Abstract
Purpose: :
The aim of the project was to investigate the potential of human Embryonic Stem (hES) cells when grafted into the developing rat eye.
Methods: :
hES cells were grafted into the vitreous of post–natal day 4 rat eyes. Tissue was fixed at 4 weeks.
Results: :
There was extensive survival of hES cells. They did not form tumors. There were three regions where the hES cells were located: within the host retina, adjacent to the host inner retina and surrounding the lens. ED1 staining for macrophages/microglia showed there were occasional cells located around the graft, but with a non–reactive morphology. The hES cells did not express the astrocytic marker GFAP, but host astrocytic process were seen entering the region of the graft. There were differences in the expression and position of markers within the graft dependent on where in the eye the graft was located. Within the host retina, hES cells expressed NF–200 and HuD, markers for mature retinal ganglion cells (RGC). However, in the inner nuclear layer, hES expressed syntaxin–1, an amacrine cell marker, with processes extending into the inner plexiform layer and RGC layer. Numerous connections between host and transplant were visualized using recoverin, tyrosine hydroxyalase and melanopsin immunohistochemistry. Interestingly, hES adjacent to the host inner retina had the appearance of an ectopic retina expressing NF–200 on the inner surface. Grafted cells around the lens appeared organized morphologically, but did not express retinal markers in layers. Instead, NF–200, map–2 and HuD staining was expressed within the transplanted hES. Host microglia also expressed recoverin when in close proximity to the transplant. There was extensive expression of nestin in the host retina by Müller glia
Conclusions: :
In conclusion, hES survive, integrate and express mature retinal markers. The pattern of expression suggests that hES are influenced by extrinsic environmental cues possibly mediated through host microglia. Although, hES may differentiate along an intrinsic developmental clock.
Keywords: transplantation • regeneration • differentiation