Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells

  • Stem Cell Res. 2021 May:53:102368. doi: 10.1016/j.scr.2021.102368.
Tessa E Dewell  1 ,  Ketrin Gjoni  1 ,  Angela Z Liu  1 ,  Ashley R G Libby  2 ,  Anthony T Moore  3 ,  Po-Lin So  4 ,  Bruce R Conklin  5
Affiliations
  • 1. Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158, USA.
  • 2. Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158, USA; Developmental and Stem Cell Biology Program, University of California, 1675 Owens St, San Francisco, CA 94158, USA.
  • 3. UCSF Department of Ophthalmology, 10 Koret Way, San Francisco, CA 94143-0730, USA.
  • 4. Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158, USA; Innovative Genomics Institute, 2151 Berkeley Way, Berkeley, CA 94720, USA; Gladstone Institutes Stem Cell Core, 1650 Owens Street, San Francisco, CA 94158, USA.
  • 5. Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158, USA; UCSF Department of Ophthalmology, 10 Koret Way, San Francisco, CA 94143-0730, USA; Innovative Genomics Institute, 2151 Berkeley Way, Berkeley, CA 94720, USA; UCSF Department of Medicine, 535 Mission Bay Blvd South, San Francisco, CA 94158, USA. Electronic address: [email protected].
PMID: 34087997 DOI: 10.1016/j.scr.2021.102368
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) is 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 Best Disease 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.

Keywords
Cell Differentiation; Induced Pluripotent Stem Cells; Retinal Diseases; Retinal Pigment Epithelium; Transcription Factors; iPSC.
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