Mesenchymal stromal cells (MSC) can be cultivated from human choroidal tissue

Purpose : In the course of studying vascular endothelial cell cultures established from human choroidal tissue, we observed the existence of significant numbers of contaminating cells with a fibroblastic morphology. We hypothesized that these fibroblastic cells represent a form of mesenchymal stromal cell (MSC). Moreover, we considered that these presumptive choroidal MSC may be used as a feeder layer to encourage the growth of choroidal vascular endothelial cells (CVEC) in vitro. Methods : Primary cultures of choroidal stromal cells (from 5 cadaveric donors) were initiated by seeding choroid tissue explants into collagen gels (1 mg/mL; type I porcine collagen; Nitta Gelatin, Japan). The emergent cells were released by digestion in collagenase and passaged twice in DMEM supplemented with 10% foetal bovine serum, 2 mM L-glutamine and antibiotics (PenStrep). The phenotype of cells in each culture was examined by flow cytometry using a standard MSC-marker panel (CD34, CD45, CD73, CD90, CD105 and HLA-DR) and by immunocytochemistry for alpha-smooth muscle actin. Pluripotency was assessed using classic tri-lineage differentiation assays in parallel with MSC cultures derived from human bone marrow. CVEC were isolated from cadaveric tissue using magnetic-assisted cell sorting (MACS; via negative selection for CD45 followed by positive selection for CD31) and cultured in the presence and absence of stromal cells that had previously been growth inactivated by treatment with mitomycin C. CVEC growth was assessed by immunostaining for von Willebrand Factor. Results : Stromal cell cultures displayed a phenotype consistent with MSC derived from bone marrow (<1% positive for HLA-DR, CD34, CD45, and >95% positive for CD73, CD90 and CD105). Low numbers of myofibroblasts were also observed (1-7%). Evidence of classic MSC-tri-lineage differentiation was observed when the choroidal MSC were cultured under adipidogenic, osteogenic and chondrogenic conditions, however, the level of differentiation was less than that observed for MSC derived from bone marrow. Contrary to our feeder layer hypothesis, growth-arrested cultures of choroidal MSC consistently inhibited growth of freshly isolated CVEC. Conclusions : Our study demonstrates for the first time the ability to grow cells with MSC properties from the human choroid. The apparent inhibition of CVEC growth observed in the presence of growth-arrested choroidal MSC requires further investigation.