Complement C6 deficiency exacerbates pathophysiology after spinal cord injury.

Historically, the membrane attack complex, composed of complement components C5b-9, has been connected to lytic cell death and implicated in secondary injury after a CNS insult. However, studies to date have utilized either non-littermate control rat models, or mouse models that lack significant C5b-9 activity. To investigate what role C5b-9 plays in spinal cord injury and recovery, we generated littermate PVG C6 wildtype and deficient rats and tested functional and histological recovery after moderate contusion injury using the Infinite Horizon Impactor. We compare the effect of C6 deficiency on recovery of locomotor function and histological injury parameters in PVG rats under two conditions: (1) animals maintained as separate C6 WT and C6-D homozygous colonies; and (2) establishment of a heterozygous colony to generate C6 WT and C6-D littermate controls. The results suggest that maintenance of separate homozygous colonies is inadequate for testing the effect of C6 deficiency on locomotor and histological recovery after SCI, and highlight the importance of using littermate controls in studies involving genetic manipulation of the complement cascade.

Novel C1q receptor-mediated signaling controls neural stem cell behavior and neurorepair.

C1q plays a key role as a recognition molecule in the immune system, driving autocatalytic complement cascade activation and acting as an opsonin. We have previously reported a non-immune role of complement C1q modulating the migration and fate of human neural stem cells (hNSC); however, the mechanism underlying these effects has not yet been identified. Here, we show for the first time that C1q acts as a functional hNSC ligand, inducing intracellular signaling to control cell behavior. Using an unbiased screening strategy, we identified five transmembrane C1q signaling/receptor candidates in hNSC (CD44, GPR62, BAI1, c-MET, and ADCY5). We further investigated the interaction between C1q and CD44 , demonstrating that CD44 mediates C1q induced hNSC signaling and chemotaxis in vitro, and hNSC migration and functional repair in vivo after spinal cord injury. These results reveal a receptor-mediated mechanism for C1q modulation of NSC behavior and show that modification of C1q receptor expression can expand the therapeutic window for hNSC transplantation.

The effects of mouse strain and age on a model of unilateral cervical contusion spinal cord injury.

There are approximately 1.2 million people currently living with spinal cord injury (SCI), with a majority of cases at the cervical level and half involving incomplete injuries. Yet, as most preclinical research has been focused on bilateral thoracic models, there remains a disconnect between bench and bedside that limits translational success. Here, we profile a clinically relevant model of unilateral cervical contusion injury in the mouse (30kD with 0, 2, 5, or 10 second dwell time). We demonstrate sustained behavioral deficits in performance on grip strength, cylinder reaching, horizontal ladderbeam and CatWalk automated gait analysis tasks. Beyond highlighting reliable parameters for injury assessment, we also explored the effect of mouse strain and age on injury outcome, including evaluation of constitutively immunodeficient mice relevant for neurotransplantation and cellular therapy testing. Comparison of C57Bl/6 and immunodeficient Rag2gamma(c)-/- as well as Agouti SCIDxRag2Gamma(c)-/- hybrid mouse strains revealed fine differences in post-injury ipsilateral grip strength as well as total number of rearings on the cylinder task. Differences in post-SCI contralateral forepaw duty cycle and regularity index as measured by CatWalk gait analysis between the two immunodeficient strains were also observed. Further, assessment of young (3-4 months old) and aging (16-17 months old) Rag2gamma(c)-/- mice identified age-related pre-injury differences in strength and rearing that were largely masked following cervical contusion injury; observations that may help interpret previous results in aged rodents as well as human clinical trials. Collectively, the work provides useful insight for experimental design and analysis of future pre-clinical studies in a translational unilateral cervical contusion injury model.

Systemic Neutrophil Depletion Modulates the Migration and Fate of Transplanted Human Neural Stem Cells to Rescue Functional Repair.

The interaction of transplanted stem cells with local cellular and molecular cues in the host CNS microenvironment may affect the potential for repair by therapeutic cell populations. In this regard, spinal cord injury (SCI), Alzheimer's disease, and other neurological injuries and diseases all exhibit dramatic and dynamic changes to the host microenvironment over time. Previously, we reported that delayed transplantation of human CNS-derived neural stem cells (hCNS-SCns) at 9 or 30 d post-SCI (dpi) resulted in extensive donor cell migration, predominantly neuronal and oligodendrocytic donor cell differentiation, and functional locomotor improvements. Here, we report that acute transplantation of hCNS-SCns at 0 dpi resulted in localized astroglial differentiation of donor cells near the lesion epicenter and failure to produce functional improvement in an all-female immunodeficient mouse model. Critically, specific immunodepletion of neutrophils (polymorphonuclear leukocytes) blocked hCNS-SCns astroglial differentiation near the lesion epicenter and rescued the capacity of these cells to restore function. These data represent novel evidence that a host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population, and support targeting the inflammatory microenvironment in combination with cell transplantation after SCI.SIGNIFICANCE STATEMENT The interaction of transplanted cells with local cellular and molecular cues in the host microenvironment is a key variable that may shape the translation of neurotransplantation research to the clinical spinal cord injury (SCI) human population, and few studies have investigated these events. We show that the specific immunodepletion of polymorphonuclear leukocyte neutrophils using anti-Ly6G inhibits donor cell astrogliosis and rescues the capacity of a donor cell population to promote locomotor improvement after SCI. Critically, our data demonstrate novel evidence that a specific host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population.

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis.

Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.

Preclinical Efficacy Failure of Human CNS-Derived Stem Cells for Use in the Pathway Study of Cervical Spinal Cord Injury.

We previously showed the efficacy of multiple research cell lines (RCLs) of human CNS neural stem cells (HuCNS-SCs) in mouse and rat models of thoracic spinal cord injury (SCI), supporting a thoracic SCI clinical trial. Experts recommend in vivo preclinical testing of the intended clinical cell lot/line (CCL) in models with validity for the planned clinical target. We therefore tested the efficacy of two HuCNS-SC lines in cervical SCI: one RCL, and one CCL intended for use in the Pathway Study of cervical SCI in man. We assessed locomotor recovery and sensory function, as well as engraftment, migration, and fate. No evidence of efficacy of the CCL was observed; some data suggested a negative impact of the CCL on outcomes. These data raise questions about the development and validation of potency/comparability assays for clinical testing of cell products, and lack of US Food and Drug Administration requirements for in vivo testing of intended clinical cell lines.

Characterization of recovery, repair, and inflammatory processes following contusion spinal cord injury in old female rats: is age a limitation?

BACKGROUND: Although the incidence of spinal cord injury (SCI) is steadily rising in the elderly human population, few studies have investigated the effect of age in rodent models. Here, we investigated the effect of age in female rats on spontaneous recovery and repair after SCI. Young (3 months) and aged (18 months) female rats received a moderate contusion SCI at T9. Behavioral recovery was assessed, and immunohistocemical and stereological analyses performed. RESULTS: Aged rats demonstrated greater locomotor deficits compared to young, beginning at 7 days post-injury (dpi) and lasting through at least 28 dpi. Unbiased stereological analyses revealed a selective increase in percent lesion area and early (2 dpi) apoptotic cell death caudal to the injury epicenter in aged versus young rats. One potential mechanism for these differences in lesion pathogenesis is the inflammatory response; we therefore assessed humoral and cellular innate immune responses. No differences in either acute or chronic serum complement activity, or acute neutrophil infiltration, were observed between age groups. However, the number of microglia/macrophages present at the injury epicenter was increased by 50% in aged animals versus young. CONCLUSIONS: These data suggest that age affects recovery of locomotor function, lesion pathology, and microglia/macrophage response following SCI.

Achieving stable human stem cell engraftment and survival in the CNS: is the future of regenerative medicine immunodeficient?

There is potential for a variety of stem cell populations to mediate repair in the diseased or injured CNS; in some cases, this theoretical possibility has already transitioned to clinical safety testing. However, careful consideration of preclinical animal models is essential to provide an appropriate assessment of stem cell safety and efficacy, as well as the basic biological mechanisms of stem cell action. This article examines the lessons learned from early tissue, organ and hematopoietic grafting, the early assumptions of the stem cell and CNS fields with regard to immunoprivilege, and the history of success in stem cell transplantation into the CNS. Finally, we discuss strategies in the selection of animal models to maximize the predictive validity of preclinical safety and efficacy studies.

Analysis of host-mediated repair mechanisms after human CNS-stem cell transplantation for spinal cord injury: correlation of engraftment with recovery.

BACKGROUND: Human central nervous system-stem cells grown as neurospheres (hCNS-SCns) self-renew, are multipotent, and have potential therapeutic applications following trauma to the spinal cord. We have previously shown locomotor recovery in immunodeficient mice that received a moderate contusion spinal cord injury (SCI) and hCNS-SCns transplantation 9 days post-injury (dpi). Engrafted hCNS-SCns exhibited terminal differentiation to myelinating oligodendrocytes and synapse-forming neurons. Further, selective ablation of human cells using Diphtheria toxin (DT) abolished locomotor recovery in this paradigm, suggesting integration of human cells within the mouse host as a possible mechanism for the locomotor improvement. However, the hypothesis that hCNS-SCns could alter the host microenvironment as an additional or alternative mechanism of recovery remained unexplored; we tested that hypothesis in the present study. METHODS AND FINDINGS: Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals. DT administration at 16 weeks post-transplant ablated 80.5% of hCNS-SCns. Stereological quantification for lesion volume, tissue sparing, descending serotonergic host fiber sprouting, chondroitin sulfate proteoglycan deposition, glial scarring, and angiogenesis demonstrated no evidence of host modification within the mouse spinal cord as a result of hCNS-SCns transplantation. Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair. However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = -0.78, p<0.05), suggesting that survival and engraftment were directly related to a quantitative measure of recovery. CONCLUSIONS: Altogether, the data suggest that the locomotor improvements associated with hCNS-SCns transplantation were not due to modifications within the host microenvironment, supporting the hypothesis that human cell integration within the host circuitry mediates functional recovery following a 9 day delayed transplant.

Deficiency in complement C1q improves histological and functional locomotor outcome after spinal cord injury.

Although studies have suggested a role for the complement system in the pathophysiology of spinal cord injury (SCI), that role remains poorly defined. Additionally, the relative contribution of individual complement pathways in SCI is unknown. Our initial studies revealed that systemic complement activation was strongly influenced by genetic background and gender. Thus, to investigate the role of the classical complement pathway in contusion-induced SCI, male C1q knock-out (KO) and wild-type (WT) mice on a complement sufficient background (BUB) received a mild-moderate T9 contusion injury with the Infinite Horizon impactor. BUB C1q KO mice exhibited greater locomotor recovery compared with BUB WT mice (p<0.05). Improved recovery observed in BUB C1q KO mice was also associated with decreased threshold for withdrawal from a mild stimulus using von Frey filament testing. Surprisingly, quantification of microglia/macrophages (F4/80) by FACS analysis showed that BUB C1q KO mice exhibited a significantly greater percentage of macrophages in the spinal cord compared with BUB WT mice 3 d post-injury (p<0.05). However, this increased macrophage response appeared to be transient as stereological assessment of spinal cord tissue obtained 28 d post-injury revealed no difference in F4/80-positive cells between groups. Stereological assessment of spinal cord tissue showed that BUB C1q KO mice had reduced lesion volume and an increase in tissue sparing compared with BUB WT mice (p<0.05). Together, these data suggest that initiation of the classical complement pathway via C1q is detrimental to recovery after SCI.

Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice.

We report that prospectively isolated, human CNS stem cells grown as neurospheres (hCNS-SCns) survive, migrate, and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord-injured NOD-scid mice. hCNS-SCns engraftment was associated with locomotor recovery, an observation that was abolished by selective ablation of engrafted cells by diphtheria toxin. Remyelination by hCNS-SCns was found in both the spinal cord injury NOD-scid model and myelin-deficient shiverer mice. Moreover, electron microscopic evidence consistent with synapse formation between hCNS-SCns and mouse host neurons was observed. Glial fibrillary acidic protein-positive astrocytic differentiation was rare, and hCNS-SCns did not appear to contribute to the scar. These data suggest that hCNS-SCns may possess therapeutic potential for CNS injury and disease.