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S. G. Lecaude, R. S. Zulliger, V. Enzmann; In vitro Transformation of Bone Marrow-Derived Stem Cells During Coculture With Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1283.
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To harvest and characterize bone marrow-derived stem cells (BMSC) and to investigate their potential to pre-differentiate into RPE-like cells.
Stem cells were prepared from the bone marrow of three weeks old GFP+ C57BL/6 mice using paramagnetic beads for lineage depletion as well as for negative CD45 selection. The Lin- CD45- BMSC were then added to murine retinal pigment epithelium (RPE) cells (ratio 1:1). After coculture for up to 21 days immunohistochemistry was used to identify lineage-specific differentiation markers on BMSC. In addition, GFP+ BMSC were sorted by flow cytometry and expression of the marker genes was analyzed by RT-PCR.
Magnetic sorting for Lin- CD45- BMSC yielded 0.78 ± 0.53% of the original population of bone marrow cells with a purity of 87%. Immunohistochemistry staining showed that these cells expressed RPE markers (MITF, RPE65) after direct coculture with RPE cells on day 7 to 21. However, neuronal markers (MAP-2) were also detectable. Sorting for GFP+ cells after coculture yielded 2.56 ± 3.39% of the originally introduced cell number. PCR results confirmed the expression pattern in the sorted BMSC after coculture.
Lin- CD45- BMSC express RPE markers in vitro after coculture with RPE cells. These cells might represent a new source of RPE-like pre-differentiated stem cells and could be possibly used for regeneration therapy in dry AMD.
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