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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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Grafted 253G1-NSs Mainly Differentiate into Neurons and Form Synapses with Host Spinal Cord Neurons(A and B) Venus+ 253G1-NSs integrated into the mouse spinal cord. Arrowheads indicate the lesion epicenter.(C–F) Representative images of Venus+ grafted cells immunostained for the markers NeuN (mature neurons) (C), βIII tubulin (all neurons) (D), GFAP (astrocytes) (E), and APC (oligodendrocytes) (F).(G) Percentages of cell-type-specific marker-positive cells among Venus+ grafted cells at 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 4 mice).(H) Most 253G1-derived neurons differentiated into GAD67+ (GABAergic) neurons.(I and J) TH+/HNu+ neurons and ChAT+/HNu+ neurons were observed, but were rare.(K) Sections were triple stained for HNu (green), βIII 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), βIII tubulin (red), and the human-specific presynaptic marker hSyn (white). βIII tubulin+/HNu− neurons represented host mouse neurons. The hSyn antibody recognized the human, but not the mouse, protein.(M) Large numbers of somatic and dendritic terminals from graft-derived nerve cells were present on host ChAT+ motor neurons at the ventral horns.(N) Electron microscopy (EM) images show synapse formation between host mouse neurons and graft-derived Venus+ (black) human neurons. Pre- and post-synaptic structures indicate transmission from a graft-derived neuron to a host neuron, and from a host neuron to a graft-derived neuron. H, host neuron; G, graft-derived neuron; arrowheads, post-synaptic density.(O) Motor function in the hind limbs was assessed weekly using the BMS score until 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 32 mice).(P) Rotarod test 47 days after transplantation. Graph shows total run time. Values are expressed as the means ± SEM (n = 10 mice).(Q) Treadmill gait analysis using the DigiGait system 47 days post-transplantation. Graph shows stride length. Values are expressed as the means ± SEM (n = 19 mice). Behavioral analyses were performed by two observers who were blinded to the treatment conditions.Scale bars, 1,000 μm in (A); 100 μm in (B); 50 μm in (J-1); 20 μm in (F-3), (F-4), (H-1), (J-2), (K-1), (L-1), and (M-1); 10 μm in (H-2), (J-3), (K-2), (L-2), and (M-2); 0.5 μm in (N). See also Figure S1.
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fig1: Grafted 253G1-NSs Mainly Differentiate into Neurons and Form Synapses with Host Spinal Cord Neurons(A and B) Venus+ 253G1-NSs integrated into the mouse spinal cord. Arrowheads indicate the lesion epicenter.(C–F) Representative images of Venus+ grafted cells immunostained for the markers NeuN (mature neurons) (C), βIII tubulin (all neurons) (D), GFAP (astrocytes) (E), and APC (oligodendrocytes) (F).(G) Percentages of cell-type-specific marker-positive cells among Venus+ grafted cells at 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 4 mice).(H) Most 253G1-derived neurons differentiated into GAD67+ (GABAergic) neurons.(I and J) TH+/HNu+ neurons and ChAT+/HNu+ neurons were observed, but were rare.(K) Sections were triple stained for HNu (green), βIII 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), βIII tubulin (red), and the human-specific presynaptic marker hSyn (white). βIII tubulin+/HNu− neurons represented host mouse neurons. The hSyn antibody recognized the human, but not the mouse, protein.(M) Large numbers of somatic and dendritic terminals from graft-derived nerve cells were present on host ChAT+ motor neurons at the ventral horns.(N) Electron microscopy (EM) images show synapse formation between host mouse neurons and graft-derived Venus+ (black) human neurons. Pre- and post-synaptic structures indicate transmission from a graft-derived neuron to a host neuron, and from a host neuron to a graft-derived neuron. H, host neuron; G, graft-derived neuron; arrowheads, post-synaptic density.(O) Motor function in the hind limbs was assessed weekly using the BMS score until 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 32 mice).(P) Rotarod test 47 days after transplantation. Graph shows total run time. Values are expressed as the means ± SEM (n = 10 mice).(Q) Treadmill gait analysis using the DigiGait system 47 days post-transplantation. Graph shows stride length. Values are expressed as the means ± SEM (n = 19 mice). Behavioral analyses were performed by two observers who were blinded to the treatment conditions.Scale bars, 1,000 μm in (A); 100 μm in (B); 50 μm in (J-1); 20 μm in (F-3), (F-4), (H-1), (J-2), (K-1), (L-1), and (M-1); 10 μm in (H-2), (J-3), (K-2), (L-2), and (M-2); 0.5 μm in (N). See also Figure S1.

Mentions: Immunodeficient (NOD-SCID) mice were used for xenograft experiments. After laminectomy, contusive SCI was induced at the Th10 level. Nine days after injury, 5 × 105 253G1-NS-derived cells, which were lentivirally transduced with the fluorescent protein Venus (an altered yellow fluorescent protein; Nagai et al., 2002) or ffLuc (Venus fused to firefly luciferase; Hara-Miyauchi et al., 2012), were injected into the lesion epicenter. Histological analyses were performed 47 days (d) after transplantation. The grafted 253G1-NSs survived, migrated into the host spinal cord (Figures 1A and 1B), and differentiated into neuronal nuclei (NeuN)+ (17.2% ± 2.6%) and β-tubulin isotype III (βIII tubulin)+ (42.2% ± 3.1%) neurons, glial fibrillary acidic protein (GFAP)+ astrocytes (15.0% ± 0.7%), and adenomatous polyposis coli CC-1 (APC)+ oligodendrocytes (2.7% ± 0.3%; Figures 1C–1G). Quantitative analysis revealed that 67% of NeuN+ mature neurons were GAD67+ GABAergic neurons (Figure 1H). Small numbers of grafted cells differentiated into tyrosine hydroxylase (TH)+ and choline acetyltransferase (ChAT)+ cholinergic neurons (Figures 1I and 1J).


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)

Grafted 253G1-NSs Mainly Differentiate into Neurons and Form Synapses with Host Spinal Cord Neurons(A and B) Venus+ 253G1-NSs integrated into the mouse spinal cord. Arrowheads indicate the lesion epicenter.(C–F) Representative images of Venus+ grafted cells immunostained for the markers NeuN (mature neurons) (C), βIII tubulin (all neurons) (D), GFAP (astrocytes) (E), and APC (oligodendrocytes) (F).(G) Percentages of cell-type-specific marker-positive cells among Venus+ grafted cells at 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 4 mice).(H) Most 253G1-derived neurons differentiated into GAD67+ (GABAergic) neurons.(I and J) TH+/HNu+ neurons and ChAT+/HNu+ neurons were observed, but were rare.(K) Sections were triple stained for HNu (green), βIII 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), βIII tubulin (red), and the human-specific presynaptic marker hSyn (white). βIII tubulin+/HNu− neurons represented host mouse neurons. The hSyn antibody recognized the human, but not the mouse, protein.(M) Large numbers of somatic and dendritic terminals from graft-derived nerve cells were present on host ChAT+ motor neurons at the ventral horns.(N) Electron microscopy (EM) images show synapse formation between host mouse neurons and graft-derived Venus+ (black) human neurons. Pre- and post-synaptic structures indicate transmission from a graft-derived neuron to a host neuron, and from a host neuron to a graft-derived neuron. H, host neuron; G, graft-derived neuron; arrowheads, post-synaptic density.(O) Motor function in the hind limbs was assessed weekly using the BMS score until 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 32 mice).(P) Rotarod test 47 days after transplantation. Graph shows total run time. Values are expressed as the means ± SEM (n = 10 mice).(Q) Treadmill gait analysis using the DigiGait system 47 days post-transplantation. Graph shows stride length. Values are expressed as the means ± SEM (n = 19 mice). Behavioral analyses were performed by two observers who were blinded to the treatment conditions.Scale bars, 1,000 μm in (A); 100 μm in (B); 50 μm in (J-1); 20 μm in (F-3), (F-4), (H-1), (J-2), (K-1), (L-1), and (M-1); 10 μm in (H-2), (J-3), (K-2), (L-2), and (M-2); 0.5 μm in (N). See also Figure S1.
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fig1: Grafted 253G1-NSs Mainly Differentiate into Neurons and Form Synapses with Host Spinal Cord Neurons(A and B) Venus+ 253G1-NSs integrated into the mouse spinal cord. Arrowheads indicate the lesion epicenter.(C–F) Representative images of Venus+ grafted cells immunostained for the markers NeuN (mature neurons) (C), βIII tubulin (all neurons) (D), GFAP (astrocytes) (E), and APC (oligodendrocytes) (F).(G) Percentages of cell-type-specific marker-positive cells among Venus+ grafted cells at 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 4 mice).(H) Most 253G1-derived neurons differentiated into GAD67+ (GABAergic) neurons.(I and J) TH+/HNu+ neurons and ChAT+/HNu+ neurons were observed, but were rare.(K) Sections were triple stained for HNu (green), βIII 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), βIII tubulin (red), and the human-specific presynaptic marker hSyn (white). βIII tubulin+/HNu− neurons represented host mouse neurons. The hSyn antibody recognized the human, but not the mouse, protein.(M) Large numbers of somatic and dendritic terminals from graft-derived nerve cells were present on host ChAT+ motor neurons at the ventral horns.(N) Electron microscopy (EM) images show synapse formation between host mouse neurons and graft-derived Venus+ (black) human neurons. Pre- and post-synaptic structures indicate transmission from a graft-derived neuron to a host neuron, and from a host neuron to a graft-derived neuron. H, host neuron; G, graft-derived neuron; arrowheads, post-synaptic density.(O) Motor function in the hind limbs was assessed weekly using the BMS score until 47 days post-transplantation. Values are expressed as the mean ± SEM (n = 32 mice).(P) Rotarod test 47 days after transplantation. Graph shows total run time. Values are expressed as the means ± SEM (n = 10 mice).(Q) Treadmill gait analysis using the DigiGait system 47 days post-transplantation. Graph shows stride length. Values are expressed as the means ± SEM (n = 19 mice). Behavioral analyses were performed by two observers who were blinded to the treatment conditions.Scale bars, 1,000 μm in (A); 100 μm in (B); 50 μm in (J-1); 20 μm in (F-3), (F-4), (H-1), (J-2), (K-1), (L-1), and (M-1); 10 μm in (H-2), (J-3), (K-2), (L-2), and (M-2); 0.5 μm in (N). See also Figure S1.
Mentions: Immunodeficient (NOD-SCID) mice were used for xenograft experiments. After laminectomy, contusive SCI was induced at the Th10 level. Nine days after injury, 5 × 105 253G1-NS-derived cells, which were lentivirally transduced with the fluorescent protein Venus (an altered yellow fluorescent protein; Nagai et al., 2002) or ffLuc (Venus fused to firefly luciferase; Hara-Miyauchi et al., 2012), were injected into the lesion epicenter. Histological analyses were performed 47 days (d) after transplantation. The grafted 253G1-NSs survived, migrated into the host spinal cord (Figures 1A and 1B), and differentiated into neuronal nuclei (NeuN)+ (17.2% ± 2.6%) and β-tubulin isotype III (βIII tubulin)+ (42.2% ± 3.1%) neurons, glial fibrillary acidic protein (GFAP)+ astrocytes (15.0% ± 0.7%), and adenomatous polyposis coli CC-1 (APC)+ oligodendrocytes (2.7% ± 0.3%; Figures 1C–1G). Quantitative analysis revealed that 67% of NeuN+ mature neurons were GAD67+ GABAergic neurons (Figure 1H). Small numbers of grafted cells differentiated into tyrosine hydroxylase (TH)+ and choline acetyltransferase (ChAT)+ cholinergic neurons (Figures 1I and 1J).

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