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Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium from Human Induced Pluripotent Stem Cells

15 Pages Posted: 7 Oct 2020 Publication Status: Published

See all articles by Tessa E. Dewell

Tessa E. Dewell

Gladstone Institutes

Ketrin Gjoni

Gladstone Institutes

Angela Z. Liu

Gladstone Institutes

Ashley R.G. Libby

University of California, San Francisco (UCSF) - Developmental and Stem Cell Biology PhD Program; University of California, San Francisco (UCSF) - Gladstone Institute of Cardiovascular Disease

Anthony T. Moore

UCSF Department of Ophthalmology

Po-Lin So

Gladstone Institutes

Bruce R. Conklin

University of California, San Francisco (UCSF) - Gladstone Institute of Cardiovascular Disease

Abstract

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) are valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

Suggested Citation

Dewell, Tessa E. and Gjoni, Ketrin and Liu, Angela Z. and Libby, Ashley R.G. and Moore, Anthony T. and So, Po-Lin and Conklin, Bruce R., Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium from Human Induced Pluripotent Stem Cells. Available at SSRN: https://ssrn.com/abstract=3701636 or http://dx.doi.org/10.2139/ssrn.3701636

Tessa E. Dewell

Gladstone Institutes ( email )

Ketrin Gjoni

Gladstone Institutes ( email )

Angela Z. Liu

Gladstone Institutes

Ashley R.G. Libby

University of California, San Francisco (UCSF) - Developmental and Stem Cell Biology PhD Program

Third Avenue and Parnassus
San Francisco, CA 94143
United States

University of California, San Francisco (UCSF) - Gladstone Institute of Cardiovascular Disease

1650 OWENS STREET
San Francisco, CA 94158
United States

Anthony T. Moore

UCSF Department of Ophthalmology

Po-Lin So

Gladstone Institutes ( email )

Bruce R. Conklin (Contact Author)

University of California, San Francisco (UCSF) - Gladstone Institute of Cardiovascular Disease

1650 OWENS STREET
San Francisco, CA 94158
United States

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