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 cMyc) 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 noncellautonomous (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.stem-cell-based medicine | cell transplantation | neurotrauma | synaptic connection S tem-cell-based approaches, such as the transplantation of neural stem/progenitor cells (NS/PCs), are promising sources of therapies for various central nervous system disorders (1-3). Previous studies reported functional recovery after transplantation of NS/PCs into the injured spinal cord of rodents and nonhuman primates (4-9). Furthermore, recent studies revealed that embryonic stem cells (ESCs) can generate neural cells including NS/PCs (10-12) and oligodendrocyte precursor cells (OPCs) (13,14). Therefore, human ESC-based therapies are moving out of the laboratory and into clinical treatments for spinal cord injury (SCI) (12,13,15). However, the use of human ESC-based therapies is complicated by ethical concerns in certain countries. To avoid the problems associated with ESCs, we previously established induced pluripotent stem cells (iPSCs) from mouse fibroblasts (16, 17) and confirmed the therapeutic potential of iPSC-derived neurospheres (iPSC-NSs) for treating SCI in animal models (18).Here, aiming at human iPSC-based therapies for SCI patients, we examined the therapeutic potential of human iPSC-NSs by transplanting them into nonobese diabetic severe combined immunodeficient (NOD-SCID) SCI model mice. We used a clone from human iPSCs (hiPSCs) that we established from adult human dermal fibroblasts by the retroviral transduction of four reprogramming factors; for the clone used in this study, 201B7, the factors were Oct3/4, Sox2, Klf4, and c-Myc (19). These grafted hiPSC-NSs survived, migrated, and differentiated in...

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. "Safe" iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT + serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPSderived neurospheres pre-evaluated as "unsafe" showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that preevaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.neural stem/progenitor cell | cell transplantation | regenerative medicine | remyelination | axonal regrowth

Ever since pioneering reports introduced mouse 1 and humaninduced 2-4 pluripotent stem cells (iPSCs) to the scientific community and the populace at large, there has been an increasing interest in applications for their use in the fields of biomedical research. These include cell therapy in regenerative medicine and modeling of human disease.

Murine and human iPSC-NS/PCs (induced pluripotent stem cell-derived neural stem/progenitor cells) promote functional recovery following transplantation into the injured spinal cord in rodents. However, for clinical applicability, it is critical to obtain proof of the concept regarding the efficacy of grafted human iPSC-NS/PCs (hiPSC-NS/PCs) for the repair of spinal cord injury (SCI) in a non-human primate model. This study used a pre-evaluated “safe” hiPSC-NS/PC clone and an adult common marmoset (Callithrix jacchus) model of contusive SCI. SCI was induced at the fifth cervical level (C5), followed by transplantation of hiPSC-NS/PCs at 9 days after injury. Behavioral analyses were performed from the time of the initial injury until 12 weeks after SCI. Grafted hiPSC-NS/PCs survived and differentiated into all three neural lineages. Furthermore, transplantation of hiPSC-NS/PCs enhanced axonal sparing/regrowth and angiogenesis, and prevented the demyelination after SCI compared with that in vehicle control animals. Notably, no tumor formation occurred for at least 12 weeks after transplantation. Quantitative RT-PCR showed that mRNA expression levels of human neurotrophic factors were significantly higher in cultured hiPSC-NS/PCs than in human dermal fibroblasts (hDFs). Finally, behavioral tests showed that hiPSC-NS/PCs promoted functional recovery after SCI in the common marmoset. Taken together, these results indicate that pre-evaluated safe hiPSC-NS/PCs are a potential source of cells for the treatment of SCI in the clinic.

BackgroundThe transplantation of neural stem/progenitor cells (NS/PCs) at the sub-acute phase of spinal cord injury, but not at the chronic phase, can promote functional recovery. However, the reasons for this difference and whether it involves the survival and/or fate of grafted cells under these two conditions remain unclear. To address this question, NS/PC transplantation was performed after contusive spinal cord injury in adult mice at the sub-acute and chronic phases.ResultsQuantitative analyses using bio-imaging, which can noninvasively detect surviving grafted cells in living animals, revealed no significant difference in the survival rate of grafted cells between the sub-acute and chronic transplantation groups. Additionally, immunohistology revealed no significant difference in the differentiation phenotypes of grafted cells between the two groups. Microarray analysis revealed no significant differences in the expression of genes encoding inflammatory cytokines or growth factors, which affect the survival and/or fate of grafted cells, in the injured spinal cord between the sub-acute and chronic phases. By contrast, the distribution of chronically grafted NS/PCs was restricted compared to NS/PCs grafted at the sub-acute phase because a more prominent glial scar located around the lesion epicenter enclosed the grafted cells. Furthermore, microarray and histological analysis revealed that the infiltration of macrophages, especially M2 macrophages, which have anti-inflammatory role, was significantly higher at the sub-acute phase than the chronic phase. Ultimately, NS/PCs that were transplanted in the sub-acute phase, but not the chronic phase, promoted functional recovery compared with the vehicle control group.ConclusionsThe extent of glial scar formation and the characteristics of inflammation is the most remarkable difference in the injured spinal cord microenvironment between the sub-acute and chronic phases. To achieve functional recovery by NS/PC transplantation in cases at the chronic phase, modification of the microenvironment of the injured spinal cord focusing on glial scar formation and inflammatory phenotype should be considered.