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. 2011 Oct 4;108(40):16825-30.
doi: 10.1073/pnas.1108077108. Epub 2011 Sep 26.

Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice

Satoshi Nori  1 , Yohei Okada, Akimasa Yasuda, Osahiko Tsuji, Yuichiro Takahashi, Yoshiomi Kobayashi, Kanehiro Fujiyoshi, Masato Koike, Yasuo Uchiyama, Eiji Ikeda, Yoshiaki Toyama, Shinya Yamanaka, Masaya Nakamura, Hideyuki Okano
Affiliations

Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice

Satoshi Nori et al. Proc Natl Acad Sci U S A. .

Abstract

Once their safety is confirmed, human-induced pluripotent stem cells (hiPSCs), which do not entail ethical concerns, may become a preferred cell source for regenerative medicine. Here, we investigated the therapeutic potential of transplanting hiPSC-derived neurospheres (hiPSC-NSs) into nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice to treat spinal cord injury (SCI). For this, we used a hiPSC clone (201B7), established by transducing four reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc) into adult human fibroblasts. Grafted hiPSC-NSs survived, migrated, and differentiated into the three major neural lineages (neurons, astrocytes, and oligodendrocytes) within the injured spinal cord. They showed both cell-autonomous and noncell-autonomous (trophic) effects, including synapse formation between hiPSC-NS-derived neurons and host mouse neurons, expression of neurotrophic factors, angiogenesis, axonal regrowth, and increased amounts of myelin in the injured area. These positive effects resulted in significantly better functional recovery compared with vehicle-treated control animals, and the recovery persisted through the end of the observation period, 112 d post-SCI. No tumor formation was observed in the hiPSC-NS-grafted mice. These findings suggest that hiPSCs give rise to neural stem/progenitor cells that support improved function post-SCI and are a promising cell source for its treatment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In vivo differentiation of hiPSC-NSs. (A and B) Venus+ hiPSC-NSs were integrated at or near the lesion epicenter (arrowheads). (Scale bars, 1000 μm in A; 100 μm in B.) (C–F) Representative images of Venus+-grafted cells labeled with the neural markers NeuN+ (mature neurons) (C); βIII tubulin+ (all neurons) (D); GFAP+ astrocytes (E); and APC+ oligodendrocytes (F). (Scale bar, 20 μm.) (G) Percentages of cell-type–specific marker-positive cells among the Venus+-grafted cells 56 d after SCI. Values are means ± SEM (n = 4). (H) Most hiPSC-derived neurons differentiated into GAD67+ (GABAergic) neurons. (Scale bars, 50 μm in H-1; 20 μm in H-2; and 10 μm in H-3.)
Fig. 2.
Fig. 2.
Evidence for synapse formation between hiPSC-derived neurons and host mouse spinal cord neurons. (A) Sections were triple-stained with HNu (green), βIII tubulin (red), and the presynaptic marker Bassoon (Bsn, white). The Bsn antibody used here recognized the rat and mouse, but not human, protein. (B) Sections triple-stained for HNu (green), βIII tubulin (red), and the human-specific presynaptic marker hSyn (white). (C) Confocal images showing a large number of somatic and dendritic terminals from graft-derived nerve cells on host motor neurons at the ventral horns. (D) Electron microscopy showing synapse formation between host mouse neurons and graft-derived Venus+ (black) human neurons: the pre- and postsynaptic structures indicated transmission from a host neuron to a graft-derived neuron (D-1) and from a graft-derived neuron to a graft-derived neuron (D-2). H, host neuron; G, graft-derived neuron; arrowheads, postsynaptic density. (Scale bars, 20 μm in A-1, A-2, B-1, B-2, C-1, and C-2; 10 μm in A-3, B-3, and C-3; and 0.5 μm in D.)
Fig. 3.
Fig. 3.
Transplanted hiPSC-NSs enhanced angiogenesis and prevented atrophic changes and demyelination after SCI. (A and B) Representative images of PECAM-1+ blood vessels. (C) Quantitative analysis of PECAM-1+ blood vessels at the lesion epicenter. Values are means ± SEM (n = 4). (D) Representative images of axial sections stained for VEGF. (E) Quantitative analysis of the VEGF+ area at the lesion epicenter. Values are means ± SEM (n = 4). (F and G) Expression of VEGF in GFAP+ astrocytes among Venus+-graft–derived human cells (yellow indicates VEGF+, GFAP+, and Venus+ cells) (F) and host mouse-derived cells (G) in the spinal cord. (H) Expression of human VEGF mRNA (VEGFA, -B, and -C are the human VEGF family members) 5 d after the hiPSC-NSs were transplanted (black bars) compared with cultured hiPSC-NSs before transplantation (gray bars). Values are means ± SEM (n = 3, each). Human VEGF expression was undetectable in the spinal cord of mice treated with PBS. (I) Expression of mouse Vegf mRNA (Vegfa, -b, and -c are the mouse Vegf family members) 5 d after hiPSC-NS transplantation (black bars) or PBS injection (gray bars) into the spinal cord. The mouse Vegf expression level was higher in the hiPSC-NS–grafted mice than in PBS-injected mice. Values are means ± SEM (n = 3, each). (J and K) Representative H&E-stained images of sagittal and axial sections at the lesion epicenter 56 d after SCI. (L) Quantitative analysis of the spinal cord area measured in H&E-stained axial sections at different regions. Values are means ± SEM (n = 6). (M and N) Representative LFB-stained images of the axial sections at the lesion epicenter 56 d after SCI. (O) Quantitative analysis of the myelinated area by LFB-stained axial sections at different regions. Values are means ± SEM (n = 6). *P < 0.05, **P < 0.01. (Scale bars, 500 μm in A, K, and M; 200 μm in B; 100 μm in D and N; 20 μm in F-1 and G; 10 μm in F-2; and 1,000 μm in J.)
Fig. 4.
Fig. 4.
Transplanted hiPSC-NSs enhanced axonal growth after SCI. (A) Representative images of sagittal sections stained for NF-H at the lesion epicenter. (B) Quantitative analysis of the NF-H+ area. Values are means ± SEM (n = 4). (C) Representative images of axial sections stained for 5HT at the lumbar intumescence. (D) Quantitative analysis of the 5HT+ area at the lumbar intumescence in axial sections. Values are means ± SEM (n = 6 each in the 7 d and control 56 d after SCI groups, and n = 5 in the hiPSC-NS (56 d after SCI) group). (E) Representative images of midsagittal sections stained for GAP43 in the ventral region 1 mm caudal to the lesion epicenter. (F) Quantitative analysis of the GAP43+ area in midsagittal sections. Values are means ± SEM (n = 4). (G and H) NF-H+ neural fibers and 5HT+ (serotonergic) fibers extended in association with GFAP+/Venus+-graft–derived human astrocytes. (I) Expression of human neurotrophic factor mRNAs (NGF, BDNF, and HGF) 5 d after hiPSC-NS transplantation (black bars) compared with cultured hiPSC-NSs before transplantation (gray bars). Values are means ± SEM (n = 3, each). (J) Expression of mouse neurotrophic factor mRNAs (Ngf, Bdnf, and Hgf) 5 d after hiPSC-NS transplantation (black bars) or PBS injection (gray bars) into the spinal cord. Values are means ± SEM (n = 3, each). *P < 0.05, **P < 0.01. (Scale bars, 100 μm in A; 50 μm in C, E, and H-1; 20 μm in G-1, H-2, and H-3; and 10 μm in G-2.)
Fig. 5.
Fig. 5.
Transplanted hiPSC-NSs promoted motor functional and electrophysiological recovery after SCI. (A) Motor function in the hindlimbs was assessed weekly by the BMS score for 56 d. Values are means ± SEM. (B) Rotarod test 56 d after SCI. Graph shows the total run time. Values are means ± SEM. (C) Treadmill gait analysis using the DigiGait system 56 d after SCI. Graph shows stride length. Values are means ± SEM. (D) Electrophysiological analysis performed 112 d after SCI. MEP waves were detected in most of the hiPSC-NS group (14 out of 17), whereas they were not detected in the control group (0 out of 15). **P < 0.01. Behavioral analyses were assessed by two observers who were blind to the treatment.
Fig. 6.
Fig. 6.
Long-term observation revealed no tumor formation after hiPSC-NS transplantation. (A) For 112 d, motor function in the hindlimbs was assessed weekly by the BMS score. Values are means ± SEM. (B) Representative H&E image of hiPSC-NS–grafted mice. (C) Boxed area in B. (C-1–C-3) Immunohistochemistry showing normal neural differentiation of the grafted cells. (D) Percentages of cell-type–specific marker-positive cells among the Venus+ human cells 56 and 112 d after SCI. Values are means ± SEM (n = 4 and 5, respectively). **P < 0.01. (Scale bars, 500 μm in B; and 50 μm in C.)
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