Journal article
Conditional Immortalization Using SV40 Large T Antigen and Its Effects on Induced Pluripotent Stem Cell Differentiation Toward Retinal Progenitor Cells
Stem cells and development, Vol.34(1-2), pp.26-34
01/2025
DOI: 10.1089/scd.2024.0124
PMCID: PMC11839531
PMID: 39611948
Abstract
Current treatments for retinal degenerative diseases are limited and cell replacement therapies, in tandem with a supportive biomaterial scaffold, serve as a promising emerging option. However, the development and in vitro testing of these therapies require large quantities of human retinal progenitor cells (RPCs) to thoroughly assess the impact of material properties, culture conditions, and surgical parameters on cell survival and fate to refine and optimize this approach. Although induced pluripotent stem cells (iPSCs) are an ideal cell source for human RPC derivation, large-scale production is resource-intensive and requires specialized expertise. In this study, our objective was to address this barrier by creating conditional, Tet-On SV40-T immortalized RPCs derived from human iPSCs. In our approach, we employ the Tet-On system to conditionally immortalize RPCs by inducing a SV40 large T (SV40-T) antigen, a gene known to influence cell cycle regulation and differentiation. We transduced human iPSCs with the Tet-On SV40-T system and analyzed their proliferation and RPC differentiation capabilities in the presence and absence of doxycycline (a tetracycline class of antibiotics). Our results revealed that while SV40-T immortalization increased cell proliferation, it adversely impacted the expression of crucial RPC markers (PAX6, SOX2, CHX10), leading to a significant loss of RPC identity and multipotency. This de-differentiation was irreversible, even after removing doxycycline, indicating permanent alterations in differentiation potential. Overall, this study highlights the challenges associated with generating and maintaining an immortal human RPC cell line, particularly with respect to balancing proliferation and differentiation. Our findings prompt further research into optimizing conditional immortalization techniques, culture conditions, and proliferation timing to maintain the integrity and functional characteristics of RPCs. Such advancements are crucial for reducing labor and costs associated with in vitro testing of therapeutics as we work toward the development of improved stem cell-based interventions for retinal disease.
Details
- Title: Subtitle
- Conditional Immortalization Using SV40 Large T Antigen and Its Effects on Induced Pluripotent Stem Cell Differentiation Toward Retinal Progenitor Cells
- Creators
- Qi Wang - University of IowaBrittany N Allen - University of IowaLaura R Bohrer - University of IowaErin R Burnight - University of IowaBudd A Tucker - University of IowaKristan S Worthington - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Stem cells and development, Vol.34(1-2), pp.26-34
- DOI
- 10.1089/scd.2024.0124
- PMID
- 39611948
- PMCID
- PMC11839531
- NLM abbreviation
- Stem Cells Dev
- ISSN
- 1547-3287
- eISSN
- 1557-8534
- Publisher
- MARY ANN LIEBERT, INC
- Grant note
- NIH: T32 GM007337, T32 HL144461 Roy J. Carver Charitable TrustUniversity of Iowa Office of the Vice President for Research
This work was funded by the NIH (T32 GM007337, Q.W.; T32 HL144461, Q.W.), the Roy J. Carver Charitable Trust, and the University of Iowa Office of the Vice President for Research.
- Language
- English
- Electronic publication date
- 11/29/2024
- Date published
- 01/2025
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; The University of Iowa Institute for Vision Research; Iowa Neuroscience Institute; Neuroscience and Pharmacology; Chemical and Biochemical Engineering; Ophthalmology and Visual Sciences
- Record Identifier
- 9984751758802771
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