Abstract
Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.
Highlights
Pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells have two capabilities: 1) pluripotency: the ability to differentiate into a variety of cells and 2) self-renewal: the ability to undergo numerous cycles of cell division while maintaining their cellular identity
It is noteworthy that clinical trials have been conducted with retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) to treat patients with dry age-related macular degeneration and Stargardt’s macular dystrophy by Advanced Cell Technology. Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate into RPE cells, which display functionality both in vitro and in vivo [7,8]
Immunocytochemical staining revealed that N-cadherin, the major cadherin expressed in RPE cells [16], showed a distribution to the tight junction of the hiPSC-derived RPE cells, which is consistent with primary RPE cells (Fig. 1C)
Summary
Pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells have two capabilities: 1) pluripotency: the ability to differentiate into a variety of cells and 2) self-renewal: the ability to undergo numerous cycles of cell division while maintaining their cellular identity. Because of these two characteristics, it has been expected that they would provide new sources for robust and continuous production of a variety of cells and tissues for regenerative medicine/cell therapy. Autologous transplant of hiPSC-derived RPE cells holds great promise in the clinical therapy of macular degeneration
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
