br Introduction Advances in stem cell based therapies
Introduction Advances in stem-cell-based therapies may help overcome CNS disorders such as spinal cord injury (SCI). Transplantation of neural stem/progenitor pppa (NS/PCs) has yielded beneficial effects and improved functional recovery in SCI animal models (Cummings et al., 2005; Hofstetter et al., 2005; Iwanami et al., 2005; Ogawa et al., 2002; Okada et al., 2005; Salazar et al., 2010; Yasuda et al., 2011). Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), can differentiate into NS/PCs (Falk et al., 2012; Fujimoto et al., 2012a; Kumagai et al., 2009; Miura et al., 2009; Nori et al., 2011; Okada et al., 2004, 2008; Tsuji et al., 2010), oligodendrocyte precursor cells (OPCs) (Keirstead et al., 2005; Wang et al., 2013), and motoneuron progenitors (Erceg et al., 2010; Lukovic et al., 2014) in vitro. Previous studies demonstrated the therapeutic potential of mouse and human iPSC-derived NS/PCs for SCI in mice and non-human primates (Fujimoto et al., 2012b; Kobayashi et al., 2012; Nori et al., 2011; Tsuji et al., 2010). However, tumorigenicity remains a major concern for clinical applications of iPSCs. Previously, we reported the safety and therapeutic potential of human iPSC-derived neurospheres (iPSC-NSs) for SCI in non-obese diabetic–severe combined immunodeficient (NOD-SCID) mice (Nori et al., 2011) using the iPSC clone 201B7 (Nori et al., 2011; Takahashi et al., 2007). Here, we aimed to characterize novel NS/PCs derived from a different iPSC clone, 253G1. We established this clone from the same adult human dermal fibroblasts used for 201B7 by transducing three reprogramming factors: OCT4, SOX2, and KLF4 (Nakagawa et al., 2008). Grafted 253G1-derived neurospheres (253G1-NSs) survived and differentiated into three neural lineages in the injured spinal cord, and some of the resultant cells formed synapses with host neurons. Motor function in grafted mice initially recovered but then gradually declined, and tumors emerged during long-term observation. These tumors consisted of undifferentiated Nestin+ cells, but not NANOG+ pluripotent cells. Late-onset activation of the OCT4 transgene (Tg) may be associated with tumor formation. Transcriptome analysis revealed altered expression of genes involved in the epithelial-mesenchymal transition (EMT), which is related to tumor invasion and progression. Moreover, canonical pathway analysis revealed upregulation of the Wnt/β-catenin signaling pathway after 253G1-NS transplantation, which played a critical role in tumor development. Thus, although 253G1-NSs conferred temporary functional recovery in mice with SCI, they later developed into tumors and worsened the overall outcome.
Discussion Because we generated the human iPSC clone 253G1 without introducing c-MYC, we initially speculated that 253G1-NSs would be less tumorigenic than 201B7-NSs. Therefore, we performed transplantation of 253G1-NSs to treat SCI in adult NOD-SCID mice. Like the 201B7-NSs, the grafted 253G1-NSs differentiated into three neural lineages, reconstructed local circuitry, and promoted angiogenesis as well as axonal regrowth (data not shown). Thus, 253G1-NS transplantation promoted motor function recovery after SCI in NOD-SCID mice. When considering the clinical use of iPSC-NSs, it is important to address safety issues, especially with regard to tumorigenicity. To this end, we extended the observation period after transplantation. Previously, we reported that 201B7-NS-grafted mice maintained functional recovery until 103 days post-transplantation, and confirmed that 201B7-NSs were non-tumorigenic based on histological findings (Nori et al., 2011). Here, we found that 253G1-NS-grafted mice exhibited temporary motor function recovery for up to 47 days post-transplantation; however, this was followed by a gradual deterioration in motor function, along with grafted cell proliferation and tumor development. Bioluminescence imaging revealed that the photon count of the grafted cells increased more than 10-fold from the initial value by 103 days after transplantation. These tumors were negative for the pluripotency marker NANOG. Tumor size correlated with the proportion of Nestin+ cells in the graft at 103 days post-transplantation. Previously, we showed that after 201B7-NS transplantation, the proportion of grafted cells that were Nestin+ decreased from 10.7% ± 2.2% at 47 days to 7.5% ± 1.0% at 103 days post-transplantation, resulting in no evidence of tumorigenicity (Nori et al., 2011). By contrast, the proportion of Nestin+ cells increased from 19.6% ± 0.5% at 47 days to 33.1% ± 7.4% at 103 days after 253G1-NS transplantation, suggesting that differentiation-resistant Nestin+ cells proliferated over time and formed tumors. Consistent with this, the proportion of Ki-67+ cells significantly increased from 1.7% ± 0.17% at 47 days to 3.0% ± 0.2% at 103 days after 253G1-NS transplantation, which is significantly higher than what was observed after 201B7-NS transplantation. These findings suggest that the proportion of proliferating cells increased over time and induced tumor formation in 253G1-NS-grafted mice in the long term.