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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.

Nori S, Okada Y, Nishimura S, Sasaki T, Itakura G, Kobayashi Y, Renault-Mihara F, Shimizu A, Koya I, Yoshida R, Kudoh J, Koike M, Uchiyama Y, Ikeda E, Toyama Y, Nakamura M, Okano H - Stem Cell Reports (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan; Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan.

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Global Human Gene-Expression Analysis(A) Hierarchical clustering analysis of mouse gene-expression data from spinal cord tissues of the PBS-5d and -103d, 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups.(B) Hierarchical clustering analysis of human gene-expression data: 253G1-NSs and 201B7-NSs, as well as spinal cord tissues of the 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups. In (A) and (B), the signal intensity of each gene is displayed as a heatmap colored according to the expression level.(C) Principal-component analysis (PCA) of human gene-expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%); z axis, component 3 (13.88%).(D) Two-dimensional PCA of human gene expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%).(E) Venn diagram of human genes whose expression increased in the 253G1-NS/TP-103d and 201B7-NS/TP-103d groups relative to 253G1-NSs and 201B7-NSs. Color key: red, 692 genes highly expressed in the 253G1-NS/TP-103d group; blue, 335 genes highly expressed in the 201B7-NS/TP-103d group; purple, 1,023 genes highly expressed in both the 253G1- and 201B7-NS/TP-103d groups.(F–L) EMT-related human gene expression in 253G1- and 201B7-NSs and the 253G1- and 201B7-NS/TP-103d groups. Values represent the means ± SEM (n = 1 each in the human iPSC-NS, n = 2 for 201B7-NS/TP-103d, and n = 3 for 253G1-NS/TP-103d; n indicates the number of independent experiments).(M–P) The expression of SNAI1, SNAI2, TWIST1, and TWIST2 mRNA in 201B7-NSs and 253G1-NSs was analyzed by RT-PCR. Data are presented as expression levels relative to the control (the U87 human glioblastoma cell line). Values represent the means ± SEM (n = 3 independent experiments).(Q) Representative H&E image of the mid-sagittal section 103 days after transplantation.(R) p-STAT3-stained image of the adjacent section of (Q). Arrow, lesion epicenter; arrowhead, distribution of grafted 253G1-NSs.(S) Boxed area in (R).Scale bar, 1,000 μm in (Q) and (R), 100 μm in (S). ∗p < 0.05, ∗∗p < 0.01. See also the mRNA-seq read distribution in Table S1.
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fig4: Global Human Gene-Expression Analysis(A) Hierarchical clustering analysis of mouse gene-expression data from spinal cord tissues of the PBS-5d and -103d, 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups.(B) Hierarchical clustering analysis of human gene-expression data: 253G1-NSs and 201B7-NSs, as well as spinal cord tissues of the 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups. In (A) and (B), the signal intensity of each gene is displayed as a heatmap colored according to the expression level.(C) Principal-component analysis (PCA) of human gene-expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%); z axis, component 3 (13.88%).(D) Two-dimensional PCA of human gene expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%).(E) Venn diagram of human genes whose expression increased in the 253G1-NS/TP-103d and 201B7-NS/TP-103d groups relative to 253G1-NSs and 201B7-NSs. Color key: red, 692 genes highly expressed in the 253G1-NS/TP-103d group; blue, 335 genes highly expressed in the 201B7-NS/TP-103d group; purple, 1,023 genes highly expressed in both the 253G1- and 201B7-NS/TP-103d groups.(F–L) EMT-related human gene expression in 253G1- and 201B7-NSs and the 253G1- and 201B7-NS/TP-103d groups. Values represent the means ± SEM (n = 1 each in the human iPSC-NS, n = 2 for 201B7-NS/TP-103d, and n = 3 for 253G1-NS/TP-103d; n indicates the number of independent experiments).(M–P) The expression of SNAI1, SNAI2, TWIST1, and TWIST2 mRNA in 201B7-NSs and 253G1-NSs was analyzed by RT-PCR. Data are presented as expression levels relative to the control (the U87 human glioblastoma cell line). Values represent the means ± SEM (n = 3 independent experiments).(Q) Representative H&E image of the mid-sagittal section 103 days after transplantation.(R) p-STAT3-stained image of the adjacent section of (Q). Arrow, lesion epicenter; arrowhead, distribution of grafted 253G1-NSs.(S) Boxed area in (R).Scale bar, 1,000 μm in (Q) and (R), 100 μm in (S). ∗p < 0.05, ∗∗p < 0.01. See also the mRNA-seq read distribution in Table S1.

Mentions: 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 (mRNA-seq) 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).


Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition.

Nori S, Okada Y, Nishimura S, Sasaki T, Itakura G, Kobayashi Y, Renault-Mihara F, Shimizu A, Koya I, Yoshida R, Kudoh J, Koike M, Uchiyama Y, Ikeda E, Toyama Y, Nakamura M, Okano H - Stem Cell Reports (2015)

Global Human Gene-Expression Analysis(A) Hierarchical clustering analysis of mouse gene-expression data from spinal cord tissues of the PBS-5d and -103d, 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups.(B) Hierarchical clustering analysis of human gene-expression data: 253G1-NSs and 201B7-NSs, as well as spinal cord tissues of the 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups. In (A) and (B), the signal intensity of each gene is displayed as a heatmap colored according to the expression level.(C) Principal-component analysis (PCA) of human gene-expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%); z axis, component 3 (13.88%).(D) Two-dimensional PCA of human gene expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%).(E) Venn diagram of human genes whose expression increased in the 253G1-NS/TP-103d and 201B7-NS/TP-103d groups relative to 253G1-NSs and 201B7-NSs. Color key: red, 692 genes highly expressed in the 253G1-NS/TP-103d group; blue, 335 genes highly expressed in the 201B7-NS/TP-103d group; purple, 1,023 genes highly expressed in both the 253G1- and 201B7-NS/TP-103d groups.(F–L) EMT-related human gene expression in 253G1- and 201B7-NSs and the 253G1- and 201B7-NS/TP-103d groups. Values represent the means ± SEM (n = 1 each in the human iPSC-NS, n = 2 for 201B7-NS/TP-103d, and n = 3 for 253G1-NS/TP-103d; n indicates the number of independent experiments).(M–P) The expression of SNAI1, SNAI2, TWIST1, and TWIST2 mRNA in 201B7-NSs and 253G1-NSs was analyzed by RT-PCR. Data are presented as expression levels relative to the control (the U87 human glioblastoma cell line). Values represent the means ± SEM (n = 3 independent experiments).(Q) Representative H&E image of the mid-sagittal section 103 days after transplantation.(R) p-STAT3-stained image of the adjacent section of (Q). Arrow, lesion epicenter; arrowhead, distribution of grafted 253G1-NSs.(S) Boxed area in (R).Scale bar, 1,000 μm in (Q) and (R), 100 μm in (S). ∗p < 0.05, ∗∗p < 0.01. See also the mRNA-seq read distribution in Table S1.
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fig4: Global Human Gene-Expression Analysis(A) Hierarchical clustering analysis of mouse gene-expression data from spinal cord tissues of the PBS-5d and -103d, 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups.(B) Hierarchical clustering analysis of human gene-expression data: 253G1-NSs and 201B7-NSs, as well as spinal cord tissues of the 253G1-NS/TP-5d and -103d, and 201B7-NS/TP-5d and -103d groups. In (A) and (B), the signal intensity of each gene is displayed as a heatmap colored according to the expression level.(C) Principal-component analysis (PCA) of human gene-expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%); z axis, component 3 (13.88%).(D) Two-dimensional PCA of human gene expression data. x axis, component 1 (41.03%); y axis, component 2 (24.21%).(E) Venn diagram of human genes whose expression increased in the 253G1-NS/TP-103d and 201B7-NS/TP-103d groups relative to 253G1-NSs and 201B7-NSs. Color key: red, 692 genes highly expressed in the 253G1-NS/TP-103d group; blue, 335 genes highly expressed in the 201B7-NS/TP-103d group; purple, 1,023 genes highly expressed in both the 253G1- and 201B7-NS/TP-103d groups.(F–L) EMT-related human gene expression in 253G1- and 201B7-NSs and the 253G1- and 201B7-NS/TP-103d groups. Values represent the means ± SEM (n = 1 each in the human iPSC-NS, n = 2 for 201B7-NS/TP-103d, and n = 3 for 253G1-NS/TP-103d; n indicates the number of independent experiments).(M–P) The expression of SNAI1, SNAI2, TWIST1, and TWIST2 mRNA in 201B7-NSs and 253G1-NSs was analyzed by RT-PCR. Data are presented as expression levels relative to the control (the U87 human glioblastoma cell line). Values represent the means ± SEM (n = 3 independent experiments).(Q) Representative H&E image of the mid-sagittal section 103 days after transplantation.(R) p-STAT3-stained image of the adjacent section of (Q). Arrow, lesion epicenter; arrowhead, distribution of grafted 253G1-NSs.(S) Boxed area in (R).Scale bar, 1,000 μm in (Q) and (R), 100 μm in (S). ∗p < 0.05, ∗∗p < 0.01. See also the mRNA-seq read distribution in Table S1.
Mentions: 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 (mRNA-seq) 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).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan; Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan.

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Related in: MedlinePlus