Highly Sensitive In Vitro Methods for Detection of Residual Undifferentiated Cells in Retinal Pigment Epithelial Cells Derived from Human iPS Cells

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.

Preclinical Studies for Induced Pluripotent Stem Cell-based Therapeutics

“…Extensive epigenetic modifications occurring during reprogramming and differentiation may make iPSCs more prone to causing cancer following transplantation. Development of highly sensitive methods for detection and efficient separation of undifferentiated cells will be needed (86,87). Among new methods potentially limiting the tumorigenicity of iPSCs are increasing the copy number of tumor suppressors (88) and the use of specific drugs such as metformin (89) and pluripotent cell-specific inhibitors (90).…”

Tumorigenicity Studies for Human Pluripotent Stem Cell-Derived Products

“…The qRT-PCR method with a specific probe and primers has been found to detect a trace amount of lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSc and 5×10 4 RPE cells. 14) As tumorigenic cells are commonly highly proliferative and immortalized, observation of the cell growth rate by culturing for a limited period also seems to be useful to detect rapidly growing contaminated immortalized cells. If combinations of these in vitro tumorigenicity-associated tests do not demonstrate the existence of both undifferentiated and immortalized cells, the tumorigenic potential of final products can be considered extremely low.…”

Impaired Autophagy in Retinal Pigment Epithelial Cells Induced from iPS Cells obtained from a Patient with Sialidosis

“…The cells exhibited epithelial sheet-like morphology and had tight junctions, which was confirmed by immunocytochemistry using anti-ZO-1 antibodies reacting with tight junction protein 1 ( Figures 3A(b) and 3A(e)). qPCR analysis showed that ZO-1-positive epithelial sheet-like cells from the control and the patient had lost the pluripotent marker Oct3/4, but expressed RPE-specific genes RPE65 and CRALP, as well as neuronal or retinal cell markers Pax6, Rx and MITF, although the expression levels slightly differed between the cell lines ( Figure 3) [15,16].…”