Abstract
Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61–77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.
Highlights
Human pluripotent stem cells, such as human induced pluripotent stem cells and human embryonic stem cells, are used as the raw materials of human cell-based therapeutic products due to their infinite self-renewal capacity and ability to differentiate into various cell types in vitro
To precisely define the number of human induced pluripotent stem cells (hiPSCs) transplanted into mice, single cell dissociation of hiPSCs would be required for cell counting
When defined numbers of dissociated single cells of the hiPSC line 201B7 were mixed with feeder cells, Matrigel and a Rho kinase inhibitor and subcutaneously transplanted into NOG mice, the TPD50 was calculated as 631
Summary
Human pluripotent stem cells (hPSCs), such as human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), are used as the raw materials of human cell-based therapeutic products (hCTPs) due to their infinite self-renewal capacity and ability to differentiate into various cell types in vitro. Several clinical trials and studies using hPSC-derived hCTPs have already started to treat spinal cord injuries, age-related macular degeneration, heart failure, and type I diabetes around the world [1]. Undifferentiated hPSCs are known to possess intrinsic tumorigenicity properties and to form teratomas in immunodeficient animals. Safety concerns associated with teratoma derived from hPSCs have been raised for development of hCTPs [2]. The possibility cannot be excluded that a trace amount of residual undifferentiated hPSCs in hCTPs leads to tumor development following transplantation. Contamination of hCTPs with residual undifferentiated hPSCs is a potential concern in terms of certain hazardous factors as well as the quality attributes of hPSC-derived hCTPs
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