Neural stem cells (NSCs) from embryonic or fetal/adult tissue sources have shown considerable promise in regenerative strategies for traumatic spinal cord injury (SCI). However, there are limitations with their use related to the availability, immunogenicity, and uncertainty of the mechanisms involved. To address these issues, definitive NSCs derived from induced pluripotent stem (iPS) cells generated using a nonviral, piggyBac transposon approach, were investigated. Committed NSCs were generated from iPS cells using a free-floating neurosphere methodology previously described by our laboratory. To delineate the mechanism of action, specifically the role of exogenous myelination, NSCs derived from wildtype (wt) and nonmyelinating Shiverer (shi) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation. Behavioral, histological, and electrophysiological outcomes were analyzed to assess the effectiveness of this treatment. The wt-and shi-iPS-NSCs were validated and shown to be equivalent except in myelination capacity. Both iPS-NSC lines successfully integrated into the injured spinal cord and predominantly differentiated to oligodendrocytes, but only the wt-iPS-NSC treatment resulted in a functional benefit. The wt-iPS-dNSCs, which exhibited the capacity for remyelination, significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk), histological outcomes, and electrophysiological measures of axonal function (sucrose gap analysis) compared with the nonmyelinating iPS-dNSCs and cell-free controls. In summary, we demonstrated that iPS cells can generate translationally relevant NSCs for applications in SCI. Although NSCs have a diverse range of functions in the injured spinal cord, remyelination is the predominant mechanism of recovery following thoracic SCI. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:743-754 SIGNIFICANCEGain-of-function/loss-of-function techniques were used to examine the mechanistic importance of graft-derived remyelination following thoracic spinal cord injury (SCI). The novel findings of this study include the first use of neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) derived using the clonal neurosphere expansion conditions, for the treatment of SCI, the first characterization and in vivo application of iPSCs from Shiverer mouse fibroblasts, and the first evidence of the importance of remyelination by pluripotent-sourced NSCs for SCI repair and regeneration.

Traumatic spinal cord injuries (SCIs) affect millions of people worldwide; the majority of whom are in the chronic phase of their injury. Unfortunately, most current treatments target the acute/subacute injury phase as the microenvironment of chronically injured cord consists of a well-established glial scar with inhibitory chondroitin sulfate proteoglycans (CSPGs) which acts as a potent barrier to regeneration. It has been shown that CSPGs can be degraded in vivo by intrathecal Chondroitinase ABC (ChABC) to produce a more permissive environment for regeneration by endogenous cells or transplanted neural stem cells (NSCs) in the subacute phase of injury. Using a translationally-relevant clip-contusion model of cervical spinal cord injury in mice we sought to determine if ChABC pretreatment could modify the harsh chronic microenvironment to enhance subsequent regeneration by induced pluripotent stem cell-derived NSCs (iPS-NSC). Seven weeks after injury—during the chronic phase—we delivered ChABC by intrathecal osmotic pump for one week followed by intraparenchymal iPS-NSC transplant rostral and caudal to the injury epicenter. ChABC administration reduced chronic-injury scar and resulted in significantly improved iPSC-NSC survival with clear differentiation into all three neuroglial lineages. Neurons derived from transplanted cells also formed functional synapses with host circuits on patch clamp analysis. Furthermore, the combined treatment led to recovery in key functional muscle groups including forelimb grip strength and measures of forelimb/hindlimb locomotion assessed by Catwalk. This represents important proof-of-concept data that the chronically injured spinal cord can be ‘unlocked’ by ChABC pretreatment to produce a microenvironment conducive to regenerative iPS-NSC therapy.

The pathology of spinal cord injury (SCI) makes it appropriate for cell-based therapies. Treatments using neural stem cells (NSCs) in animal models of SCI have shown positive outcomes, although uncertainty remains regarding the optimal cell source. Pluripotent cell sources such as embryonic stem cells (ESCs) provide a limitless supply of therapeutic cells. NSCs derived using embryoid bodies (EB) from ESCs have shown tumorigenic potential. Clonal neurosphere generation is an alternative method to generate safer and more clinically relevant NSCs without the use of an EB stage for use in cell-based therapies. We generated clonally derived definitive NSCs (dNSCs) from ESC. These cells were transplanted into a mouse thoracic SCI model. Embryonic stem cell-derived definitive neural stem cell (ES-dNSC)-transplanted mice were compared with controls using behavioral measures and histopathological analysis of tissue. In addition, the role of remyelination in injury recovery was investigated using transmission electron microscopy. The SCI group that received ES-dNSC transplantation showed significant improvements in locomotor function compared with controls in open field and gait analysis. The cell treatment group had a significant enhancement of spared neural tissue. Immunohistological assessments showed that dNSCs differentiated primarily to oligodendrocytes. These cells were shown to express myelin basic protein, associate with axons, and support nodal architecture as well as display proper compact, multilayer myelination in electron microscopic analysis. This study provides strong evidence that dNSCs clonally derived from pluripotent cells using the default pathway of neuralization improve motor function after SCI and enhance sparing of neural tissue, while remaining safe and clinically relevant.

Cell-based therapies using neural stem cells (NSCs) have shown positive outcomes in various models of neurological injury and disease. Induced pluripotent stem cells (iPSCs) address many problems associated with NSCs from various sources, including the immune response and cell availability. However, due to inherent differences between embryonic stem cells (ESCs) and iPSCs, detailed characterization of the iPS-derived NSCs will be required before translational experiments can be performed. Murine piggyBac transposon iPSCs were clonally expanded in floating sphere colonies to generate primitive NSCs initially with serum-free media (SFM) containing the leukemia inhibitory factor and followed by SFM with the fibroblast growth factor-2 (FGF2) to form colonies of definitive NSCs (dNSCs). Primitive and definitive clonally derived neurospheres were successfully generated using the default conditions from iPSCs and ESCs. However, the iPSC-dNSCs expressed significantly higher levels of pluripotency and nonectoderm lineage genes compared to equivalent ESC-dNSCs. The addition of the bone morphogenetic proteins antagonist, Noggin, to the media significantly increased primary neurosphere generation from the iPSC lines, but did not affect the dNSC sphere colonies generated. The induction of the NOTCH pathway by the Delta-like ligand 4 (DLL4) improved the generation and quality of dNSCs, as demonstrated by a reduction in pluripotency and nonectodermal markers, while maintaining NSCspecific gene expression. The iPS-dNSCs ( + DLL4) showed functional neural differentiation by immuncytochemical staining and electrophysiology. This study suggests the intrinsic differences between ESCs and iPSCs in their ability to acquire a dNSC fate that can be overcome by inducing the NOTCH pathway.