Progenitor Cells Derived From Embryonic Stem Cells Promote Functional Recovery After Transplantation Into Injured Spinal Cord of Nonhuman Primates.

Published Ahead of Print on May 27, 2015 as 10.5966/sctm.2014-0215.

Pluripotent Stem Cells

PLURIPOTENT STEM CELLS Allogeneic Neural Stem/Progenitor Cells Derived From Embryonic Stem Cells Promote Functional Recovery After Transplantation Into Injured Spinal Cord of Nonhuman Primates

Key Words. Embryonic stem cells x Neural stem cells x Spinal cord injury x Cell transplantation x Allograft x Primates Departments of aOrthopaedic Surgery and bPhysiology, Keio University School of Medicine, Tokyo, Japan; c Central Institute for Experimental Animals, Kawasaki, Japan; dGenomic Science Laboratories, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan; e Department of Immunology, Juntendo University, Tokyo, Japan Correspondence: Hideyuki Okano, M.D., Ph.D., Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Telephone: 813-5363-3747; E-Mail: hidokano@ a2.keio.jp; or Masaya Nakamura, M.D., Ph.D., Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Telephone: 813-5363-3812; E-Mail: masa@a8. keio.jp Received September 26, 2014; accepted for publication March 25, 2015.

ABSTRACT Previous studies have demonstrated that neural stem/progenitor cells (NS/PCs) promote functional recovery in rodent animal models of spinal cord injury (SCI). Because distinct differences exist in the neuroanatomy and immunological responses between rodents and primates, it is critical to determine the effectiveness and safety of allografted embryonic stem cell (ESC)-derived NS/PCs (ESC-NS/PCs) in a nonhuman primate SCI model. In the present study, common marmoset ESC-NS/PCs were grafted into the lesion epicenter 14 days after contusive SCI in adult marmosets (transplantation group). In the control group, phosphate-buffered saline was injected instead of cells. In the presence of a low-dose of tacrolimus, several grafted cells survived without tumorigenicity and differentiated into neurons, astrocytes, or oligodendrocytes. Significant differences were found in the transverse areas of luxol fast blue-positive myelin sheaths, neurofilament-positive axons, corticospinal tract fibers, and platelet endothelial cell adhesion molecule-1-positive vessels at the lesion epicenter between the transplantation and control groups. Immunoelectron microscopic examination demonstrated that the grafted ESC-NS/PC-derived oligodendrocytes contributed to the remyelination of demyelinated axons. In addition, some grafted neurons formed synaptic connections with host cells, and some transplanted neurons were myelinated by host cells. Eventually, motor functional recovery significantly improved in the transplantation group compared with the control group. In addition, a mixedlymphocyte reaction assay indicated that ESC-NS/PCs modulated the allogeneic immune rejection. Taken together, our results indicate that allogeneic transplantation of ESC-NS/PCs from a nonhuman primate promoted functional recovery after SCI without tumorigenicity. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1–12

SIGNIFICANCE This study demonstrates that allogeneic embryonic stem cell (ESC)-derived neural stem/progenitor cells (NS/PCs) promoted functional recovery after transplantation into the injured spinal cord in nonhuman primates. ESC-NS/PCs were chosen because ESC-NS/PCs are one of the controls for induced pluripotent stem cell-derived NS/PCs and because ESC derivatives are possible candidates for clinical use. This translational research using an allograft model of a nonhuman primate is critical for clinical application of grafting NS/PCs derived from various allogeneic pluripotent stem cells, especially induced pluripotent stem cells, into injured spinal cord at the subacute phase.

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INTRODUCTION Neural stem/progenitor cells (NS/PCs) show promise in the treatment of several central nervous system diseases [1–3]. Previous studies have demonstrated that NS/PC transplantation promotes functional recovery in spinal cord injury (SCI) animal models, prompting several investigators to proceed to clinical applications [4–10]. Some clinical trials

of NS/PC transplantation for SCI patients have recently begun, although some trials have been discontinued. Almost all trials of stem/progenitor cell transplantation for SCI, including the trial by Geron using human embryonic stem cell (ESC)derived oligodendrocyte progenitor cell (OPC)like cells [11] were initiated by doing basic research using rodent models. However, distinct differences exist in the neuroanatomy and immunological

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HIROKI IWAI,a,b HIROKO SHIMADA,b SORAYA NISHIMURA,a,b YOSHIOMI KOBAYASHI,a GO ITAKURA,a,b KEIKO HORI,a,b KEIGO HIKISHIMA,b,c HAYAO EBISE,d NAOKO NEGISHI,e SHINSUKE SHIBATA,b SONOKO HABU,e YOSHIAKI TOYAMA,a MASAYA NAKAMURA,a HIDEYUKI OKANOb


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MATERIALS AND METHODS Animals Adult female common marmosets aged 18 months and older were purchased from Clea Japan, Inc. (Tokyo, Japan). The animals were maintained at 26°C with 65% humidity and 12 hours of light daily. All the animals had free access to food and water. All experiments were performed in accordance with the Laboratory Animal Welfare Act, Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD), Guidelines and Policies for Animal Surgery (Animal Study Committee of the Central Institute for Experimental Animals and Keio University), and guidelines outlined by the Weatherall report [19]. The Animal Study Committee of the Central Institute for Experimental Animals approved the present study (approval nos. 12018 and 13014), according to which, the animals were fed after being purchased from Clea Japan, Inc. until sacrifice after conducting the motor functional assessments, SCI, and transplantation experiments.

Culture and Differentiation of ESC-NS/PCs Marmoset ESCs (line no. 20), established previously [20], were used. The cell culture and neural induction of marmoset ESCs were performed, as described previously [18], to induce oligodendrocyte differentiation. In this protocol, retinoic acid and purmorphamine were added to the culture media during embryoid body formation, and purmorphamine, platelet-derived growth factor (PDGF)-AA and other molecules were added to the culture medium during neurosphere formation. The neurospheres were plated onto poly-L-ornithine/laminin (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com)-coated cover glasses and allowed to differentiate into oligodendrocytes for 30–35 days [18]. A lentivirus was prepared and transduced into neurospheres


to detect grafted cells in the host spinal cord, as described previously [8]. In brief, ESC-derived secondary neurospheres were dissociated and infected with a lentivirus expressing a venus fluorescent protein [21] modified from green fluorescent protein (GFP) under the control of the cytomegalovirus promoter after passage [22]. This vector allowed grafted cells expressing venus to be detected in spinal cord sections using an anti-GFP antibody [21]. The secondary neurospheres were then passaged into tertiary neurospheres and used for transplantation.

Microarray Analysis Microarray analyses of ESCs (n = 1) and secondary neurospheres (n = 1) were performed, as described previously [18]. Total RNA was isolated from these cells using an RNeasy Plus Micro kit (Qiagen, Valencia, CA, http://www.qiagen.com). RNA was reverse transcribed, biotin-labeled, and hybridized for 16 hours to Marmo2, a marmoset genome oligonucleotide custom array [23], which was subsequently washed and stained in a Fluidics Station 450 (Affymetrix Japan, Tokyo, Japan, http://www.affymetrix. com) according to the manufacturer’s instructions. The microarrays were scanned using a GeneChip Scanner 3000 7G (Affymetrix Japan), and the MAS5 algorithm (Affymetrix Japan) was used to normalize the raw image files.

Contusive SCI and Transplantation in Common Marmosets Adult female common marmosets (300–350 g) were anesthetized with an intramuscular injection of ketamine and xylazine, followed by inhalation of isoflurane. Eight marmosets received a moderate contusive SCI using a modified NYU impactor (New York University, New York, NY), as previously reported [8, 12, 13, 24–26]. After laminectomy, a 17-g weight was dropped from a height of 50 mm onto the dura mater at the C5 level. At 14 days after injury, partially dissociated marmoset ESC-NS/PCs (approximately 1 3 106 cells per 5 ml of growth factor-free culture medium) were transplanted into the lesion epicenter [27] using a glass pipette fitted to a 25-ml Hamilton syringe and a stereotaxic microinjector (KDS 310; Muromachi Kikai Co., Ltd., Tokyo, Japan, http://www.muromachi.com) (n = 4) [8, 12, 24]. An equal volume of phosphate-buffered saline (PBS, vehicle) was injected into the lesion in the control marmosets (n = 4). Manual bladder expression was performed until the urinary reflexes were re-established. Manual manipulation of the joints in all four extremities was also conducted twice a day. The paralyzed animals were supplied with sufficient amounts of food and water until they recovered their ability to ingest food and water without assistance. All animals received a daily dose of ampicillin (100 mg/kg) for 1 week after cell engraftment and a subcutaneous injection of tacrolimus (0.05 mg/kg; Astellas Pharma, Inc., Tokyo, Japan, http://www. astellas.com) [28] until they were sacrificed. At 10 weeks after transplantation, magnetic resonance imaging (MRI) was performed with the marmosets under general anesthesia, as described previously [18]. At 12 weeks after transplantation, the animals were injected with sodium pentobarbital (100 mg/kg) and euthanized for histological analysis.

Behavioral Analyses All behavioral examinations were performed until 14 weeks after SCI. The original open field rating scale was used to evaluate open field locomotion, as described previously [26]. All marmosets S TEM C ELLS T RANSLATIONAL M EDICINE


responses between rodents and primates [12, 13]. Therefore, we believe it is critical to determine the effectiveness and safety of allografted ESC-derived NS/PCs (ESC-NS/PCs) in a nonhuman primate SCI model before initiating clinical trials. The optimal therapeutic time-window for NS/PC transplantation is the subacute phase of SCI, when the acute inflammatory response has subsided and cystic cavities and glial scars have not yet had time to form [14–16]. Recently, we examined the gene expression profile of the injured spinal cord of common marmosets using microarray and nextgeneration sequencing analyses and found that 2–4 weeks after SCI is the optimal therapeutic time-window for NS/PC transplantation in a nonhuman primate SCI model [17]. On the basis of this finding, we decided to graft marmoset ESCNS/PCs into the allogeneic injured spinal cord at 14 days after injury in the present study. Human fetal NS/PCs and human induced pluripotent stem cell (iPSC)-NS/PCs were transplanted into a marmoset SCI model at 9 days after injury in our previous studies [8, 12]. Furthermore, we had previously developed a protocol to isolate OPC-enriched marmoset ESC-NS/PCs, which are capable of differentiating into neurons, astrocytes, and oligodendrocytes, both in vitro and in vivo [18]. The purpose of the present preclinical study was to determine the safety and effectiveness of allogeneic transplantation of ESC-NS/PCs for SCI in adult common marmosets.

ESC-NS/PCs Promote Recovery in Primate SCI Models


Iwai, Shimada, Nishimura et al.

were individually tested for 5 minutes on the floor and then in the cage to obtain the overall score (maximum score, 30 points). The test was performed 3 times each week until 4 weeks after SCI and twice a week thereafter. The motor function of the upper extremities was also evaluated using a bar grip strength test, which assessed the gripping reflex. The test was performed three times a day, and the maximal daily grip strength was expressed as a percentage of the preinjury grip strength [13].

For a preclinical trial, only a noninvasive device can be used to evaluate the efficiency of ESC-NS/PCs in remediating spinal cord injury. Therefore, MRI, a noninvasive method that can detect hemorrhage, edema, and cavity formation after SCI, was performed at 10 weeks after transplantation [29, 30]. Using a 7.0Tesla superconducting magnet (Bruker Biospin GmbH, Ettlingen, Germany, http://www.bruker.com) fitted with a 62-mm inner diameter volume coil, myelin mapping was performed with the marmoset under general anesthesia, as previously described [8] (n = 3 for each group).

Histological Analyses At 12 weeks after transplantation, the animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA). The spinal cords were removed, postfixed overnight in 4% PFA, soaked overnight in 10% sucrose and then 30% sucrose, embedded in optimal cutting temperature (OCT) compound, frozen, and sectioned in the sagittal plane at a thickness of 16 mm using a model CM3050 cryostat (Leica Microsystems, Wetzlar, Germany, http://www.leica-microsytems.com). The spinal cord sections were stained with H&E and luxol fast blue (LFB) for general histological examination. For immunohistochemistry, the sections were stained with the following primary antibodies: anti-GFP (rabbit IgG, 1:500; Frontier Institute Co., Ltd., Hokkaido, Japan, http://www.frontier-instute.com; goat IgG, 1:500; Abcam, Cambridge, U.K., http://www.abcam.com), anti-Hu (human IgG, 1:1000, a gift from Dr. Robert Darnell, Rockefeller University, New York, NY), anti-glial fibrillary acidic protein (GFAP) (rat IgG, 1:500; Invitrogen, Carlsbad, CA http://www.invitrogen.com), anti-adenomatous polyposis coli antigen (APC) (mouse IgG, 1:500; Calbiochem, San Diego, CA, http://www.emdbiosciences. com), anti-PDGF receptor (PDGFR) (rabbit IgG, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, http://www.scbt.com), antimyelin basic protein (MBP) (rat IgG, 1:200; AbD Serotec, Raleigh, NC, http://www.ab-direct.com), anti-neurofilament 200 kDa (NF-H) (mouse IgG, 1:200; EMD Millipore, Billerica, MA, http://www. emdmillipore.com), anti-calcium/calmodulin-dependent protein kinase IIa (CaMKIIa) (mouse IgG, 1:100; Invitrogen), and antiplatelet endothelial cell adhesion molecule-1 (PECAM-1) (mouse IgG, 1:50; Dako, Carpinteria, CA, http://www.dako.com). For the differentiation efficiency of the grafted cells, the samples were incubated with host-specific secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen) at 1:1,000 for 2 hours at room temperature after incubation with primary antibodies at 4°C overnight. Nuclei were stained with Hoechst 33258 (10 mg/ml; Sigma-Aldrich). For CaMKIIa staining, only Alexa Fluor 555 (Invitrogen) at a dilution of 1:1,000 was used as a secondary antibody. For immunohistochemistry with anti-NF-H and anti-PECAM-1 antibodies, the sections were exposed to

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0.3% hydrogen peroxide to inactivate endogenous peroxidases and then treated with a biotinylated secondary antibody (Jackson ImmunoResearch, Inc., West Grove, PA, http://www. jacksonimmuno.com). The signals were enhanced using the VECTASTAIN ABC kit (Vector Laboratories, Inc., Burlingame, CA, http://www.vectorlabs.com). All images were obtained using a model BZ 9000 fluorescence microscope (Keyence Co., Osaka, Japan, http://www.keyence.com) or a model LSM 7000 confocal laser scanning microscope (Carl Zeiss, GmbH, Munich, Germany, http://www.zeiss.com). ImageJ software (NIH) was used to quantify the cells of interest in H&E-, LFB-, NF-H-, CaMKIIa-, and PECAM-1-stained sections.

Immunoelectron Microscope Analysis Venus-positive transplanted cells were fixed with 4% PFA in PBS overnight, followed by incubation with 10% and 30% sucrose. The OCT compound-embedded frozen blocks were sectioned at a thickness of 16 mm and incubated with 5% BLOCK ACE (AbD Serotac) with 0.1% saponin in 0.1 M phosphate buffer [31] for 1 hour. The sections were stained with primary anti-GFP antibody (rabbit IgG, 1:100; Medical and Biological Laboratories Co., Ltd., Nagoya, Japan, http://www.mbl.co.jp) for 72 hours and Alexa Fluor 488 and nanogold conjugated anti-rabbit secondary antibody (1:100, Invitrogen) for 24 hours at 4°C. After fixation with 2.5% glutaraldehyde, nanogold signals were enhanced with the HQ-Silver kit (Nanoprobes, Inc., Yaphank, NY, http://www.nanoprobes.com) for 10 minutes. The sections were fixed with 0.5% osmium tetroxide, dehydrated through a graded series of ethanol baths, and embedded in Epon. Ultrathin sections (70 nm) were prepared with a model UC7 ultramicrotome (Leica Microsystems, Tokyo, Japan, http://www. leica-microsytems.com), and stained with uranyl acetate and lead citrate. The sections were observed under a model 1400 plus transmission electron microscope (JEOL, Tokyo, Japan, http://www.jeol.co.jp).

Mixed Lymphocyte Reaction The mixed lymphocyte reaction (MLR) assay was performed to evaluate the effect of ESC-NS/PCs on the allogeneic lymphocyte reaction. Marmoset host lymphocytes were collected from spleens immediately after sacrifice using Lymphoprep (AxisShield PoC AS, Oslo, Norway, http://www.axis-shield.com), according to the manufacturer’s instructions. Tertiary neurospheres from marmoset ESC-NS/PCs at 10 days after the last passage, the adequate period for cell engraftment, were passaged and diluted in Roswell Park Memorial Institute medium (SigmaAldrich) containing 10% fetal bovine serum. The cells received 20 Gy using a CellRad x-ray irradiation system (Faxitron, Tucson, AR, http://www.faxitron.com), and 1 3 105 ESC-NS/PCs were cocultured with an equivalent number of lymphocytes from each recipient in 200 ml of medium in ultra-low-cluster 96-well plates (Corning Inc., Rochester, NY, http://www.corning.com). Lymphocytes from the marmoset host were used as stimulators. These cultures were incubated for 5 days at 37°C in a humidified atmosphere of 5% carbon dioxide. As a positive control, the lymphocytes were cocultured with irradiated allogeneic marmoset lymphocytes from another recipient spleen. During the last 8 hours, 1 mCi [methyl-3H]-thymidine was added to the culture medium. Subsequently, the cells were harvested with a FilterMate cell harvester (PerkinElmer, Boston, MA, http://www.perkinelmer. com) and counted in a TopCount NXT radioactivity counter (PerkinElmer) after the addition of 50 ml of scintillation fluid.



Magnetic Resonance Imaging

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Cell proliferation was calculated as the mean count per minute (CPM) of triplicate samples. The CPM ratio was defined as the CPM in lymphocytes cocultured with ESC-NS/PCs or allogeneic or autologous lymphocytes divided by the CPM in lymphocytes only.

Statistical Analysis

RESULTS Differentiation and Microarray Assays of ESC-NS/PCs In Vitro To detect selectively grafted cells and distinguish them from the host cells, NS/PCs derived from marmoset ESCs were labeled by transfecting them with a lentivirus expressing a fluorescent protein, venus (Fig. 1A). The in vitro differentiation assays revealed that ESC-NS/PCs differentiated into bIII-tubulin-positive neurons (21.8% 6 2.3%), GFAP-positive astrocytes (28.6% 6 3.9%), and 29,39-cyclic nucleotide 39-phosphodiesterase (CNPase)positive oligodendrocytes (25.3% 6 3.6%). Nestin-positive undifferentiated NS/PCs were also detected (17.7% 6 2.7%) (Fig. 1B, 1C). Microarray analysis revealed that the gene expression of various trophic factors, including vascular endothelial growth factor (VEGF)-A, insulin-like growth factor-1 (IGF-1), fibroblast growth factor-1 (FGF-1), and PDGF-C, was significantly higher in the secondary neurospheres of ESC-NS/PCs than in the original ESCs before neural differentiation using the cutoff values of the expression levels (.10) and fold changes (.5) (Fig. 1D).

Differentiation of Transplanted ESC-NS/PCs Into Three Neural Lineages in the Injured Spinal Cord Anti-GFP immunohistochemistry, which detected venus-positive cells [21], was performed to investigate the survival of grafted cells. Immunohistological analysis showed that allogeneic grafted ESC-NS/PCs survived well in the injured spinal cord, indicating that immunorejection was prevented, even in the presence of a low dose of tacrolimus. Grafted cells migrated extensively into both the gray and white matter at the lesion epicenter (Fig. 2A). Quantitative analyses revealed that the population of differentiated cells derived from ESC-NS/PCs also differentiated into Hu-positive neurons (39.0% 6 7.3%), GFAP-positive astrocytes (24.3% 6 4.2%), APC-positive oligodendrocytes (32.1% 6 5.1%), and PDGFR-positive OPCs (5.8% 6 0.4%), which were negative for APC staining (Fig. 2B, 2C). Nestin-positive cells were not clearly detected. In addition, MBP-positive cells derived from grafted ESCNS/PCs were detected (Fig. 2D). Low magnification images of the spinal cord showed that Hu- and APC-positive grafted cells were mostly located in the white matter, an area distant from that of the lesion. In contrast, GFAP-positive graft cells were located close to the lesion (Fig. 2E).


ESC-NS/PC-Derived Oligodendrocytes Contributed to Remyelination After SCI To demonstrate the capacity of grafted cells to prevent demyelination or to promote remyelination after SCI, LFB staining was performed at 14 weeks after injury (Fig. 3A). The LFB-positive areas were measured at sites that were 4 mm rostral and 4 mm caudal to the lesion. The LFB-positive areas within the lesion epicenter were significantly larger in the NS/PC group than in the control group (Fig. 3B). Furthermore, noninvasive MRI of myelin (myelin map) [8] was conducted at 10 weeks after transplantation to assess myelination by noninvasive imaging (Fig. 3C). The myelin map showed that the myelin-positive areas were significantly larger at the lesion epicenter and at the site 1.5 mm rostral to the lesion epicenter in the grafted group than in the control group (Fig. 3D), similar to the histological findings of LFB-positive myelinated areas after SCI. Although some discrepancy between the myelin map and LFB-based histological findings was observed, this could be explained by the technical differences, including problems with voxel sizes and resolutions. Moreover, immunoelectron microscopic examination was performed to determine whether the grafted ESC-NS/PCs alone remyelinated the demyelinated axons. A number of myelin sheathes with different numbers of lamellae were located adjacent to the GFP-positive grafted cells that covered the spared axons (Fig. 3E). A higher magnification image obtained by transmission electron microscopy (TEM) showed not only immature myelin sheathes but also mature myelin structures, which had more than 10 lamellae originating from the grafted cells (Fig. 3F). Consistent with the finding that grafted ESC-NS/PCs differentiated into MBP-positive cells, the grafted ESC-NS/PCs contributed to the remyelination of the demyelinated axons after SCI.

ESC-NS/PC Transplantation Prevented Atrophy of the Injured Spinal Cord and Promoted Angiogenesis H&E staining was performed to investigate the effect of transplanted NS/PCs on the atrophy and cystic cavities of the injured spinal cord (Fig. 4A). The H&E-positive areas were quantified, not only at the lesion epicenter, but also at sites that were 2 and 4 mm rostral and caudal to the lesion epicenter, respectively. In the transplantation group, the H&E-positive areas in the region of the lesion epicenter, but not at the rostral and caudal sites, were significantly larger than those in the control group (Fig. 4B). In addition, the cystic cavities in the transplantation group were significantly smaller than those in the control group (Fig. 4C). NS/PC transplantation prevented spinal atrophy and cavity formation after contusive injury. Furthermore, the ESC-NS/PCs did not result in any microscopic tumors at 12 weeks after transplantation (Fig. 4A). To evaluate the effect of ESC-NS/PC transplantation on angiogenesis after SCI, immunohistochemical analyses for PECAM-1 were performed on day 84 after transplantation (Fig. 4D). Quantitative analysis revealed that the areas of PECAM-1-positive vessels were also significantly larger at the lesion epicenter in the grafted group than in the control group (Fig. 4E).

Grafted ESC-NS/PCs Enhanced Axonal Regrowth and Formed Neuronal Interactions With Host Cells We also performed immunohistochemical analyses for NF-H and CaMKIIa on day 84 after transplantation to assess the effect of the grafted ESC-NS/PCs on spared neuronal fibers in the injured spinal cord (Fig. 5A, 5B). Quantitative analysis showed that not S TEM C ELLS T RANSLATIONAL M EDICINE


All data are reported as the mean 6 SEM. For all histological examinations, an unpaired two-tailed Student’s t test was used for single comparisons between transplantation and vehicle control groups and for comparisons of the results of the MLR assay. The results of the open field test and bar grip test were analyzed using the Mann-Whitney U test. In each case, p , .05 was considered statistically significant.




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ESC-NS/PCs Inhibited the Allogeneic Lymphocyte Reaction The MLR assay demonstrated that marmoset lymphocytes significantly responded to stimulator allogeneic lymphocytes (allo-MLR) but not to stimulator autologous lymphocytes (auto-MLR) (Fig. 7A, 7B). In contrast, the CPM ratio of the responder lymphocytes from marmosets 1–3 was slightly, but significantly, reduced when ESC-NS/PCs were used as stimulators but not when autologous lymphocytes alone were used. In marmoset 4, the uptake of the tracer in the cocultured group decreased moderately, but not significantly, compared with that in the other animals (Fig. 7B). These findings indicate that the marmoset recipient lymphocytes did not respond to allogeneic ESC-NS/PCs.

DISCUSSION Figure 1. Marmoset embryonic stem cell-derived neural stem/progenitor cells (ESC-NS/PCs) differentiated in vitro into three neural lineages and expressed various growth factors. (A): Phase contrast (left) and fluorescence (right) images of venus-positive neurospheres derived from marmoset ESCs. (B): After passage 3, neurospheres differentiated into bIII-tubulin-positive neurons, GFAP-positive astrocytes, and CNPase-positive oligodendrocytes. (C): Quantitative analysis of the differentiated ESC-NS/PC phenotypes (n = 3). (D): Microarray analysis of secondary neurospheres from ESC-NS/PCs and ESCs. The expression levels of VEGF-A, IGF-1, PDGF-C, FGF-1, and FGF-9 were higher in the secondary neurospheres than those in the ESCs, limiting the expression level (.10) and fold change (.5) compared with ESCs (n = 1). Scale bars = 100 mm (A) and 20 mm (B). Abbreviations: CNPase, 29,39-cyclic nucleotide 39-phosphodiesterase; FGF, fibroblast growth factor; GFAP, glial fibrillary acid protein; IGF-1, insulin-like growth factor-1; PDGF-C, platelet-derived growth factor-C; VEGF-A, vascular endothelial growth factor-A.

only the NF-H-positive areas but also the CaMKIIa-positive areas, a marker of the corticospinal tract (CST), at the lesion epicenter were significantly larger in the transplantation group than in the control group (Fig. 5C, 5D). Immunoelectron microscopy revealed that the transplanted cells formed synaptic connections with host axons (Fig. 5E). In addition, GFP-positive neuronal fibers were detected around the transplanted region in the injured spinal cord. They were myelinated by host myelin sheathes that had less than 10 (Fig. 5F) and more than 10 (Fig. 5G) lamellae.

ESC-NS/PC Transplantation Promoted Functional Recovery After SCI Motor function was evaluated using the original open field rating scale score for general motor function and the bar grip test for grip

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Previous studies have demonstrated that NS/PC transplantation for SCI affects histological and/or functional outcomes in several processes, including neurotrophic support [32, 33], angiogenesis [34, 35], axonal regrowth [24, 36], neural circuit reconstruction [37–39], and remyelination [40, 41]. Most of these previous studies involved the use of rodent SCI models. The advantage of using common marmosets instead of rodents is the similarity between their neuroanatomy and immunological responses and those of humans. For example, in nonhuman primates and humans, most CST fibers cross to the contralateral side through pyramidal decussation and descend the lateral funicles, and CST fibers descend the dorsal funicles in rodent spinal cord [42–45]. Immunological responses to allografts in primates also differ from those in rodents. Regulatory T cells (Treg) play an important role in allogeneic immunity, and the Treg functions and features in humans are similar to those in nonhuman primates but quite different from those in rodents [46]. Moreover, CD40/CD40 ligand interactions [47] and major histocompatibility (MHC) complexity [48, 49] differ between primates, including common marmosets and rodents. Many reports have referred to the benefit of using nonhuman primate models in translational research of spinal cord injury or allogeneic transplantation [42, 46, 50, 51]. Thus, it is critical to determine whether allogeneic transplantation of NS/PCs can promote functional recovery in nonhuman primates as a part of the translational research required for clinical trials. In the present study, we demonstrated that ESC-NS/PCs grafted into the injured spinal cord of marmosets resulted in an improvement in motor functional recovery at an early, as well a late, phase after transplantation compared with the motor functional recovery in the control animals. Neurotrophic support might underlie the mechanism that provokes functional recovery



strength [28]. The score in the original open field rating scale decreased to near zero shortly after contusive SCI. The ESC-NS/PC transplantation group showed rapid functional recovery in the open field rating scale score compared with the control group. The difference was statistically significant at 4 weeks after transplantation. In contrast, the control animals showed a gradual increase in the open field rating scale score after SCI, with a plateau at approximately 8 points (Fig. 6A). The bar grip test also yielded similar results to the open field rating scale scores. Contusive SCI sharply decreased the grip strength in the bar grip test to approximately 0%, which then gradually recovered. A significant difference was found in the bar grip strength between the transplantation and control groups at 9 weeks after transplantation (Fig. 6B).


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at the early phase after NS/PC transplantation [32], because grafted ESC-NS/PCs enhanced tissue sparing, angiogenesis, and axonal regrowth, including that of CST fibers after SCI. This concept is consistent with our previous xenograft studies using human fetal NS/PCs [12] and human iPSC-NS/PCs [8] in a common marmoset SCI model. Moreover, immunoelectron microscopy demonstrated the rafted cells could integrate the synaptic relay with host neuronal cells. The host-graft interactions of neuronal regeneration accelerated recovery [33, 38].


IGF-1 promotes axonal outgrowth and survival of corticospinal motor neurons [52], and FGF-1 and FGF-9 exert beneficial effects on SCI and axon regeneration [53, 54]. In addition, PDGF induces the remyelination of NS/PCs into oligodendrocytes [55]. Finally, VEGF facilitates angiogenesis at the lesion site by inhibiting cell death [56]. The mRNA levels of these five growth factors were higher in ESC-NS/PCs at the transplantation stage than in the original ESCs before neural differentiation. Thus, a relatively higher expression level of these S TEM C ELLS T RANSLATIONAL M EDICINE


Figure 2. Grafted marmoset embryonic stem cell-derived neural stem/progenitor cells (ESC-NS/PCs) survived and differentiated into trineural lineages after spinal cord injury. (A): Immunohistochemical analyses revealed that a large number of GFP-positive grafted cells survived. The cells that survived migrated at least 2 mm rostral and caudal to the lesion epicenter. (B): The grafted marmoset ESC-NS/PCs differentiated into Hupositive neurons, GFAP-positive astrocytes, APC-positive oligodendrocytes, and PDGFR-positive oligodendrocytes progenitor cells. (C): Quantitative analysis of the grafted cell phenotypes at 12 weeks after transplantation in the marmoset injured spinal cord (n = 3). (D): MBP-positive grafted cells were observed. (E): In low-magnification images of spinal cord in the lesion epicenter, GFAP-positive grafted cells were detected in the gray matter, which is located close to the lesion, and Hu- and APC-positive cells were observed at a relatively distant location from the lesion. Scale bars = 1,000 mm (A, E), 20 mm (B), and 10 mm (D). Abbreviations: APC, adenomatous polyposis coli antigen; GFAP, glial fibrillary acid protein; GFP, green fluorescent protein; Hu, human; MBP, myelin basic protein; PDGFR, platelet-derived growth factor receptor.



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Figure 3. Transplanted marmoset ESC-NS/PCs enhanced myelinated areas in part because they remyelinated demyelinated axons. (A): LFB staining was performed 12 weeks after transplantation. (B): In the grafted group, LFB-positive regions were significantly prominent (p, p , .05) at the lesion epicenter compared with the control group (n = 3). (C): Myelin map imaging was performed at 10 weeks after transplantation. (D): In the grafted group, the myelin-positive areas were significantly larger (p, p , .05) at the lesion epicenter than those in the lesion epicenter of the control group (n = 3). (E): Immunoelectron microscopic analyses revealed lamellae in the myelin sheath at different thicknesses located next to green fluorescent protein-positive cells. (F): A high magnification image showing a 16-layer myelin sheath by transmission electron microscopy. Scale bars = 1,000 mm (A, C), 5 mm (E), and 200 nm (F). Abbreviations: ESC-NS/PCs, embryonic stem cell-derived neural stem/progenitor cells; LFB, luxol fast blue.

neurotrophic factors of the ESC-NS/PCs might have contributed to the functional recovery observed in the early phase after transplantation by a trophic mechanism similar to that reported previously [32].

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In contrast, remyelination is one of the potential mechanisms for functional recovery, especially at the late phase after NS/PC transplantation [40, 41, 57, 58]. However, it is difficult to obtain oligodendrocytes derived from human NS/PCs [12] and human



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iPSC-NS/PCs [33] in vitro. We could not obtain any oligodendrocytes from fetal NS/PCs of the common marmoset in vitro [59]. Therefore, we established a culture method to enrich OPCs from ESC-NS/PCs, which were then used in the present study [18]. These ESC-NS/PCs differentiated more efficiently into CNPasepositive oligodendrocytes (25.3% 6 3.6%) in vitro than did fetal


Figure 5. Grafted marmoset ESC-NS/PCs improved axonal regrowth, formed synaptic connections with host cells, and differentiated into neurons myelinated by host cells. (A): Anti-NF-H of axial sections of the spinal cord on day 84 after transplantation. Positively stained areas were quantified at the lesion epicenter. A high magnification image shows the lateral column (a, c) and ventral column (b, d). (C): In the grafted group, NF-H-positive regions were significantly larger (p, p , .05) at the lesion epicenter than at the lesion epicenter of the control group. (B): CaMKIIa immunohistochemistry of axial sections of the spinal cord at the 84 days after transplantation. Positively stained areas were quantified at the center of the lesion. A high magnification image shows corticospinal tract fibers were more prominent in the grafted group than in the control groups. (D) In the grafted group, the areas of CaMKIIa-positive areas were also significantly larger (p, p , .05) in the NS/PC group than in the control group (n = 3). (E): Immunoelectron microscopy demonstrated synaptic connectivity between host neurons (H) and grafted green fluorescent protein (GFP)-positive neurons (G), indicating transmission from a transplanted neuron to a host neuron. (F): GFP-positive graft neurons myelinated by host myelin sheathes that had more than five lamellae. (G): Mature myelination of transplanted neurons shown in host spinal cord with myelin having more than 10 lamellae. Scale bars = 1,000 mm (A, B), 200 nm (E), and 500 nm (F, G). Abbreviations: CaMKIIa, calcium/calmodulin-dependent protein kinase IIa; ESC-NS/ PCs, embryonic stem cell-derived neural stem/progenitor cells; NF-H, neurofilament 200 kDa.

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Figure 4. Engrafted marmoset ESC-NS/PCs prevented atrophy without inducing tumor formation and enhanced angiogenesis. (A): Representative images of H&E-stained axial sections at the lesion epicenter in control and ESC-NS/PCs groups. In the grafted group, no obvious tumor formation was present at 12 weeks after transplantation. (B): H&E-positive areas were significantly larger (p, p , .05) at the lesion epicenter in the transplantation group than in the lesion epicenter of the control group. (C): In addition, cystic cavities were significantly wider in the control group than in the grafted group. (D): Anti-PECAM-1 immunohistochemistry of axial sections of the spinal cord at 84 days after transplantation. We quantified the positively stained areas in the lesion epicenter. A high magnification image shows neovascular vessels around a cystic cavity in both groups. (E): In the grafted group, the area occupied by PECAM-1-positive vessels was also significantly larger (p, p , .05) in the NS/PC group than in the control group (n = 3). Scale bars = 1,000 mm (A, D). Abbreviations: ESC-NS/PCs, embryonic stem cell-derived neural stem/progenitor cells; PECAM-1, platelet endothelial cell adhesion molecule-1.




NS/PCs [59]. Likewise, quantitative immunohistological analyses demonstrated a significantly higher survival rate for APC-positive oligodendrocytes derived from ESC-NS/PCs (32.1% 6 5.1%) than for APC-positive oligodendrocytes derived from marmoset fetal NS/PCs (fNS/PCs) (7.3% 6 1.7%) (in preparation) at 84 days after transplantation into the marmoset injured spinal cord. Thus, a higher yield of oligodendrocytes was obtained in vivo by the transplantation of OPC-enriched nonhuman primate ESC-NS/PCs [18]. No significant difference was found in the proportion of GFAPpositive astrocytes between the ESC-NS/PCs (24.3% 6 4.2%) and fNS/PCs (21.7% 6 4.5%). In contrast, the percentage of Hupositive neurons was lower among ESC-NS/PCs (39.0% 6 7.3%) than among fNS/PCs (58.1% 6 3.5%), although the difference was not significant (in preparation). In addition, several MBPpositive mature oligodendrocytes derived from grafted ESC-NS/ PCs were detected by immunohistochemical analyses. Furthermore, TEM also demonstrated different numbers of lamellae in the grafted cells, which wrapped the host axons in the injured spinal cord, indicating that remyelination by exogenous ESC-NS/PCs had

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Figure 7. Embryonic stem cell-derived neural stem/progenitor cells (ESC-NS/PCs) inhibited the allogeneic lymphocyte reaction in vitro. (A): MLR assay results show that no significant difference was found in the uptake of 3H-thymidine between responder isolated-cultured lymphocytes and responders cocultured with autologous irradiated lymphocytes, which exhibit auto-MLR. The CPM ratio, defined as the CPM in the responder lymphocytes mixed with stimulator divided by the CPM in the responders only, was significantly lower (p, p , .05) in responder lymphocytes than in responder lymphocytes stimulated with allo-MLR (n = 3). (B): A significant difference shown in the CPM ratio between responders alone of marmosets 1–3 and those of responders cocultured with stimulator marmoset ESC-NS/PCs. In marmoset 4, the incorporation of the tracer in the cocultured group decreased moderately, but not significantly, compared with that in the other marmosets. These results indicate that marmoset ESCNS/PCs do not trigger an allogeneic reaction by host marmoset lymphocytes or suppress the allogeneic lymphocyte reaction in vitro (n = 3). Abbreviations: allo-MLR, allogeneic lymphocytes in allogeneic MLR; auto-MLR, autologous MLR; CPM, count per minute; MLR, mixed lymphocyte reaction.

occurred. The present study is the first to report remyelination by allotransplanted ESC-NS/PCs in a nonhuman primate with SCI, as predicted by Shimada et al. [18]. ESC-NS/PCs differentiated into oligodendrocytes and contributed to remyelination after SCI. In addition, LFB staining and myelin map analysis with MRI demonstrated that the myelin-positive areas were significantly larger at the lesion epicenter in the grafted group than those at the lesion epicenter in the control group. Taken together, the transplantation of ESC-NS/PCs, which enhances remyelination while suppressing endogenous demyelination [60, 61], resulted in larger myelinated areas in the spinal cord than in the control spinal cord.



Figure 6. Engraftment of ESC-NS/PCs promoted motor functional recovery. (A): The original open field rating scale score (maximum score, 30 points) decreased to near zero immediately after spinal cord injury. The score gradually increased in the transplantation group until 98 days after injury, without reaching a plateau. In contrast, the behavior score in the control group also gradually increased after transplantation, reaching a plateau at 8 points. The score differed significantly (p, p , .05) between the transplantation and control groups at 4 weeks after transplantation (n = 4). (B): The grip strength in the bar grip test decreased to approximately 0%. At 9 weeks after transplantation, the strength was significantly greater (p, p , .05) in the transplantation group than in the control group (n = 4). Abbreviation: ESC-NS/PCs, embryonic stem cell-derived neural stem/progenitor cells.

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allogeneic immune responses [65]. Also, allogeneic iPSC-NS/PCs are more efficient in inducing the migration of CD45-positive cells near the site of the grafted cells than autologous cells, and the allograft showed better survival than the autograft. In contrast to the report by Morizane et al. [65], we used an immunosuppressive agent, namely, tacrolimus, at 0.05 mg/kg/day with subcutaneous injection. This dose is lower than the maintenance dose used for human organ transplantation [66–68]. Our results revealed that numerous grafted ESC-NS/PCs survived in the presence of a lowdose of tacrolimus after SCI. As a next step, additional validation using allograft and autograft models in the absence of an immunosuppressant will be required to determine whether immunosuppression is required for allogeneic iPSC-NS/PCs transplantation after SCI [65].

CONCLUSION In the present study, we demonstrated that allogeneic transplantation of ESC-NS/PCs from a nonhuman primate improved functional recovery after SCI without tumorigenicity. In particular, the grafted ESC-NS/PCs, which efficiently differentiated into oligodendrocytes after transplantation, contributed to the remyelination of the demyelinated host axons. In addition, a large number of allogeneic grafted ESC-NS/PCs survived in the presence of a low dose of tacrolimus, in part because the ESC-NS/PCs of nonhuman primates can suppress immune rejection. Our findings support the use of allogeneic multipotent stem cell derived-NS/PCs for the treatment of SCI, not only in nonhuman primates but also in patients.

ACKNOWLEDGMENTS We appreciate the help of Drs. A. Iwanami, K. Fujiyoshi, O. Tsuji, K. Kitamura, Y. Takahashi, M. Shinozaki, A. Yasuda, S. Nori, T. Konomi, M. Takano, R. Zhang, S. Tashiro, S. Kawabata, Y. Nishiyama, M. Ozaki, T. Iida, and K. Matsubayashi, members of the spinal cord research team in the Department of Orthopaedic Surgery, Rehabilitation Medicine and Physiology, Keio University School of Medicine. We also thank N. Hashimoto, T. Ishibuchi, T. Inoue, N. Okahara, S. Miyao, and M. Mizutani for their assistance with the experiments and animal care. This work was supported by grants from the Japan Science and Technology-California Institute for Regenerative Medicine Collaborative Program; Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (SPS) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); a Grant-in-Aid for Research Center Network for Realization of Regenerative Medicine from the JST to H.O.; Keio Gijuku Academic Development Funds; the Funding Program for World-leading Innovative R&D on Science and Technology; and a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the MEXT.

AUTHOR CONTRIBUTIONS H.I.: conception/design, data analysis and interpretation, manuscript writing, final approval of manuscript, performed experiments; H.S.: conception/design, manuscript writing, performed experiments; S.N. and Y.K.: conception/design, performed experiments; G.I. and K. Hori: performed experiments; K. Hikishima and H.E.: data analysis and interpretation, performed experiments; N.N.: S TEM C ELLS T RANSLATIONAL M EDICINE


The original open field rating scale score demonstrated that motor function in the ESC-NS/PC group recovered immediately after transplantation. Moreover, the recovery did not reach a plateau but continued even 70 days after transplantation. Remyelination does not occur easily at the early phase after transplantation. For example, Yasuda et al. reported that remyelination only starts 6 weeks after transplantation [41]. It probably takes longer for marmoset ESC-NS/PCs to differentiate into mature oligodendrocytes than for mouse NS/PCs. It might also take longer for marmoset ESC-NS/PCs to form synapse connections with the host and grafted neurons before recovery can occur. Nori et al. reported synaptic connections from a host neuron to a graft-derived neuron and from a graft-derived neuron to a graft-derived neuron [33]. However, functional recovery in the late phase was not detected, although they observed recovery at 104 days after transplantation [33]. Therefore, remyelination might be one of the mechanisms responsible for functional recovery in the late-onset phase after transplantation. In a previous study, we transplanted human NS/PCs into injured spinal cords at 9 days after injury in common marmosets [8, 12, 24]. In the present study, ESC-NS/PCs were engrafted at 14 days after injury, and the transplantation group showed significant motor functional recovery compared with the control group. Although a different immunosuppressant to that used in a previous study was used in the present study, it is significant that the ESC-NS/PCs group showed the same degree of motor functional recovery to that of the transplantation groups in the previous study [8, 12]. Considering the time course of the changes in the microenvironment of injured spinal cords of the common marmosets demonstrated in our previous study, observed by performing microarray and next-generation sequencing [17], we expect engraftment at 28 days after injury to also be efficient. It would be tremendously beneficial for SCI patients if the optimal therapeutic time-window could be prolonged for NS/PC transplantation. Additional studies to determine the optimal time for cell transplantation are necessary before clinical trials can be initiated. In the present study, we performed allogeneic transplantation of ESC-NS/PCs for SCI in a nonhuman primate, which is important for assessing the immunological responses of the allogeneic graft. To our knowledge, although the whole genome sequence has been recently published [62], the details of MHC complex genes of the common marmoset have not been reported, which prevented us from performing precise genotyping of the MHC genes in this model. Instead, an MLR assay was performed to validate the allogeneic immune responses. Responder lymphocytes in the MLR assay with ESC-NS/PCs showed a significantly lower response than allo-MLR and responder lymphocytes alone. These findings suggest that marmoset ESC-NS/PCs do not induce allogeneic reactions by recipient lymphocytes; rather, they inhibit the allogeneic response in vitro similar to human NS/PCs derived from the first trimester forebrain [63, 64]. This indicates that ESC-NS/PCs might also suppress allogeneic immune rejection in vivo and that ESC-NS/PCs can survive without any immunosuppressive agent after transplantation. Morizane et al. transplanted allogeneic and autologous iPSC-derived neural cells into the putamen in cynomolgus monkeys without immunosuppression and reported that allografts with mismatched MHC molecules survived 6 months after transplantation, although the cells elicited




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data analysis and interpretation, performed experiments; S.S.: manuscript writing, data analysis and interpretation, performed experiments; S.H.: manuscript writing, final approval of manuscript; Y.T.: final approval of manuscript; M.N. and H.O.: conception/ design, manuscript writing, final approval of manuscript.

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H.O. is a compensated scientific consultant of SanBio, Inc. and Daiichi Sankyo Co., Ltd. H.E. is employed by Dainippon Sumitomo Pharma Co., Ltd. The other authors indicated no potential conflicts of interest.

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Progenitor Cells Promote Functional Recovery through Synapse Reorganization with Spared Host Neurons after Spinal Cord Injury.

Schwann cells generated from neonatal skin-derived precursors or neonatal peripheral nerve improve functional recovery after acute transplantation into the partially injured cervical spinal cord of the rat.

Migration of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells toward An Injured Spinal Cord.

Neuroprotective effects of human spinal cord-derived neural precursor cells after transplantation to the injured spinal cord.

Transplantation of Embryonic Spinal Cord Derived Cells Helps to Prevent Muscle Atrophy after Peripheral Nerve Injury.

Polarized Macrophages Have Distinct Roles in the Differentiation and Migration of Embryonic Spinal-cord-derived Neural Stem Cells After Grafting to Injured Sites of Spinal Cord.

Transplanted Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Do Not Promote Functional Recovery of Pharmacologically Immunosuppressed Mice With Contusion Spinal Cord Injury.

Engrafted peripheral blood-derived mesenchymal stem cells promote locomotive recovery in adult rats after spinal cord injury.

Expression of neurotrophic factors in injured spinal cord after transplantation of human-umbilical cord blood stem cells in rats.

Plasticity and Recovery After Dorsal Column Spinal Cord Injury in Nonhuman Primates.

Differentiation of Donor-Derived Cells Into Microglia After Umbilical Cord Blood Stem Cell Transplantation.

Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats.

Transplantation of choroid plexus epithelial cells into contusion-injured spinal cord of rats.

Sequential Differentiation of Embryonic Stem Cells into Neural Epithelial-Like Stem Cells and Oligodendrocyte Progenitor Cells.

Derivation of cardiac progenitor cells from embryonic stem cells.

Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons.

Neurogenic differentiation from adipose-derived stem cells and application for autologous transplantation in spinal cord injury.

Oct4-induced oligodendrocyte progenitor cells enhance functional recovery in spinal cord injury model.

CXCR4 axis promotes recovery after spinal cord injury by mediating bone marrow-derived from mesenchymal stem cells.

progenitor cells to promote tissue repair and forelimb functional recovery in cervical spinal cord injury.

Transplantation of Induced Pluripotent Stem Cell-Derived Neural Stem Cells Mediate Functional Recovery Following Thoracic Spinal Cord Injury Through Remyelination of Axons.

Extracellular matrix-regulated neural differentiation of human multipotent marrow progenitor cells enhances functional recovery after spinal cord injury.