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Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions.

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.

SUBMITTER: Regent F

PROVIDER: S-EPMC6650487 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions.

Regent Florian F Morizur Lise L Lesueur Léa L Habeler Walter W Plancheron Alexandra A Ben M'Barek Karim K Monville Christelle C

Scientific reports 20190723 1

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 ...[more]

PMID: 31337830

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Project description:UnlabelledThe derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications.SignificanceIn the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs), followed by the differentiation of these cells toward a retinal lineage, including photoreceptors, retinal ganglion cells, and retinal pigment epithelium, has been demonstrated. The use of mRNA reprogramming to yield pluripotency represents a unique ability to derive pluripotent stem cells without the use of DNA vectors, ensuring the lack of genomic integration and constitutive expression. The studies reported in the present article serve to establish a more reproducible system with which to derive retinal cell types from hiPSCs through the prevention of genomic integration of delivered genes and should also eliminate the risk of constitutive expression of these genes. Such ability has important implications for the study of, and development of potential treatments for, retinal degenerative disorders and the development of novel therapeutic approaches to the treatment of these diseases.

Dataset Information

Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions.

Publications

Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions.

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