Mural Cell Differentiation in Human Retina and Choroid: Role of CD44+ Stem Cells in Mural Cell Formation and the Cellular Basis for Blood Flow Regulation

Abstract

Purpose: : During development, smooth muscle cells (SMCs) and pericytes are thought to play an important role in regulating endothelial proliferation, vascular remodelling, vessel stabilisation and synthesis of the extracellular matrix. The origin of mural cells and their differentiation in human retina and choroid remain unclear. We sought to examine normal mural cell differentiation and its relationship to vascular formation and related our findings to the different ability of the retina and choroid to autoregulate.

Methods: : Mural cells were investigated following triple-label immunohistochemistry applied to human retinal and choroidal wholemounts and histological cross sections, aged 8 to 40 weeks gestation (WG). Antibodies to αSMA, Desmin, NG2, Calponin, Caldesmon, CD34 and CD39 were used to visualise the relationship between smooth muscle cells, pericytes and the forming vasculature. Further, mural cell markers were combined with stem cell markers to determine the role of stem cells in mural cell formation.

Results: : SMA⁺ mural precursor cells were found scattered and isolated over the primordial vascular tree at 12WG. Retinal vessels were ensheathed with numerous NG2⁺ pericytes during their formation and remodelling. αSMA⁺ SMCs were restricted to major artery/vein pairs radiating from the optic nerve head. CD44⁺ /αSMA⁺ smooth muscle cells and CD44+/CD39+ vascular endothelial cells were observed on large choroidal arteries during human fetal development. Non vascular-associated αSMA⁺/CD34^+/--/ NG2⁺/desmin^- presumed mural precursor cells (MPCs) were observed associated with immature choroidal blood vessels at 12, 18 and 20 WG. αSMA was evident along arteries from 15WG and increased centripedally with maturation. αSMA was localised only on medium and large vessels, being stronger on arteries. Similarly, calponin and caldesmon (calcium regulating proteins) were expressed only on the large vessels. Even at 36WG gestation, SMA ensheathment of large arteries was complete in the retina, but less organised in the choroid. Remarkably, pericytes, as indicated by desmin and NG2 labelling, were absent on human choroidal vessels.

Conclusions: : We have shown for the first time that CD44⁺ stem cells give rise to αSMA⁺ smooth muscle cells and CD39⁺ vascular endothelial cells in the human choroid. Moreover, we have provided evidence of a marked difference in pericyte ensheathment between choroidal and retinal vessels, underlying the marked difference in auto-regulatory ability reported between the two vascular plexii.

Keywords: choroid • retinal development • age-related macular degeneration