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November 14, 2013

Culturing Primary Cells

A small molecule can reversibly proliferate primary RPE cells isolated from human donors.

Culturing Primary Cells

A supply of RPE cells would be useful not only for research but potentially as a therapy for eye diseases such as age-related macular degeneration. [ggw/Fotolia.com]

  • The ability to work with primary cells ex vivo can enable the development of more physiologically relevant assays. Primary retinal epithelial cells (RPE) typically enter senescence after six passages. A supply of RPE cells would be useful not only for studying the pathways involved with RPE cell health but potentially as a therapy for ocular diseases such as age-related macular degeneration (AMD) through transplantation of healthy cells into the eye. This article* demonstrates that a small molecule (W3), originally identified as a proliferative molecule for β cells, can promote culturing of RPE cells through 15 passages.

  • Click Image To Enlarge +
    Figure. (A,B) WS3-expanded fetal human retinal pigment epithelial (RPE) cells grown in the presence of WS3 and differentiated for 5 weeks. (C–F) Staining of differentiated fetal RPE cells show the formation of tight junctions, expression of mature RPE markers, and function in vitro as determined by phagocytosis of internalized opsin.

    After approximately 15 passages the RPE cells entered senescence without evidence of cell death. Optimal proliferation of RPE cells occurred when W3 was added prior to passage 6. Cytogenic analysis of passage 10 RPE cells did not show karyotype aberrations. Additionally, when the RPE cells were allowed to reach confluence, they differentiated into a cobblestone monolayer and became pigmented, and cell staining showed the presence of tight junctions (Figure). RPE cells are one of the most active phagocytic cells known and this activity is required to recycle the discs containing visual pigments at outer photoreceptor segments.

    The W3-expanded and differentiated RPE cells were found to be phagocytic as measured by the internalization of oposin (Figure). The target of W3 in β cells is Erb3 binding protein 1 (Ebp1), a tyrosine kinase related to the epidermal growth factor receptor (EGFR) receptor tyrosine kinase family. Pull-down experiments using a biotinylated analog of W3 supports that Ebp1 is also a primary target of W3 in RPE cells.

    To demonstrate that the W3-expanded RPE cells are functional in vivo, the authors transplanted these cells into rats harboring a mutation that inactivates photoreceptor outer segment phagocytosis resulting in a decrease in visual acuity with age. The RPE grafted eyes showed improved luminance threshold values and optokinetic responses. Additionally, P90 rats showed four to six layers of photoreceptors compared to only one found in untreated rats. The fact that W3 acts as proliferative molecule in both β cells and RPE cells with a common target suggest that this could be useful for the expansion of other primary cell types.

  • *Abstract from ACS Chemical Biology 2013, Vol. 8: 1407–1411

    Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for transplantation.

    Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.

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