Abstract
Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeutic products (CTPs). In the development of hPSC-derived CTPs, it is imperative to ensure that they do not form tumors after transplantation for safety reasons. Because cellular immortalization is a landmark of malignant transformation and a common feature of cancer cells, we aimed to develop an in vitro assay for detecting immortalized cells in CTPs. We employed retinal pigment epithelial (RPE) cells as a model of hPSC-derived products and identified a gene encoding slow skeletal muscle troponin T (TNNT1) as a novel marker of immortalized RPE cells by comprehensive microarray analysis. TNNT1 mRNA was commonly upregulated in immortalized RPE cells and human induced pluripotent stem cells (hiPSCs), which have self-renewal ability. Additionally, we demonstrated that TNNT1 mRNA expression is higher in several cancer tissues than in normal tissues. Furthermore, stable expression of TNNT1 in ARPE-19 cells affected actin filament organization and enhanced their migration ability. Finally, we established a simple and rapid qRT-PCR assay targeting TNNT1 transcripts that detected as low as 3% of ARPE-19 cells contained in normal primary RPE cells. Purified hiPSC-derived RPE cells showed TNNT1 expression levels below the detection limit determined with primary RPE cells. Our qRT-PCR method is expected to greatly contribute to process validation and quality control of CTPs.
Highlights
Human embryonic stem cells[1] and human induced pluripotent stem cells[2] are regarded promising cell sources for transplantation in regenerative medicine
The following three immortalized retinal pigment epithelial (RPE) cell lines were used: ARPE-19, a spontaneously immortalized cell line[21]; ARPE-19/HPV-16, an ARPE19 cell line transfected with DH-5 HPV-16; h1RPE7, a cell line derived from primary RPE cells transfected with a SV40 large T antigen[24]
Tumorigenicity, which is attributed to residual undifferentiated cells and transformation of cells, is one of the major concerns in developing Human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs)-derived cell-based therapeutic products (CTPs)
Summary
Human embryonic stem cells (hESCs)[1] and human induced pluripotent stem cells (hiPSCs)[2] are regarded promising cell sources for transplantation in regenerative medicine. Several methods for detecting a small population of residual undifferentiated cells in hPSC-derived CTPs have been reported: (1) in vivo tumorigenicity tests that detect teratoma formation in severe immunodeficient NOG mice[12], (2) detection of LIN28 mRNA as an undifferentiated cell marker using quantitative reverse transcription (qRT-)PCR and droplet digital PCR13, 14, and (3) a highly efficient culture method for residual undifferentiated hiPSCs in products[15]. Assay methods have been developed for the detection of small numbers of transformed cells in CTPs: (1) in vivo tumorigenicity tests with NOG mice[16], (2) digital analysis of soft agar colony formation[17], and (3) cell growth analysis[18]. Our study identified a gene encoding slow skeletal muscle troponin T (TNNT1) as a suitable marker of immortalized RPE cells contained in hPSC-derived CTPs, and we propose a qRT-PCR method for the detection of this marker
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