39 and CXCR4 as they differentiate and participate in vasculogenesis in the fetal human. They retain expression of CD39 as endothelial cells in the newly formed retinal vasculature but they down-regulate CXCR4 expression.

Fig. 1

Sections from the developing optic nerve head region of a 6-WG embryonic eye (optic nerve to the right). A: The hematoxylin and eosin (H&E) -stained section shows a few cells in the innermost retina and within the developing nerve (arrowheads). B: CD31 was only present in the developing choriocapillaris (bottom right) (arrow indicates the bleached RPE in all panels). C: CD39 staining was observed in a few round-shaped cells in the inner neuroblastic layer and in cells in the developing nerve fiber layer (arrowheads). CD39 was also present in the developing choriocapillaris (bottom). D: CD45⁺ cells were scattered throughout choroid (asterisks). E: CXCR4 was observed throughout the inner neuroblastic layer and within the inner retina and developing optic nerve head (arrowheads). F: Ki67 was only associated with cells within the outermost neuroblastic layer. B-F: APase reaction product.

Fig. 2

Sections from the optic nerve head region of a 7-WG embryonic eye (optic nerve to the right). A: The H&E-stained section shows round and spindle-shaped cells in the innermost retina and within the developing nerve fiber layer (arrowheads). B: CD31 was only present in the developing choriocapillaris (arrow indicates the bleached RPE in all panels). C: CD39 was present in cells in the nerve fiber layer (arrowheads). Staining was also seen in the developing choriocapillaris. D: CD45⁺ cells were scattered throughout choroid and some in the vitreous cavity. E: CXCR4 was observed throughout the inner neuroblastic layer and within cells of the nerve fiber layer (arrowheads), similar to those in the H&E (A) and the cells labeled with CD39 (C). F: Ki67 was only associated with cells within the outermost portion of the neuroblastic layer of retina and within the choroid. B–F: APase reaction product.

Fig. 3

Sections from the peripapillary region of a 12-WG fetal eye (optic nerve to the right and out of the field). A: The H&E-stained section shows scattered cells within the developing nerve fiber layer (arrowheads) and a demarcation between the inner and outer neuroblastic layer. B: CD31 was only present in the developing choriocapillaris (arrow indicates the bleached RPE in all panels). C: CD39 staining was observed in scattered cells in the nerve fiber layer (arrowheads) and some in the inner portion of the neuroblastic layer. Staining was also seen in the developing choriocapillaris. D: CD45 labels cells in the nerve fiber layer but the number of cells was less then those stained with CD39. E: CXCR4 was observed within the inner neuroblastic layer and in scattered cells within the nerve fiber layer (arrowhead), similar to those in the H&E (A) and the cells labeled with CD39 (C). F: Ki67 staining was only associated with cells within the outermost portion of the neuroblastic layer of retina and within the choroid. B–F: APase reaction product.

Fig. 4

Sections from the peripapillary region of a 14-WG fetal eye at the onset of retinal vascularization (optic nerve to the right and out of the field). A: The H&E-stained section shows a well-developed nerve fiber layer, and a clearly defined inner and outer neuroblastic layer separated by the inner plexiform layer. B: CD31 was present in the developing peripapillary retinal vessels (arrow indicates the edge of formed vessels in all panels). C: CD39 staining was observed in formed retinal vessels and in scattered cells in advance. D: CD45 labeled single cells in the nerve fiber layer but the number of cells was less then those stained with CD39. E: CXCR4 was observed within the apparent ganglion cell layer in this region and not within the formed vessels. In midperipheral regions, however, CXCR4⁺ cells were observed throughout the inner retina (arrowhead in inset). F: Ki67 was now associated with cells in the vicinity of the developing retinal vessels and in the outer neuroblastic layer. G: Pax-2 labeling was also evident in the region of developing vessels as was GFAP staining (H). B–H: APase reaction product.

Fig. 5

Sections from the peripheral edge of formed vasculature of a 16-WG fetal eye. A: The H&E-stained section shows a well-developed nerve fiber layer, and a clearly defined inner and outer neuroblastic layer separated by the inner plexiform layer. B: CD31 was present in the formed retinal vessels (arrow indicates the edge of formed vessels in all panels). C: CD39 staining was observed in formed retinal vessels (arrow) and in scattered cells in advance (arrowhead). D: Single CD45⁺ cells were scattered cells in the nerve fiber layer. E: CXCR4 was observed within the apparent ganglion cell layer and to a lesser degree within spindle-shaped cells in advance of formed vessels (paired arrows). F: Ki67 was associated with cells in the vicinity of the edge of developing retinal vessels, some slightly in advance (asterisk) and in the outer neuroblastic layer. G: Pax-2 labeling was also evident at the edge of developing vessels and in cells slightly in advance (asterisk). H: GFAP staining clearly lagged behind the edge of formed vessels. B–H: APase reaction product; small arrows indicate the RPE in all panels.

Fig. 6

Sections from the peripheral edge of formed vasculature of a 20-WG fetal eye. A: The H&E-stained section shows a well-developed nerve fiber and ganglion cell layers, and a clearly defined developing inner and outer nuclear layers separated by the outer plexiform layer. B: CD31 was present in the formed retinal vessels (arrow indicates the edge of formed vessels in all panels). C: CD39 was observed in formed retinal vessels and in scattered cells in advance (arrowheads). D: CD45⁺ cells were scattered cells in the nerve fiber layer but they are rare in this region. E: CXCR4 was observed within the apparent ganglion cell layer and to a lesser degree within spindle-shaped cells in advance of formed vessels (arrowheads). F: Ki67 was associated with cells in the vicinity of the edge of developing retinal vessels and in the outer neuroblastic layer. G: Pax-2 labeling was also evident at the edge of developing vessels and in cells slightly in advance. H: GFAP staining clearly lagged behind the edge of formed vessels. B–H: APase reaction product; small arrows indicate the RPE in all panels.

Fig. 7

Multispectral confocal analysis of Ki67 (green arrows), CD34 (red arrows in A), Pax-2 (red arrows in B), and GFAP (red arrows in C) colocalization was performed in sections from a 20-WG human fetal retina. All sections were counterstained with mounting media containing DAPI (blue). Spectral analysis revealed the presence of dual Pax-2-positive and GFAP-positive cells (yellow arrows in B,C) at the edge of forming vasculature. The majority of Pax-2-labeled cells were proliferating as determined by cell-specific spectral analysis. No CD34/Ki67 double-labeled cells were observed in these sections. D: Double labeling with Ki67 (green) and CD39 (red) in the nerve fiber layer of peripapillary region of a 14-WG retina demonstrates that adjacent cells and not angioblasts express Ki67. E: Ki67 (green) and CXCR4 (red) in the peripapillary retina of a 7-WG eye demonstrates that proliferation in this region is in the posterior region of the neuroblastic layer (double arrow), while CXCR4 is the inner portion of the neuroblastic layer and inner retina.

Fig. 8

Double labeling of embryonic/fetal retina in sections and wholemounts. A–C: Section from nerve head region of a 7-WG embryonic retina double labeled with CD39 (red) and CXCR4 (green). In A, both channels are shown while CXCR4 (B) and CD39 (C) are shown independently. The arrows in all indicate double-labeled CD39⁺/CXCR4⁺ cells, some having a spindle-shaped morphology. D–G: Section from the peripheral edge of forming vasculature in a 23-WG retina double labeled with CD39 (red) and CXCR4 (green) and counterstained with DAPI (blue). Double-labeled CD39⁺/CXCR4⁺ angioblasts (arrows in all) are shown migrating from the ganglion cell layer to the inner retina. In D, all channels are shown. In E, both the red and green channels are shown, while in F and G the red (CD39) and green (CXCR4) channels are shown independently. H,I: Retinal whole mount from a 22-WG specimen double labeled with CD39 (red) and CXCR4 (green). At the edge of forming vasculature (H), double-labeled CD39⁺/CXCR4⁺ elongated angioblasts with processes (arrows) are seen aligning with formed lumen. In the avascular retina (I), double-labeled CD39⁺/CXCR4⁺ angioblasts with a round morphology are shown (arrows)

Fig. 9

Retinal whole mount from a 22-WG specimen double labeled with CD39 (red) and CXCR4 (green). A: With only the red channel shown (CD39), the newly formed retinal vasculature is apparent. Angioblasts are assembling at the tips of the new blood vessels while many individual angioblasts are apparent in avascular retina. B: At the edge of forming vasculature, double-labeled CD39⁺/CXCR4⁺ elongated angioblasts with processes are seen aligning to form a vascular cord (arrows).