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Jing Hua, Takaaki Hattori, Sandhya Sharma, Francisco Amparo, Narghes Calcagno, Daniel Saban, Reza Dana; Ex Vivo Expansion Of Bone Marrow Derived Cells From Normal Human Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1116.
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© ARVO (1962-2015); The Authors (2016-present)
Hematopoietic bone marrow derived cells differentiate into leukocyte types, e.g. antigen presenting cells (APC), and then migrate through or take up and residence in peripheral tissue, e.g. cornea. Interestingly, a considerable number of bone marrow derived cells in the normal cornea exhibit a ‘precursor’ phenotype (e.g., CD14+, Sca-1+, CD34+); however, their capacity to expand and/or differentiate into APCs is incompletely understood and we thus began to investigate this here.
Human corneas were obtained, pooled and separated into cornea, limbus and bulbar conjunctiva. Tissues were collagenase digested into single-cells and respectively plated (10^5/ml). Expansion was driven by addition of hematopoietins (50ng/ml GMCSF and 50ng/ml IL-4, replenished every 48-72hr) in complete RPMI for 2 weeks. Negative controls were plated in the absence of hematopoietins. Another negative control used were human dermal fibroblasts plated in the presence of hematopoietins. The positive control was served by plating human peripheral blood monocytic cells (PBMC) in the presence of hematopoietins. Cultures were followed via inverted microscopy for evidence of expansion, and cells were harvested after 14d and assessed via FACS for relevant APC markers.
Limbal cells plated with hematopoietins did not show significant expansion since percent CD45+ cells increased only marginally, relative to limbal cells plated without hematopoietins (5.9% vs. 2.1%, respectively). Similarly, while no significant expansion was observed in conjunctival cells plated with hematopoietins, a 10-fold increase in CD45+ cells (22%) was observed for corneal cells plated with hematopoietins. These CD45+ cells from cornea cultures were also CD11b+ CD11c+, thus suggesting a myeloid dendritic cell (DC) differentiation, and expressed HLA DR+ CD86+. Interestingly, however, the mean fluorescence intensity of HLA DR+ CD86+ was considerably lower than levels expressed by CD11b+ CD11c+ DCs differentiated from PBMC, even after 24hr LPS stimulation.
These data suggest that cornea possesses bone marrow derived cells at a functional precursor state, and capable of expanding and differentiating into DCs. Furthermore, consistent with the function of corneal DCs in vivo, putative ex vivo expanded DCs also appear to be resistant to phenotypic maturation.
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