Background Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (and expression levels increased when 3 dosages of AF (10%, 20%, 30%) were used to treat cultures, when compared to FBS cultures and that this elevation was highest when using 30% AF. Surprisingly, expression levels in DMEM/F12-treated cultures (control) increased several-fold compared to those of AF- and FBS-treated cells (Figure ?(Figure66 Left). Figure 6 Relative retinal progenitor gene expression in AF-treated cultures (10%, 20%, 30%), FBS-treated cultures (10%) and DMEM/F12 (control)-treated cultures. RPE cell preparation and RNA extraction were performed as described in the methods. Relative gene expression … expression was not detected when 10% and 20% concentrations of AF were used but was present in 30% AF-treated cells in which expression was significantly increased when compared to FBS-treated 540769-28-6 IC50 cultures (5.55 fold). Similar to expression, control cultures also displayed a much greater increase in expression levels (70.85 fold) (Figure ?(Figure66 Center). Although expression levels increased with increasing doses of AF (10% and 20%), there was a slow decrease in expression levels in 30% AFexpression than FBS-treated cells. In the control cultures, the trend in level of expression was similar to that of and (Figure ?(Figure66 Right). Discussion The results presented in this study show that AF is a robust promoter of growth for retinal progenitor cells. AF has an approximately neutral pH (7.2), and its osmotic pressure is in the physiological range, thus providing a suitable and appropriate environment for cell growth and proliferation. Previous studies have been carried out to identify the content of the AF proteome. Cho et al identified the 15 most abundant proteins in AF at gestational ages of 1618 weeks which included albumin, fibronectin, serotransferrin, complement C3, ceruloplasmin and TGF- [27]. Previous works have also suggested the ability of AF to initiate regeneration in damaged cells [25,26], thus confirming the data obtained in this study. The innate 540769-28-6 IC50 capacity of adult somatic cells has many potential applications in regenerative medicine [28]. The retinal pigment epithelium begins as a plastic tissue, capable, in some species, of generating lens and retina but differentiates early in development and normally remains nonproliferative [29]. Our results show that RPE cells cultured on AF-coated surfaces displayed an organized alignment when compared to the disorganized spread on FBS-coated dishes. Although proteomic analysis of AF has not been the focus of this work, it seems that fibronectin, as the 3rd most abundant protein in AF in the 16th week of gestation [27], plays a pivotal role in making this organized alignment. Fibronectin is an extracellular matrix protein that has an essential role in cell attachment, polarity and migration [30]. The results also indicate that RPE cells grown in AF-containing medium require only 15 min for their initial attachment compared to at least 90 min required for those cultured in FBS-supplemented medium. Consistent with this observation, the report by Heth et al [31] demonstrates that cells grown on fibronectin- and laminin-coated microfilters required much less time to reach confluency when compared to collagen I-coated microfilters. Also, cell morphology was maintained better on fibronectin-coated microfilters, similar to our own observations in Mouse monoclonal to AKT2 this study of AF-treated cells. The increase in RPE cell proliferation and retinal progenitor gene expression levels in AF-supplemented medium were found to be dose-dependent. The cell proliferation ELISA, immunocytochemistry and RT-PCR data showed the ability of AF to induce retinal progenitor genes and thus convert an RPE culture into an invaluable source of retinal progenitor cells. It is likely that such a dose-dependent increase in proliferation and regeneration is due to the presence of growth factors whose concentration correlates with cell proliferation and regeneration. The effect on RPE cells of several of these growth factors, including TGF-, complement C3, 540769-28-6 IC50 albumin, plasminogen, ceruloplasmin and serotransferrin, has been examined previously. As suggested by Saika, following the formation of a wound in the tissue, the TGF- factor is activated, turning on a series of signaling pathways involved in proliferation and regeneration [32]. There are several other reports indicating the role of TGF- in epithelial mesenchymal transition (EMT), cell migration to the area of damage and the establishment of regeneration [33]. Complement C3 is another factor in AF that has been found to be responsible for the regeneration of damaged 540769-28-6 IC50 tissue. Kimura et al suggested that complement C3 plays a role in inducing cell proliferation and is specifically.