Traumatic spinal cord injury (SCI) results in partial or complete paralysis along with sensory loss below the level ofinjury. The pathology of SCI is characterized by the loss of neurons and oligodendrocytes, axonal injury, and demyelination/dysmyelination of spared axons. Therapeutic transplantation of stem cell populations may promote functional recovery by providing trophic support, modifying the host environment to create a permissive environment for endogenous regeneration/repair, or by replacing neurons and/or oligodendrocytes 
SCI therapies can target acute, sub-acute, or chronic time points post-injury. The continuum from acute to chronic injury both in animal models and clinically is defined by the transition from a dynamic to a relatively stable injury environment, and when behavioral recovery reaches a plateau 
. In rodent contusion injury models these criteria are met beginning at approximately 30 days post-injury (dpi) 
. There are over 1,275,000 individuals living with chronic SCI in the U.S. alone (Christopher & Dana Reeve Foundation Paralysis Resource Center); thus, a chronic transplantation model is highly clinically relevant.
Several studies have investigated chronic SCI models using whole tissue grafts and peripheral nervous system (PNS) cells. Transplantation of fetal spinal tissue, fetal brain cortex, olfactory ensheathing cells (OECs), peripheral nerve grafts, and Schwann cells after SCI have all been shown to improve locomotor recovery 
, suggesting that the chronic post-injury period may be a feasible target for repair.
In contrast, the few studies that have compared sub-acute and chronic transplantation of CNS cell populations such as human oligodendrocyte progenitor cells (OPCs) and mouse neural stem cells (NSCs) in chronic SCI models have not reported improved locomotor recovery 
. Human OPCs transplanted 7 dpi survived and promoted locomotor recovery; however, at 10 months post-injury, OPCs survived but failed to improve locomotor recovery 
. Mouse NSCs transplanted 2 weeks post-SCI survived and improved locomotor recovery; however, at 2 months post-SCI, NSCs neither survived nor improved locomotor recovery 
Thus, while whole tissue grafts and PNS cells have shown some capacity for chronic stage repair (≥4 weeks post-SCI in rodents), CNS cell populations have thus far failed in the chronic setting. These studies suggest that the mechanism of cell transplant-mediated repair, the properties of specific cell transplant populations, and/or the microenvironment of the injured niche during the acute, sub-acute, and chronic periods may influence the potential to impact recovery post-SCI. Defining the potential window for successful engraftment and recovery in animal models with specific cell populations, particularly CNS populations, is therefore a critical step to developing therapeutics for chronic injuries.
We have previously reported that NOD-scid
mice, which are constitutively immunodeficient, lacking a normal T-cell, B-cell, and complement response, exhibit similar SCI pathology and cellular innate immune response to other mouse strains (C57Bl/6 and BUB/BnJ) 
. Accordingly, NOD-scid
mice provide an excellent experimental model to investigate the potential of transplanted human cell populations to engraft and promote histological and locomotor recovery following SCI without a xenograft rejection response 
. Furthermore, NOD-scid
mice have been used as a host for induced pluripotent cells in the CNS as an assay for tumor formation and NSC transplantation studies 
. Hence, stem cell transplantation in the CNS using the NOD-scid
model can provide tumorigenicity information. We have previously reported on the sub-acute transplantation of human neural stem cells (hCNS-SCns), which are lineage restricted to generate neurons, oligodendrocytes, and astrocytes, into a NOD-scid
SCI model. hCNS-SCns are prospectively isolated based on fluorescence-activated cell sorting (FACS) for a CD133+
population from fetal brain and grown as neurospheres 
hCNS-SCns transplanted sub-acutely 9 dpi in immunodeficient NOD-scid
mice successfully engrafted and improved long-term locomotor recovery compared to vehicle and human fibroblast (hFbs) control groups 
. Notably, recovery was abolished following selective ablation of hCNS-SCns using diphtheria toxin, demonstrating that survival of hCNS-SCns was required to sustain locomotor recovery 
. The majority of hCNS-SCns exhibited differentiation to oligodendrocytes, and a smaller percentage differentiated into neurons, but few exhibited evidence of astrocytic fate 
. This is in contrast to many studies that have demonstrated predominant astroglial fate or differentiation failure following acute or sub-acute NSCs transplantation, 
. Furthermore, immuno-electron microscopy in the sub-acute study revealed that human cells remyelinated mouse host axons and formed putative synapses with mouse neurons, suggesting that transplanted hCNS-SCns had stably integrated within the functional cytoarchitecture of the host CNS 
In the present study, we tested whether hCNS-SCns transplanted into immunodeficient NOD-scid mice at an early chronic time point (30 dpi), survived, differentiated, and promoted locomotor recovery. We also investigated whether the animals experienced mechanical allodynia, and stereologically quantified the number of engrafted cells, lesion volume, spared tissue volume, and glial scar area. Our results reveal that hCNS-SCns have the ability to survive, migrate, differentiate, and promote improved locomotor recovery when transplanted in the early chronic SCI microenvironment.