The embryonic human choriocapillaris develops by hemo-vasculogenesis

Abstract

The purpose of this study was to characterize normal human choroidal vascular development from 6-23 weeks gestation (WG). Markers of endothelial cells (EC) (CD34, CD31, vWf), angioblasts and EC (CD39), leukocytes (CD45), erythroblasts (epsilon chain of hemoglobin, Hb-e), proliferating cells (Ki67), and VEGFR-2 were employed. At 6-7 WG, many erythroblasts were observed within islands of precursor cells in the choriocapillaris layer and others were independent from the islands. Many erythroblasts (Hb-epsilon(+)) were also positive for EC markers and/or VEGFR-2. By 8-12 WG, most of the Hb-epsilon cells had disappeared and vascular lumens became apparent. At 14-23 WG, some EC were proliferating on the scleral side of choriocapillaris in association with forming deeper vessels. In conclusion, embryonic choriocapillaris appears to form initially by hemo-vasculogenesis (blood vessels and blood cells form simultaneously from common precursors) while angiogenesis appears to be the mode of intermediate and large choroidal vessel development in the fetus.

Fig. 1

Glycol methacrylate sections from a 6-WG embryonic human eye. A: At low magnification (A), the lens (L), retina (R), optic nerve (ON), and choroid (C) are apparent. Vitreous (V) fills the chamber between lens and retina. B: Magnification of the boxed area in A, where the relationship between retina, the monolayer of retinal pigment epithelial (RPE) cells, and choroid can be seen. Arrows point to the forming choriocapillaris. The separation between retina and RPE is artifactual. A,B: Hematoxylin and eosin. Serial, Wright’s Giemsa-stained, sections of retinal pigment epithelial cells (RPE) (top in all) and developing choriocapillaris (below RPE) are shown in C–K. C: A single cell with moderately acidophilic (pink) cytoplasm is present just posterior to the pigmented RPE cells (top). D,E: Nucleated cells aggregate in this area that have intensely acidophilic cytoplasm, which is a characteristic of embryonic hemoglobin staining in erythroblasts. F–H: Mesenchymal-like cells with spindle-shaped nuclei become part of the aggregate or island of cells and in some areas appear to surround the erythroblasts while in other areas the erythroblasts form the outside of the aggregate (F). I–K: The island divides into several clusters of cells and individual erythroblasts as well. L,M: At higher magnification, the lumen-like nature of the center of the aggregate becomes more apparent and the presence of several types of cells in the center of the island is more obvious.

Fig. 2

Wright’s Giemsa staining of 2.5-μm plastic sections showing an island of cells in the choroid at 6 WG. Serial sections (A–I) confirm that the structure is isolated and separated from any blood vessels at 6 WG. The arrow shows the same position in each panel. J,K: High-magnification photos of D and E, respectively. Wright’s Giemsa staining shows acidophilic cytoplasm indicative of hemoglobin in pink and basophilic nuclei in blue. One cell outside of structure (double arrow in K) shows the changing of cytoplasmic staining pattern from acidophilic into basophilic, which suggests that the cell has both the characteristics of hematopoietic cells and endothelial cells. In J, the erythroblasts appear to form the lumen.

Fig. 3

Comparison of vascular markers (A–D), and choroidal vascular development with CD34 labeling (E–G) and the presence of CD45 (H–J) by age. CD39 (A) shows a layer of positive cells in the periphery of choriocapillaris (long arrow) at 6 WG. VEGFR-2 (B), CD34 (C), and CD31 (D) show the similar staining of the choriocapillaris layer (long arrow). At 7 WG (E), CD34-positive cells are only in the choriocapillaris layer (long arrow). At 12 WG (F), CD34 shows deeper vessels forming (arrowheads). At 23 WG (G), CD34 shows the three vascular layers that are present in adult choriocapillaris (long arrow), Sattler’s layer (arrowhead), and the outermost large vessels of Haller’s layer (short arrow). CD45 is localized to single cells associated with developing choriocapillaris and larger choroidal vessels at 7 (H), 12 (I), and 23 (J) WG. The RPE cells are present above the choriocapillaris in all plates, but the pigment has been bleached. APase reaction product in all.

Fig. 4

The developing choroidal vasculature contains CD31/Hb-∈ double positive cells. A–D: At 6 WG, clusters of CD31/Hb-∈ (red/green) positive cells (arrows) are visible in the choriocapillaris layer, whereas in the choroidal stroma, there are isolated cells (double arrow) that are also double labeled. E–H: At 7 WG, there are isolated cells (arrows) that are CD31/Hb-∈ positive, which appear attached to the choriocapillaris. These CD31/Hb-∈-positive cells have characteristics of both hematopoietic and endothelial cells. B and F are merged images of the single color images C, D, and G, H, respectively. A and E are images showing nuclear counter staining with DAPI (blue) merged with B and F, respectively.

Fig. 5

VEGFR-2/Hb-∈ double labeling. At 6 WG (A–D), isolated cells (arrows in the middle and bottom) that were double stained by VEGFR-2 (C, Red) and Hb-∈ (D, Green) were found in the choroidal stroma. More VEGFR-2⁺/Hb∈⁺ cells (arrow at top) were located in the choriocapillaris layer. B is a merged image of single images in C and D. A shows nuclear counterstaining (blue) combined with both colors.

Fig. 6

CD34/vWf/Hb-∈ triple labeling. A: Merged image of all colors with DAPI nuclear counter-staining (gray). B: CD34 staining in red. C: vWf staining in blue. D: Hb-∈ staining is green. At 6 WG (A–L), double-labeled cells (G, arrowhead) for CD34 (B, arrowhead) and Hb-∈ (D, arrowhead) are observed in the choroid. Some of CD34⁺/Hb-∈⁺ are triple-labeled cells (H, arrow) for CD34 (B, arrow), vWf (C, arrow), and Hb-∈ (D, arrow). I–L: A triple-stained cell is shown at high magnification (arrow) within the vascular structure. E,I: Merged images of CD34 (red) and vWf (blue). F,J: Merged images of vWf (blue) and Hb-∈ (green). G,K: Merged images of CD34 (red) and Hb-∈ (green). H,L: Merged images of CD34 (red), vWf (blue), and Hb-∈ (green). Arrows show the same cell in A–L.

Fig. 7

Quantification of Hb-∈ positive cells in the choroids and CD31/Hb-∈ double staining with age. A: After 7 WG, the number of Hb-∈-positive cells decreased rapidly and Hb-∈-positive cells were gone by 14 WG. B: At 7 WG, Hb-∈-positive, round shaped cells (arrows), were common in the choroids examined. C: At 8.5 WG, the number of Hb-∈-positive cells decreased and the morphology was smaller and more oval. D: At 16 WG, there were no detectable Hb-∈-positive cells in the sections examined.

Fig. 8

CD34/Ki67 double staining. CD34 reaction is shown in red, Ki67 reaction is shown in green. CD34⁺/Ki67⁺ cells are not observed at 6 WG (A,B), while CD34⁺/Ki67⁺ cells (arrow) appear to be budding from choriocapillaris to form a diving vessel at 12 WG (C,D). B and D are merged images of CD34 (red) and Ki67 (green). A and C are the result of merging B and D with nuclear counterstaining shown in blue.

Fig. 9

Flatmounts of CD39 labeled choroids from 5.5, 7.5, 8.5, and 9.5 WG. The choriocapillaris at 5.5 WG is very cellular with only a few vascular segments and no obvious pattern. At 7.5 WG, wide vascular segments are apparent but not contiguous and the “segments” are filled with CD39⁺ cells, perhaps representing components of the vascular islands. At 8.5 WG, a choriocapillaris-like pattern of choroidal capillaries is present. At 9.5 WG, the pattern appears to be stretched, having wider intercapillary septa (*) and undergoing remodeling (narrow vascular segments, arrows).

Fig. 10

An area in the posterior pole of a whole-mounted choroid stained with CD39. At 12 WG, the flat-mounted choroid shows choriocapillaris in the superficial layer (arrowheads) (A) and large vessels in the deeper layer (arrows) (B). C: A cross-section of the area shows these two layers of blood vessels, choriocapillaris (arrowhead) and deeper vessels (arrow). Red APase CD39 reaction product with toluidine blue counterstain in C.