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X. Deng, D. Zhu, C. Spee, D. R. Hinton; A 3d Co-Culture System Facilitates Photoreceptor-Like Differentiation of Human Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5950.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) supports and maintains the function of mature photoreceptor cells and other neural retina cells in vivo. However, its detailed effect on the developmental differentiation and maturation of photoreceptors remains to be elucidated. In this study, we developed a novel 3D co-culture system, observed and evaluated the in vitro effect of polarized RPE on the growth and differentiation of human retinal progenitor cells (RPC).
1). Polarized RPE culture: Human fetal RPE (fRPE) cells and human embryonic stem cell derived RPE (hES-RPE) cells were cultured on fibronectin-coated transwells until trans-epithelial resistance (TER) reached 500Ω/cm2. 2). RPC isolation: Retinas were dissected from human fetal eyes (18-20 gestational weeks) and digested with trypsin. The isolated RPCs were suspension-cultured in RPC medium to form neurospheres. 3). 3D culture: Retinal neurospheres were laid and cultured directly on top of polarized RPE, indirectly on RPE with the matrix gel (Invitrogen) between the RPE and neurospheres, and on top of RPE-free matrix gel as a control. The RPE and neurospheres inside the matrix gel were co-cultured for 2 to 4 weeks. 4). Immunohistochemistry was used to identify the expression of neuronal and photoreceptor specific genes.
RPC cells grew well in the matrix gel for more than 30 days under the RPC-RPE co-culture condition. With immunohistochemical staining assay, more MAP2 and Recoverin positive cells were observed in RPC-RPE co-culture system than in RPC alone system. Furthermore, many more Recoverin-positive rosette-like cells were formed in the novel 3D cultured system than in traditional 2D culture system, indicating that this 3D culture system increases photoreceptor-like cell differentiation.
The novel 3D culture system effectively facilitates the interaction between RPE and RPC. RPE can promote the differentiation of RPC into retinal neuronal and photoreceptor-like cells.
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