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Long-Term Safety Issues of iPSC-Based Cell Therapy in a Spinal Cord Injury Model: Oncogenic Transformation with Epithelial-Mesenchymal Transition

Summary Previously, we described the safety and therapeutic potential of neurospheres (NSs) derived from a human induced pluripotent stem cell (iPSC) clone, 201B7, in a spinal cord injury (SCI) mouse model. However, several safety issues concerning iPSC-based cell therapy remain unresolved. Here, we investigated another iPSC clone, 253G1, that we established by transducing OCT4, SOX2, and KLF4 into adult human dermal fibroblasts collected from the same donor who provided the 201B7 clone. The grafted 253G1-NSs survived, differentiated into three neural lineages, and promoted functional recovery accompanied by stimulated synapse formation 47 days after transplantation. However, long-term observation (for up to 103 days) revealed deteriorated motor function accompanied by tumor formation. The tumors consisted of Nestin+ undifferentiated neural cells and exhibited activation of the OCT4 transgene. Transcriptome analysis revealed that a heightened mesenchymal transition may have contributed to the progression of tumors derived from grafted cells.

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  using the iPSC clone 201B7 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 . 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 epithelialmesenchymal transition (EMT), which is related to tumor invasion and progression. Moreover, canonical pathway analysis revealed upregulation of the Wnt/b-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.

Grafted 253G1-NS-Derived Neurons Form Synaptic Connections with Host Neurons
We performed triple immunostaining for human nuclear protein (HNu) and two neuronal markers, bIII tubulin and the presynaptic protein Bassoon (Bsn). Because the anti-Bsn antibody selectively recognized the mouse and rat epitopes, but not the human epitopes ( Figure S1), we were able to evaluate the ability of 253G1-NS-derived neurons to integrate with the host neural circuitry using this approach. Grafted bIII tubulin + /HNu + cells in parenchymal locations were contacted by synaptic boutons of host neurons (Figure 1K). Moreover, triple immunostaining for HNu, bIII tubulin, and human-specific synaptophysin (hSyn) revealed dense terminal fields of human-derived boutons apposed to host neurons ( Figure 1L). Host ChAT + neurons in the ventral gray matter were contacted by the hSyn + graft-specific terminals ( Figure 1M). Immuno-electron microscopy also revealed Venus + human pre-and post-synaptic structures, as well as synapse formation between host neurons and Venus + 253G1-NSderived neurons ( Figure 1N).

Grafted 253G1-NSs Promote Motor Function Recovery after SCI
We assessed motor function recovery using the Basso mouse scale (BMS) score, Rotarod test, and DigiGait system. According to the BMS score, the 253G1-NS-grafted group exhibited significantly better functional recovery than the PBS-injected control group R12 days after transplantation (BMS score = 3.2 ± 0.1 at 12 days post-transplantation and 3.3 ± 0.2 at 47 days post-transplantation; Nori et al., 2011; Figure 1O). In the Rotarod test, 253G1-NS-grafted mice remained on the rod significantly longer (61.1 ± 7.1 s) than the control group (33.0 ± 7.3 s; Nori et al., 2011) at 47 days post-transplantation ( Figure 1P). Gait performance was evaluated using the DigiGait image analysis system. All 253G1-NS-grafted mice could walk on the treadmill at 8 cm/s, whereas some control mice (4/16) could not. Stride length was significantly longer in the 253G1-NS-grafted group (4.2 ± 0.1 cm) than in the control group (2.2 ± 0.1 cm; Nori et al., 2011; Figure 1Q).

Tumors Form after 253G1-NS Transplantation, Resulting in Deteriorated Motor Function
We extended the follow-up period to 103 days post-transplantation to investigate the long-term safety of the grafted 253G1-NSs. Although recovery of motor function persisted for up to 47 days post-transplantation, 253G1-NS-grafted mice exhibited gradual deterioration of hind limb motor function thereafter (Figure 2A). To monitor the survival and growth of the grafted cells in the mouse spinal cord, we lentivirally transduced 253G1-NSs with ffLuc, which allowed us to identify grafted cells by their bioluminescent luciferase signals and fluorescent Venus signals. The photon count of grafted 253G1-NSs decreased within the first week post-transplantation, but gradually increased at 14 days post-transplantation and thereafter, demonstrating the survival and growth of the grafted cells. Between 42 and 70 days post-transplantation, the photon counts sharply increased ( Figures 2B and 2C), consistent with the deterioration of the BMS score shown in Figure 2A. At 103 days post-transplantation, the photon count of the grafted 253G1-NSs increased more than 10-fold from its initial value ( Figure 2C).
Histological analyses revealed tumors in 253G1-NSgrafted spinal cords. These tumors were divided into three groups based on the diameter of the lesion (small-tumor group, f < 200 mm; medium-tumor group, 200 < f < 700 mm; large-tumor group, 700 mm < f). Some of the tumors (12/22) exhibited microcystic masses consisting of HNu/Nestin double-positive human-derived bipolar cells with hair-like processes. These masses were observed in all tumors from the large-tumor group (7/7; Figures 2D and 2E show representative images of microcystic masses from the large-tumor group). Such masses were also present (I and J) TH + /HNu + neurons and ChAT + /HNu + neurons were observed, but were rare. (K) Sections were triple stained for HNu (green), bIII tubulin (red), and the presynaptic marker Bassoon (Bsn, white). The Bsn antibody recognized the mouse, but not the human, protein.
(L) Sections triple stained for HNu (green), bIII tubulin (red), and the human-specific presynaptic marker hSyn (white). bIII tubulin + / HNu À neurons represented host mouse neurons. The hSyn antibody recognized the human, but not the mouse, protein. in the majority of the medium tumors (5/7), whereas none were found in the small-tumor group (0/8). In medium tumors that exhibited microcystic masses, some grafted cells underwent normal neural differentiation ( Figures 2F and  2G). However, a portion of the grafted cells formed microcystic masses positive for Nestin ( Figures 2H and 2I), and Nestin + cells were also observed outside the microcystic mass. Significantly higher percentages of Nestin + cells (34.2% ± 7.3%) and APC + oligodendrocytes (5.4% ± 0.7%), as well as significantly lower percentages of bIII tubulin + neurons (32.1% ± 5.1%), were observed at 103 days relative to 47 days ( Figure 2J). In addition, 15.8% ± 2.7% of grafted cells had differentiated into astrocytes at 103 days. Taken together, these data indicate that approximately 87.5% of grafted cells differentiated toward neural lineages. We observed no NANOG + pluripotent cells in grafted 253G1-NSs at 103 days post-transplantation ( Figure S2).
Next, we examined the correlation between tumor diameter and the percentages of Nestin + , Ki-67 + , or OCT4 + cells among HNu + grafted cells ( Figure 3A). Statistical analysis revealed a significant correlation between tumor diameter and the percentage of Nestin + /HNu + cells ( Figure 3B). Compared with the small-tumor group, the large-and medium-tumor groups contained significantly higher percentages of Ki-67 + /HNu + cells and OCT4 + /HNu + cells ( Figures 3C and 3D). However, there was no significant correlation between tumor diameter and the percentage of OCT4 + /HNu + cells at 103 days post-transplantation (Figure 3E). Meanwhile, significantly more OCT4 + /HNu + cells were observed at 103 days than at 47 days ( Figure 3F); thus, the percentage of OCT4 + /HNu + cells correlated positively with post-transplant duration.

Transcriptomic Differences between 253G1-NSs and 201B7-NSs Post-Transplantation
Comparative transcriptome analyses of grafted cells and surrounding host cells can reveal information regarding the differentiation status of the grafted cells and the effects of the graft on the host tissue. mRNA sequencing (mRNAseq) enables one to analyze the global expression of individual human and mouse genes from a mixture of human and mouse cells (Bradford et al., 2013). Here, we sought to measure expression in mouse spinal cord tissue containing human cells derived from grafted human iPSC-NSs. To analyze mRNA expression in both grafted human iPSC-NSs and host spinal cord tissue, we analyzed the mRNA from NSs of 253G1 and 201B7 cells, as well as mouse spinal cord tissues containing grafted 253G1-NSs and 201B7-NSs, which were harvested at 5 and 103 days post-transplantation (PBS-5d and 103d, 253G1-NS/TP-5d and 103d, and 201B7-NS/TP-5d and 103d). The ratio of human and mouse mRNA-seq reads derived from epicenter segments (8 mm in length) of iPSC-NS-grafted spinal cord tissue was considered to reflect the ratio of human and mouse cells (Table S1). The global gene-expression patterns of these tissues were hierarchically clustered into 5-day (PBS-5d, 253G1-NS/TP-5d, and 201B7-NS/TP-5d) and 103-day groups (PBS-103d, 253G1-NS/TP-103d, and 201B7-NS/TP-103d), which may reflect time-dependent changes in the spinal cord microenvironment following SCI ( Figure 4A). Similarly, the gene-expression profiles of the grafted iPSC-NSs clustered on the basis of time post-transplantation (NS, 5 and 103 days) rather than clonal (253G1 and 201B7) origin ( Figure 4B). However, the profiles of the two clones diverged at 103 days post-transplantation. Furthermore, the gene-expression profiles of 253G1-NS/TP-103d and 201B7-NS/TP-103d differed significantly ( Figures 4C and 4D).

Ingenuity Pathway Analysis Revealed Pathways that Differed Significantly between 253G1-NS and 201B7-NS Transplantation
We used Ingenuity Pathway Analysis (IPA) to identify pathways differentially expressed between the 253G1-NS/TP-103d and 201B7-NS/TP-103d groups. IPA detected a total of 56 significantly altered (p < 0.05) pathways. Figure 5 shows the ten most altered pathways, all of which were significantly upregulated in the 253G1-NS/TP-103d group relative to iPSC-NSs prior to transplantation.

DISCUSSION
Because we generated the human iPSC clone 253G1 without introducing c-MYC, we initially speculated that  Table S1. 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 . Here, we found that 253G1-NS-grafted mice exhibited temporary motor func-tion 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 10fold 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 . 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.
Tumor diameter exhibited no significant correlation with the proportion of OCT4 + /HNu + cells. Instead, the proportion of grafted cells that were OCT4 + was solely dependent on the post-transplantation observation period. In our previous study, we observed no evidence of OCT4 + /HNu + cells or tumor formation in 201B7-NS-grafted mice after longterm observation . It is likely that the chronologically increasing OCT4 expression in the grafted 253G1-NSs is related to tumor formation, consistent with the results of RT-PCR, which showed that the expression of OCT4-Tg at 103 days post-transplantation was significantly higher than that in untransplanted 253G1-NSs. OCT4 is a key regulator of self-renewal and plays a critical role in maintaining ESC pluripotency (Niwa, 2007). In addition, OCT4 is sometimes re-expressed in somatic cells during carcinogenesis (Monk and Holding, 2001). OCT4 is also highly expressed in human gliomas and glioma cell lines, and OCT4 overexpression in glioma cells induces Nestin expression by inhibiting glioma cell differentiation (Du et al., 2009;Ikushima et al., 2011). These findings are consistent with the results we describe here, even though the tumors observed in the present study were not pathologically identical to gliomas. We also observed that KLF4-Tg was activated in 253G1-NSs, and that its expression increased after transplantation. KLF4 is highly expressed in primary breast ductal carcinoma and oral squamous carcinoma (Foster et al., 2005; Pandya et al., 2004). Previous reports also showed that KLF4 overexpression promotes self-renewal in ESCs , and KLF4 is an essential reprogramming factor that functions by binding at Oct4 loci to initiate Oct4 transcription and complete the reprogramming process (Wei et al., 2013). These results suggest that KLF4 plays an important role in maintaining PSCs, and that the tumor formation we observed could be related to KLF4-Tg activation. In a previous study, the molecular properties of engrafted NS/PCs changed dramatically depending on the spinal cord microenvironment (Kumamaru et al., 2012). Here, we analyzed the changes in the microenvironment of mouse spinal cord tissues affected by iPSC-NS transplantation. We observed no significant differences in the mouse gene-expression profiles of spinal cord tissues among the 253G1-NS/TP, 201B7-NS/TP, and control groups, suggesting that mouse gene expression did not widely affect tumorigenesis in grafted iPSC-NSs. However, human gene-expression profiles from the 253G1-and 201B7-NS/ TP groups differed in a time-dependent manner, which may have been related to tumor formation in the 253G1-NS/TP group. GO analysis revealed increased progression of EMT in the 253G1-NS and 253G1-NS/TP groups relative to the 201B7-NS and 201B7-NS/TP groups.
During EMT, epithelial cells lose their epithelial characteristics and acquire the properties of migratory mesenchymal cells. This process is associated with the early stages of carcinogenesis, cancer invasion, and recurrence (Hay, 1995;Thiery, 2002). Brain tumors, including some human gliomas, also contain stem-like cells with both neural and mesenchymal potential that are implicated in both tumor progression and invasiveness (Ricci-Vitiani et al., 2008). STAT3 is frequently overexpressed in cancers, including gliomas (Abou-Ghazal et al., 2008), and STAT3 phosphorylation leads to the transcriptional activation of genes involved in processes such as cell proliferation, apoptosis suppression, and angiogenesis (Bowman et al., 2001). Moreover, STAT3 regulates the EMT gene TWIST (Cheng et al., 2008), consistent with the more active EMT progression and IPA revealed upregulation of human ESC pluripotency pathways in post-transplantation 253G1-NSs. This phenomenon might be related to the population of differentiation-resistant Nestin + cells that proliferated over time and formed tumors. Furthermore, the Wnt/b-catenin signaling pathway was significantly upregulated in the 253G1-NS/ TP group. Previous studies showed that Wnt/b-catenin signaling is critical for regulating the self-renewal, proliferation, and differentiation of NS/PCs in the brain (Gong and Huang, 2012), and that this pathway leads to enhanced TERT expression in human cancers, resulting in the stabilization of telomeres, a hallmark of tumorigenesis (Hoffmeyer et al., 2012). In part, the tumorigenicity of 253G1-NSs could be related to upregulation of the Wnt/b-catenin signaling pathway. Moreover, consistent with GO analysis, regulation of the EMT pathway was significantly upregulated in the 253G1-NS/TP group relative to the 207B7-NS/ TP group.
Recently, several clinical trials of stem-cell-based therapy for SCI using either human NS/PCs (Cummings et al., 2005;Salazar et al., 2010) or human ESC-derived OPCs (Strauss, 2010) have been initiated. Compared with these stem cells, iPSCs raise fewer ethical concerns in certain countries Tsuji et al., 2010). On the other hand, the use of iPSC-derived cells risks tumorigenesis (Miura et al., 2009;Tsuji et al., 2010). The present study demonstrates that even unsafe iPSC-NSs can confer therapeutic benefits against SCI, at least in the short term. However, long-term observation is required to assess the safety of iPSC-NSs, because slow-growing tumors could cause motor function to deteriorate over longer periods of time. In the present study, we used retrovirally generated iPSCs Takahashi et al., 2007) and showed that activation of OCT4-and KLF4-Tg might be related to tumor formation. Thus, from a clinical-applications perspective, NS/PCs derived from integration-free iPSCs (Okita et al., , 2011 should be chosen to avoid Tg-induced tumorigenesis. Recently, a pilot clinical study of integration-free iPSC-based therapy for age-related macular degeneration was approved following review by the Japanese government (Garber, 2013;Kamao et al., 2014). As a step toward clinical applications in the SCI field, we have already initiated integration-free iPSC-NS transplantation in the NOD-SCID mouse SCI model. At the same time, transplantation into immune-deficient animals, accompanied by subsequent long-term observation, should be used to determine the safety and effectiveness of these cells (Okano et al., 2013).

EXPERIMENTAL PROCEDURES
Additional details regarding several of the protocols used in this work are provided in Supplemental Experimental Procedures.
Cell Culture, Neural Differentiation, and Lentiviral Transduction Cell culture, neural differentiation of human iPSCs (253G1), and lentiviral transduction of neurospheres were performed as described previously Okada et al., 2008). Briefly, 253G1-derived primary neurospheres were dissociated and infected with lentivirus expressing Venus or ffLuc under the control of the EF promoter. Primary neurospheres were passaged into secondary and tertiary neurospheres. The ffLuc vector enabled detection of grafted cells as strong bioluminescent ffLuc signals in live SCI mice, and as fluorescent Venus signals in fixed spinal cord sections.

Animal Model and Cell Transplantation
Adult female NOD-SCID mice (20-22 g) were anesthetized via intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After laminectomy, contusive SCI was induced at the Th10 level using an IH impactor (60 kdyn; Precision Systems and Instrumentation) as described previously (Scheff et al., 2003). Nine days after SCI, 253G1-NSs (5 3 10 5 cells) were transplanted into the lesion epicenter of each mouse (n = 37) using a glass micropipette and stereotaxic injector (KDS310; Muromachi Kikai). The 253G1-NSs were transplanted at approximately the same time as the PBS injection and 201B7-NS transplantation described in our previous report .

Bioluminescence Imaging
A Xenogen-IVIS spectrum-cooled, charge-coupled device optical macroscopic imaging system (Summit Pharmaceuticals International) was used for bioluminescence imaging to confirm the survival of the grafted cells as previously described (Itakura et al., 2014;Okada et al., 2005;Takahashi et al., 2011). Monitoring was performed for 103 days post-transplantation.

Motor Function and Histological Analyses
Motor function was evaluated using the BMS, Rotarod apparatus (Muromachi Kikai), and DigiGait system (Mouse Specifics). For histological analyses, the animals were anesthetized and transcardially perfused with 4% paraformaldehyde (pH 7.4). Spinal cords were removed and sectioned in the sagittal/axial plane on a cryostat. All motor function and histological analyses were conducted by observers blinded to the treatment conditions. All animal experiments (approval number 09169) were performed under the control of the Keio University Institutional Animal Care and Use Committee in accordance with the Institutional Guidelines on Animal Experimentation at Keio University, the Japanese Government Law Concerning the Protection and Control of Animals, and the Japanese Government Notification of Feeding and Safekeeping of Animals, and approved by the ethics committee of Keio University (IRB approval number 09091-8).

RT-PCR
RNA isolation and RT-PCR were performed as previously described Okada et al., 2008).

Transcriptome Analysis
Total RNA from each sample was purified as previously described (Okada et al., 2008). mRNA libraries were prepared according to the TruSeq RNA sample prep kit protocol and sequenced using a Genome Analyzer IIx (Illumina). Mouse and human mRNA sequences were separated using Xenome software (Conway et al., 2012), and separated mRNA-seq data were mapped to the corresponding genomic DNA sequences (human [hg19] and mouse [mm9]) using TopHat software (Trapnell et al., 2009). The mapped sequences were normalized by trimmed mean of M values (TMM) and analyzed using Avadis NGS software (Agilent Technologies). All software used default parameters. For principal component analysis (PCA) and clustering analysis, the normalized data were narrowed down to 13,693 genes using a cutoff value for expression levels (reads per kilobase of exon per million mapped reads [RPKM] > 100). A Venn diagram was constructed to visualize the 1,715 genes that were upregulated in the 253G1-NS/TP-103d group and the 1,358 genes that were upregulated in the 201B7-NS/TP-103d group (RPKM > 100, fold change > 5.0 versus each NS group). GO analysis was performed using gene lists from the overlapping area, as well as from each separate area, in the Venn diagram. For GO analysis, p values were calculated using Fisher's exact test. Subsequently corrected p values were applied for multiple testing corrections using the Benjamini-Yekutieli method with a cutoff at p = 0.05. Pathway analysis was performed via IPA (Ingenuity Systems) using genes that were up-or downregulated in the 253G1-NS/TP-103d group versus the 201B7-NS/TP-103d group, as well as in 253G1-NS versus 201B7-NS (RPKM > 100, fold-change > 3.0). The genes were overlaid on the Ingenuity Knowledge Database and networks were algorithmically generated based on their connectivity. The p values were determined using Fisher's exact test and p = 0.05 was interpreted as indicating statistical significance.

Statistical Analyses
An unpaired, two-tailed Student's t test was used to assess the 253G1-NS differentiation efficacy. One-way ANOVA followed by the Tukey-Kramer test for multiple comparisons was used for the Ki-67, Rotarod, and DigiGait analyses. Repeated-measures, two-way ANOVA followed by the Tukey-Kramer test was used for the BMS analysis. The Kruskal-Wallis non-parametric test and Scheffe's test were used to analyze tumor diameter and the percentage of HNu + grafted cells that were Nestin + , Ki-67 + , or OCT4 + . Statistical significance was determined as *p < 0.05, **p < 0.01.

Supplemental Information includes Supplemental Experimental
Procedures, two figures, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr. 2015.01.006.