This study directly assessed the potential for hESC-derived OPCs to provide at-level benefit to cervical SCI. Our results complement other reports of cervical transplantation research 29
and suggest that outcome from treatment can differ from that in the thoracic spinal cord. These findings further our understanding of the pathology and functional outcomes of cervical SCI, and are the first to demonstrate anatomical and functional benefit following transplantation of a hESC-derivate to cervical SCI.
Cell-based therapeutics have proven successful in pre-clinical SCI models 30
at least in part due to their ability to address multiple features of SCI such as cell loss, demyelination, or homeostatic loss. Several cell replacement strategies have emerged to treat SCI, including O-2A progenitors 31, 32
, Schwann cells 29, 33, 34
, or neural stem cells 35, 36
. Several recent studies indicate that myelinogenic transplants elicit histologic repair and functional recovery following SCI 36
, validating demyelination as a therapeutic target for SCI 14, 37–39
. Nistor et alias 18
and subsequently Izreal et alias 40
described protocols to direct the differentiation of hESCs into high purity OPC populations, and demonstrated their myelination potential. The use of hESCs as a source for human transplant populations offers advantages over other cell types, including the inherently broad capacity for expansion and differentiation 41
The ability of non-myelinating cells such as bone marrow stromal cells to improve the outcome of SCI exemplifies therapeutic benefits of a non-myelinogenic transplant 42, 43
. In an analogous manner, hESC-derived OPCs might contribute to at-level histological and functional outcomes via a non-myelin mechanism. A recent report identified 49 neurotrophic factors expressed by hESC-derived OPCs 22
, including the neurotrophic factors insulin-like growth factor 1, brain-derived neurotrophic factor, NT-3, nerve growth factor, and transforming growth factor-β1. Co-cultures of hESC-derived OPCs with cortical neurons enhanced neurite outgrowth from the cortical neurons, indicating that the secreted factors can affect surrounding cells 21, 22
. These results suggest that hESC-derived OPCs might produce beneficial effects in SCI aside from remyelination, such as neuroprotection, suppression of inflammation, promotion of axonal regeneration, and/or homeostatic maintenance.
Our methods for cervical contusion produced cavitation and pathological features consistent with moderate to severe bilateral injuries reported by Schrimsher and Reier 15
and Pearse et al. 13
. Here, the lesion emanated a distance of less than 2mm from the epicenter and quantitative analysis revealed extensive remyelination. This is in contrast to thoracic injuries of the same force, which resulted in lesions extending 6–12mm either side of the lesion epicenter 14
with substantial demyelination and remyelination. This difference in remyelination is consistent with results of Franklin et alias 44
that found the migratory potential of endogenous myelinogenic cells is restricted to approximately 2 mm either side of a region of demyelination. The robust endogenous remyelination in this cervical model might also reflect enhanced axonal survival as a result of the axotomies being relatively closer to the cell bodies of origin 45
. Such potential asymmetries between cervical and thoracic injuries underscore the need to test the effectiveness of potential treatments for SCI in both injury models.
Most of the transplanted hESC-derived OPCs localized to the lesion epicenter. However, some transplanted cells were found up to 2 mm away from the site of injection. The expression of CXCR4 by hESC-derived OPCs and the detection of human-CXCR4 positive cells within areas of rat-CXCL12 reactivity outside of the injury epicenter suggest that hESC-derived OPCs can follow migratory cues. These findings cannot rule out the possibility that dispersion during the transplant injection can also contribute to the cell distribution. Quantification of Olig1 and APC/CC1 positive cells in regions of high concentrations of human nuclei-positive cells within injury epicenter suggests that a lack of niche prevents maturation of a high percentage of the transplanted cells. This feature is supported by evidence that within the injured white matter niche, the percentage of APC/CC1 and human nuclei co-labeled cells is increased. The persistent detection of Olig1 or APC/CC1 on human cells localized to the injury epicenter suggests that transplanted hESC-derived OPCs retained an oligodendroglial lineage despite the derivation method and diverse molecular milieu. These data, together with the absence of human cells expressing astrocyte, neuronal, or embryonic markers, suggests that the transplant population did not trans-differentiation or become pluripotent.
Although transplanted hESC-derived OPCs differentiated into mature oligodendrocytes within white matter tracts, no significant difference in remyelination was detected between the transplanted and non-transplanted groups. This result is not unexpected given the extensive endogenous remyelination evidenced in the control group. Importantly, the treatment group had significantly more normally myelinated axons and fewer Schwann cell and demyelinated axons relative to control, suggesting that fewer axons were demyelinated in the treatment group. This would potentially present less substrate for remyelination and thereby reduce the number of remyelinated axons in the treatment group. Comparison of oligodendrocyte remyelination efficiency supports this interpretation, as the treatment group showed an increased efficiency relative to control.
The forelimb outcome measures used in this study to determine locomotor recovery were based on the results of anatomical tracing of C5 afferents to forelimb musculature by McKenna et alias 17
. In this cervical contusion model, animals achieved a high level of recovery of the affected limbs at an earlier timepoint, as compared to that reported for thoracic contusion animals 19
. The transplant group had an increase of stride length, proximal forelimb range of motion, specifically in the step lift-off, and a relative increase in frequency of passed-perpendicular steps. Thus, the improvement in forelimb function in the transplant group was observed across multiple forelimb gait parameters relevant to the level of injury.
Morphometry and cell quantification analyses revealed a number of significantly improved histological outcomes in the transplants compared to controls. Correlation of the different histological outcomes with a measure of proximal forelimb function in both transplants and non-transplants indicated a statistically significant correlation of forelimb function with spared motor neurons and gray matter sparing, underscoring the importance of preserving at-level motor neuron function in cervical SCI. Interestingly, no significant correlations were present when forelimb functional outcomes were compared to maximal cavitation or maximal cord areas, indicating that the reduction in cavitation alone was insufficient to account for the improved locomotion in the transplant group. Although this is consistent with hESC-derived OPC-mediated neuroprotection of cultured neurons21, 22
, the differential gene expression results demonstrate that transplantation of OPCs reduce markers of SCI-induced apoptosis and inflammation, and support neuron survival in vivo
during the acute phase after injury. Having identified significant gene expression changes at 21 days post-injury, we expect it will be possible to further investigate these pathways to form a better understanding how hESC-derived OPCs interact or interfere with inflammation and cell death mechanism, such as TNFα–induced excitotoxicity 46
or microglial activation 47
, to promote tissue sparing.
These findings demonstrate that transplantation of human OPCs into acute cervical SCI improves histological outcomes that correlate with improved recovery. Importantly, our data indicate that cervical SCI presents a distinct lesion pathogenesis and that the mechanism and outcome of treatment can differ from that in the thoracic spinal cord. These findings underscore the importance of using cervical injury models in addition to thoracic models for the preclinical development of therapeutics for SCI, in order to better address the human SCI population.