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Olivier Goureau, Amelie Slembrouck, Angelique Terray, Giuliana Gagliardi, Celine Nanteau, Jose Alain Sahel, Sacha Reichman; Scalable and reliable generation of retinal cells from human transgene-free induced pluripotent stem cells under defined xeno-free and feeder-free conditions. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3577.
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© ARVO (1962-2015); The Authors (2016-present)
For retinal cell therapy based on human induced pluripotent stem (iPS) cells, one of the major challenges is to develop essential culture conditions for the use of these cells for future clinical purposes. Until recently, iPS cell culture (maintenance and/or differentiation) has been carried out using feeder cells and/or culture media that contain animal products. Here, we adapted our new retinal differentiation method using confluent human iPS cells, bypassing cell clumps or embryoid body formation and in absence of Matrigel or serum (Reichman et al. PNAS 2014; 111:8518), in a well-defined xeno-free / feeder-free (XF/FF) system
Integration-free iPS cells cultured on mouse embryonic fibroblasts were transferred onto vitronectin-coating plates and cultured with xeno-free medium. Confluent iPS cells obtained in these XF/FF conditions were directed toward a retinal lineage in a serum free proneural medium containing N2 supplement. Emergent neural retina (NR)-like structures were isolated and cultured in floating conditions for their maturation with a serum free proneural medium. Capacity for retinal differentiation was determined by immunohistochemistry and qRT-PCR analysis triggering specific developmental and mature retinal markers
In less than one month, confluent iPS cells are able to generate self-forming NR-like structures containing multipotent retinal progenitor cells (RPCs). Floating cultures of isolated neuroretinal tissue enabled the differentiation of RPCs into all types of retinal cells. Early-born retinal cells (i.e. ganglion, amacrine and horizontal cells) were identified after one month in culture, and late-born retinal cells (i.e. photoreceptors, Muller glial and bipolar cells) started to appear after two months
These data demonstrate that human iPS cell lines can be maintained and directed to differentiate into retinal cell types under XF/FF conditions that are required for translation to clinical applications. In this context the reliable generation of retinal ganglion cells and photoreceptor precursors could find important applications in regenerative medicine
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