Quality assessment tests for tumorigenicity of human iPS cell-derived cartilage

Abstract

Articular cartilage damage does not heal spontaneously and causes joint dysfunction. The implantation of induced pluripotent stem cell (iPSC)-derived cartilage (iPS-Cart) is one candidate treatment to regenerate the damaged cartilage. However, concerns of tumorigenicity are associated with iPS-Cart, because the iPSC reprogramming process and long culture time for cartilage induction could increase the chance of malignancy. We evaluated the tumorigenic risks of iPS-Cart using HeLa cells as the reference. Spike tests revealed that contamination with 100 HeLa cells in 150 mg of iPS-Cart accelerated the cell growth rate. On the other hand, 150 mg of iPS-Cart without HeLa cells reached growth arrest and senescence after culture, suggesting less than 100 tumorigenic cells, assuming they behave like HeLa cells, contaminated iPS-Cart. The implantation of 10,000 or fewer HeLa cells into joint surface defects in the knee joint of nude rat did not cause tumor formation. These in vitro and in vivo studies collectively suggest that the implantation of 15 g or less iPS-Cart in the knee joint does not risk tumor formation if assuming that the tumorigenic cells in iPS-Cart are equivalent to HeLa cells and that nude rat knee joints are comparable to human knee joints in terms of tumorigenicity. However, considering the limited immunodeficiency of nude rats, the clinical amount of iPS-Cart for implantation needs to be determined cautiously. Cartilage consists of chondrocytes embedded in abundant cartilage extracellular matrix (ECM). Articular cartilage covers the ends of bones and constitutes the joint surface, providing lubrication in diarthrodial joints. Articular cartilage has only limited capacity for intrinsic repair, and its damage due to trauma hardly heals, leading to debilitating conditions such as osteoarthritis. The implantation of cells that can induce cartilage is an expected treatment against the focal damage of articular cartilage. Cell sources for such cell therapy include chondrocytes, mesenchymal stromal/stem cells (MSCs), and pluripotent stem cells (PSCs). Autologous chondrocytes are limited in number, and thus need to be expanded in culture prior to implantation, which causes the loss of their chondrocytic properties 1. On the other hand, MSCs and PSCs are subjected to expansion and chondrogenic differentiation before implantation. Induced PSCs (iPSCs) are somatic cells that have been reprogrammed to a PSC state, but unlike other PSCs such as embryonic stem cells (ESCs), they are free from ethical issues such as the sacrifice of embryos. Clinically applicable iPSCs have been produced by the transient expression of reprogramming factors using episomal vectors, and the resultant iPSCs are free from footprints. Laboratories including ours reported protocols for the creation of cartilage from human iPSCs 2. In these protocols, iPSCs were differentiated to chondrocytes, followed by transfer to 3D culture to make the chondrocytes produce ECM for the creation of iPSC-derived cartilage (iPS-Cart). Cartilage and iPS-Car...

Transplantation of Human Autologous Synovial Mesenchymal Stem Cells with Trisomy 7 into the Knee Joint and 5 Years of Follow-up

“…In addition, synovial MSCs with trisomy 7 did not induce scaffold‐independence or clonal proliferative potential, nor did they express oncogenes or show an increase in specific senescent cells. Furthermore, similar to the safety test for iPS cells, 33 synovial MSCs with trisomy 7 did not form tumors after transplantation into the subcutaneous region and knees of NOD/SCID mice. Our intensive analysis of synovial MSCs with trisomy 7 for tumorigenesis did not reveal any specific features.…”

Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells

“…To assess these possibilities, we subjected chondrocytes isolated from cartilage and generated from a clinical‐grade iPSC line to expansion culture for a prolonged period. Notably, we found that the chondrocytes reached cell senescence, suggesting the cartilage was not contaminated with transformed cells (Takei et al., 2020).…”

“…To measure this number, we used HeLa cells as model transformed cells and transplanted them into nude rat osteochondral defects of 1 mm diameter and 0.5 mm deep. Tumors were not observed when 10 4 or fewer HeLa cells were transplanted, but they did appear if 10 5 HeLa cells were transplanted (Takei et al., 2020). These results indicate that the implantation of up to 10 4 transformed cells in the knee joint does not risk tumor formation if assuming the transformed cells are equivalent to HeLa cells and rat knee is equivalent to human knee.…”

Epigenetic reprogramming of cell identity: lessons from development for regenerative medicine

“…Another important aspect for clinical application of these cells is regarding the safety including long-term behavior of these cells and tumorigenic potential once they are transplanted back into the patients [113,114]. There have also been efforts to generate the iPSCs without viral genome integration or even without the use of viruses for delivery of the transcription factors in the cell as the integration of viral genome in recipient cell is associated with tumorigenic consequences [115].…”