Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells

Ken Kono,Akifumi Matsuyama,Rumi Sawada,Yoji Sato,Satoko Matsuyama,Satoshi Yasuda,Hiroyuki Mizuguchi,Takuya Kuroda

doi:10.1038/s41598-018-36848-5

Ken Kono, Akifumi Matsuyama + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-36848-5

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Journal: Scientific Reports	Publication Date: Mar 6, 2019
Citations: 6	License type: open-access

Affiliation: Fujita Health University

Abstract

Cell-processed therapeutic products (CTPs) derived from human pluripotent stem cells (hPSCs) have innovative applications in regenerative medicine. However, undifferentiated hPSCs possess tumorigenic potential; thus, sensitive methods for the detection of residual undifferentiated hPSCs are essential for the clinical use of hPSC-derived CTPs. The detection limit of the methods currently available is 1/105 (0.001%, undifferentiated hPSCs/differentiated cells) or more, which could be insufficient for the detection of residual hPSCs when CTPs contain more than 1 × 105 cells. In this study, we developed a novel approach to overcome this challenge, using adenovirus and adeno-associated virus (AdV and AAV)-based selective cytotoxic vectors. We constructed AdV and AAV vectors that possess a suicide gene, iCaspase 9 (iCasp9), regulated by the CMV promoter, which is dormant in hPSCs, for the selective expression of iCasp9 in differentiated cells. As expected, AdV/CMV-iCasp9 and AAV/CMV-iCasp9 exhibited cytotoxicity in cardiomyocytes but not in human induced pluripotent stem cells (hiPSCs). The vectors also induced apoptosis in hiPSC-derived cardiomyocytes, and the surviving cells exhibited higher levels of hPSC marker expression. These results indicate that the AdV- and AAV-based cytotoxic vectors concentrate cells expressing the undifferentiated cell markers in hiPSC-derived products and are promising biological tools for verifying the quality of CTPs.

Highlights

Human cell-processed therapeutic products are expected to provide novel breakthrough therapies for life-threatening or incurable diseases
We have developed several methods for detecting a trace amount of undifferentiated human pluripotent stem cells (hPSCs) in hCTPs8–10, some of which have been implemented for the assessment of Human cell-processed therapeutic products (hCTPs) quality[11]
The detection limits of our methods and those developed by other groups are 0.001% or more, which could be sufficient for the quality control of hCTPs containing fewer than 1 × 105 cells

Summary

Introduction

Human cell-processed therapeutic products (hCTPs) are expected to provide novel breakthrough therapies for life-threatening or incurable diseases. In addition to somatic and somatic stem cells, human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), have been used as new sources of hCTPs. Since hPSCs possess tumorigenic potential, there is a potential risk of tumor formation if the products contain residual undifferentiated hPSCs1. In addition to gene expression analyses for detection of undifferentiated cell markers, a highly efficient amplification method using a laminin-521-based cell culture system with Essential 8 medium directly detects a trace amount of hPSCs (0.001%)[9]. The establishment of new methods that overcome the detection limit of 0.001% is essential for the clinical use of hCTPs. In this study, we developed a novel approach using adenovirus and adeno-associated virus (AdV and AAV)-based selective cytotoxic vectors. The vectors could be a potential biological tool for overcoming the detection limit (0.001% or more) of the test methods for residual hPSCs in hPSC-derived hCTPs

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Conclusion