Abstract
Purpose: :
To examine pig aqueous humor for stimulatory activity on embryonic rat retinal progenitor cells (RPCs) in vitro and partly to identify the components that contribute to this activity.
Methods: :
RPCs was prepared from the retina of embryonic day 19 Sprague Dawley rats and were cultured in medium consisting of DMEM:F12 together with N2–supplement, epidermal growth factor(EGF) in the presence or absence of aqueous humor for 0 to 4 days. Cell proliferation and cluster formation was quantified by 3H–thymidine assay and morphometric analysis, respectively. Antibodies used to characterize the RPCs included markers for neural progenitors (Nestin and Sox–2) and astrocytes (glial fibrillary acidic protein [GFAP] ) by the use of flow cytometry. Active fractions of porcine aqueous humor were identified by the use of size exclusion gel chromatograghy.
Results: :
Addition of aqueous humor to the culture medium enhanced both the number and size of clusters formed with 66% and 29 % within 4 days, respectively (p<0.01). This was further supported by an increased cell proliferation demonstrated with 3H–thymidine assay (p<0.01). By use of gelfiltration chromatography it was possible to demonstrate fractions containing the growth promoting activities. 22%, and 16% of the primary cells was found to express Nestin and Sox–2, respectively. GFAP was not detected.
Conclusions: :
The results demonstrate that aqueous humor contain factors which can stimulate formation and growth of RPC clusters in vitro. By size exclusion gel chromatography it is possible to identify fractions with specific growth promoting activity.