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C. M. Sheridan, D. Pattwell, S. Thompson, S. Mason, D. L. Kent, I. Grierson; Differentiation Potential of Mesenchymal Stem Cells to RPE Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):502.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether human Mesenchymal Stem Cells (hMSC) could differentiate to human Retinal Pigmented Epithelium (hRPE)-like cells and therefore serve as a potential source of RPE cells for translational studies.
Female primary hRPE cells were cultured for up to 5 days in 4 well lab-teks in HAMS F10 medium + 20% FCS @ 37°C, 5% CO2. Male hMSC, were sorted using specific positive markers; CD73, CD105, and CD166 were negative for haematopoetic markers CD34, CD45 and CD14. hMSC cells were cultured for up to 5 days in MSC basal medium + 20% FCS in 4 well lab-teks @ 37°C, 5% CO2. immunocytochemistry (ICC) for primary antibodies against STRO-1, cytokeratin, RPE-65, MITF, Chx,10 Nestin and Pax-6 was compared in both cells types. For co-culture experiments, hMSC cells were grown in 25cm2 flasks for 5 days until confluent, trypsinised and loaded with 10mM Cytotracker Green (Molecular Probes). hMSC cells were added to monolayers of hRPE cells in 4 well lab-teks and co-cultured for one week in a 50:50 mixture of HAMS F10, MSC basal medium + 2% FCS. As a control group, hMSC cells were seeded in 4 well lab-teks and grown in conditioned medium from hRPE cells (50:50 mixture of HAMS F10, MSC basal medium + 2% FCS ) for the same period of time. Cells were examined for cytokeratin expression.
Under standard culture conditions hMSC cells were shown by ICC to express STRO-1 hMSC cells in culture whether in standard medium or in a 50:50 mix of HAMS F10, MSC basal medium + 2% FCS retained a monolayer appearance.hRPE cells were also found to express STRO-1 and only hRPE cells expressed cytokeratin. hRPE cells also retained a normal monolayer formation during culture under standard or co-culture conditions. Both cell types were positive for MITF expression, but negative for Chx10 expression. RPE-65 expression was only found in hRPE cell. hRPE cells and hMSC cells were both negative for the expression of neuroepithelium markers, nestin and Pax6. hMSC cells grown in conditioned medium expressed STRO-1 and MITF but did not express cytokeratin, RPE-65, Chx10, Nestin or Pax6. Results under co-culture conditions showed that 3 populations of cells existed, hRPE (positive for cytokeratin alone), hMSC (positive for the expression of Cytotracker green alone) and a population of cells that was positive for the cytotracker green and cytokeratin.
Co-culture conditions demonstrated a population of green labelled hMSC cells also positive for cytokeratin expression, whilst this was not seen in conditioned media experiments indicating cell-cell contact is required.
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