Abstract
Purpose: :
Adult Stem/progenitor cell differentiation represents a potential source of cells to repair or regenerate numerous cellular structures within the eye. RPE replacement therapy represents one such unrealised potential. We are investigating whether a population of adult progenitor/stem cells located within the eye, have the ability to differentiate into the RPE cells and thus act as a potential source of autologous cells for transplantation.
Methods: :
Pigmented epithelium from the CB of human donor eyes was seeded in serum free medium containing FGF2. Single spheres were transferred to medium containing 10% serum and these adherent cultures grown in the presence of TGF-beta superfamily members for 7 days. Markers of the neural retina, RPE and Retinal Progenitor Cells (RPCs) were then analysed by immunocytochemistry and QRT-PCR. To investigate which BMP4 pathway was being activated inhibitors of the smad and p38 pathway were added independently to the cells along with BMP4. To confirm the results Western blot analysis was carried out on protein from progenitor cells exposed to BMP4 and phospho p38 and phospho smad1/5 antibody used to investigate activation of theses respective pathways.
Results: :
Spheres from donors up to 97 yrs and up to 48 hours postmortem survived in serum free culture for several months. Removal of FGF2 was sufficient to reduce expression of the RPC marker Nestin but the additional presence of BMP4 was required to produce RPE marker expression in these retinal progenitor cells (RPCs) (an increase from less then 1% to 21% of cells expressing pan-cytokeratin). This was effect was concentration dependent up to 200ng/ml BMP4. Activin A had a much smaller effect and TGFbeta1 had no noticeable effect. Work with pathways inhibitors indicated that both the p38 and the smad pathway were being activated by BMP4 but Western blot analysis showed that BMP4 was acting mainly via its smad pathway.
Conclusions: :
This work demonstrates that BMP4 is a promising molecule for directing retinal progenitor cells toward an RPE phenotype and therefore may play a role in subsequent use in autologous cell transplantation.
Keywords: retinal pigment epithelium • differentiation • plasticity