Abstract
Dysfunction or death of retinal pigment epithelial (RPE) cells is involved in some forms of Retinitis Pigmentosa and in age-related macular degeneration (AMD). Since there is no cure for most patients affected by these diseases, the transplantation of RPE cells derived from human pluripotent stem cells (hPSCs) represents an attractive therapeutic alternative. First attempts to transplant hPSC-RPE cells in AMD and Stargardt patients demonstrated the safety and suggested the potential efficacy of this strategy. However, it also highlighted the need to upscale the production of the cells to be grafted in order to treat the millions of potential patients. Automated cell culture systems are necessary to change the scale of cell production. In the present study, we developed a protocol amenable for automation that combines in a sequential manner Nicotinamide, Activin A and CHIR99021 to direct the differentiation of hPSCs into RPE cells. This novel differentiation protocol associated with the use of cell culture robots open new possibilities for the production of large batches of hPSC-RPE cells while maintaining a high cell purity and functionality. Such methodology of cell culture automation could therefore be applied to various differentiation processes in order to generate the material suitable for cell therapy.
Highlights
Human pluripotent stem cells, including human embryonic stem cells and human induced pluripotent stem cells are characterized by unlimited self-renewal and their ability to differentiate into any cell type
In an effort to simplify previous directed differentiation protocols for automation, we evaluated whether the simple use of NIC, Activin A and Chir99021 in a sequential manner improves retinal pigment epithelial (RPE) cell differentiation of adherent human embryonic stem cells (hESCs) enough to bypass manual enrichment
The use of NIC for the first 7 days of differentiation significantly enhanced the transient expression of the early eye field transcription factors SIX homeobox 3 (SIX3) and Retinal homeobox (RAX) concomitantly to a higher decrease of the expression of the pluripotency marker NANOG at mRNA level when compared to the spontaneous protocol (p < 0.01; Fig. 1B)
Summary
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are characterized by unlimited self-renewal and their ability to differentiate into any cell type. Www.nature.com/scientificreports replacement material in on going and planned clinical trials[12,13,14,15,16] This spontaneous method remains fastidious, inefficient and time consuming (8 to 12 weeks of hPSCs differentiation) making it incompatible with the industrial large-scale production which is required to treat the potential millions of patients. Following data demonstrating that RPE and neural retina progenitors (NRPs) have the same embryonic origin, they combined a protocol allowing the efficient differentiation of NRPs23 with previously described RPE inducing factors such as Nicotinamide (NIC) and Activin A24, a member of the TGF-β super family Using this method, they obtained a large majority of cells expressing the pigmentation marker PMEL17 after 14 days of differentiation allowing bypassing manual enrichment of pigmented cells. The recent development of protocols allowing efficient differentiation of hPSCs into RPE cells offers the possibility to automate the production of these cells
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