Differentiation of human protein-induced pluripotent stem cells toward a retinal pigment epithelial cell fate
Open accessAbstract
Compared with many induced pluripotent stem cell (iPSC) lines generated using retrovirus and other non-integrating methods, the utilization of human protein-induced iPSC (piPSC) lines may provide a safer alternative for the generation of retinal pigment epithelial (RPE) cells for transplantation in retinal degenerative diseases. Here we assess the ability of piPSCs to differentiate into RPE cells, and to perform native RPE cell behavior. piPSCs were seeded in 6-well low-attachment plates to allow embryoid body formation, and then analyzed for pluripotent stem cell markers NANOG, SSEA4 and TRA-1-60 by immunofluorescence. Following colony formation, piPSCs were assessed for confirmation of RPE cell differentiation by staining for zonula occludens (ZO-1), bestrophin, microphthalmia-associated transcription factor (MITF) and retinal pigment epithelium specific protein-65 (RPE65). To evaluate piPSC-RPE cell phagocytic ability, adult bovine photoreceptor rod outer segments (ROS) were fed to piPSC-RPE cells, which were analyzed by fluorescent microscopy and flow cytometry. Undifferentiated piPSCs expressed all pluripotent markers assessed and formed embryoid body aggregates after 7 days. Differentiated piPSC-RPE cells expressed ZO-1, bestrophin, MITF and RPE65, typical RPE cell markers. Flow cytometry revealed robust ingestion of fluorescently-labeled ROS by piPSC-RPE cells, which was over four-times greater than that of undifferentiated piPSCs and comparable to that of an immortalized RPE cell line. Phagocytosis activity by piPSC-RPE cells was significantly reduced after the addition of anti-integrin αVβ5. In conclusion, piPSCs can be differentiated toward an RPE cell fate, expressing RPE cell markers and resembling native RPE cells in behavior. These results demonstrate that piPSCs can be differentiated into RPE-like cells using a method that has an increased safety profile, a critical consideration for the development of better treatments for retinal degenerative diseases such as age-related macular degeneration (AMD).
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Published protocols using directed approaches provide relatively efficient differentiation into cultures of cells reported to possess a wide range of RPE characteristics, including gene expression, polarized organization, and physiological and cell biological functions.
“There are now published protocols using directed approaches that provide relatively efficient differentiation into cultures of cells, reported to possess a wide range of RPE characteristics, including gene expression, polarized organization, and physiological and cell biological functions (Idelson et al., 2009; Buchholz et al., 2013; Gong et al., 2015; Maruotti et al., 2015; Hazim et al., 2017).”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Gong et al., 2015
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References 49 · Cited by 21
- Authors
- Gong, J., Fields, M. A., Moreira, E. F., Bowrey, H. E., Gooz, M., Ablonczy, Z., & Del Priore, L. V.
- Affiliations
- Medical University of South Carolina (US)
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2015-11-25
- Volume
- 10
- Issue
- 11
- Pages
- e0143272
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC4659559
- OpenAlex
- W2172916341
- Cited by
- 21
- References
- 49
- Citations by year
- 2025: 1 · 2023: 2 · 2022: 1 · 2021: 2 · 2020: 2 · 2019: 3 · 2018: 1 · 2017: 7 · 2016: 2
- FWCI
- 1.45
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Research to Prevent Blindness
- Medical University of South Carolina
- University of South Carolina
- National Cancer Institute (P30CA138313)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
- Keywords
- retinal pigment epithelium differentiation, integrin αvβ5, retinal degeneration, age-related macular degeneration, embryoid body formation, RPE65, MITF, bestrophin, ZO-1
- MeSH terms
- Cell Self Renewal, Animals, Cattle, Cell Differentiation, Cell Line, Transformed, Epithelial Cells, Humans, Phagocytosis, Biomarkers, Immunophenotyping, Retinal Rod Photoreceptor Cells, Retinal Pigment Epithelium, Induced Pluripotent Stem Cells
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