Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.

Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell transplantation (HSCT) alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet such clinical demand, a fast MSCs expansion method is required. In the present study, we examined the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. The cells were cultured in a rotary bioreactor with Myelocult� medium containing a combination of supplementary factors, including stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). After 8 days of culture, total cell numbers, Stro-1+CD44+CD34- MSCs and CD34+CD44+Stro-1- HSCs were increased 9, 29, and 8 folds respectively. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type II collagen (chondrogenesis) and C/EBPα (adipogenesis) were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes and adipocytes. Taken together, we conclude that the rotary bioreactor with the modified Myelocult� medium reported in this study may be used to rapidly expand MSCs.