Human Neural Stem Cell Transplantation in Chronic Cervical Spinal Cord Injury

AIS: American Spinal Injury Association Impairment Scale CNS: central nervous system GRASSP: Graded Redefined Assessment of Strength, Sensibility, and Prehension HuCNS-SC: human fetal-derived central nervous system neural stem cell IOUSG: intraoperative ultrasonography IRB: institutional review board ISNCSCI: International Standards for Neurological Classification of spinal cord injury MRI: magnetic resonance imaging NSC: neural stem cells SCI: spinal cord injury Spinal cord injury (SCI) affects roughly 10 000 people per year,1 with a prevalence of over one million in the North American population and 10 to 83 per million worldwide.2 Historically, prior treatments have focused on limiting the extent of secondary injury after acute traumatic SCI. Concurrent advancements in understanding of embryonic stem cells, neural stem cells (NSC) and their differentiation into progenitors (NPCs), and therapeutic interventions have more recently made human fetal-derived central nervous system (CNS) neural stem cell (HuCNS-SC) transplantation a potential future treatment for SCI. Human embryonic stem cells are pluripotent and have been shown to be capable of reliably differentiating into potential cellular stocks of neurons, oligodendrocytes, and astrocytes.3-9 Another potential source of human CNS-derived NSCs (HuCNS-SC®, Stemcells, Inc, Newark, California) is from fetal CNS tissue, which is relatively abundant in NSCs. NSCs are multipotent or lineage-restricted to CNS cellular populations, and have been demonstrated in rodent SCI transplant models to facilitate recovery, both functionally and anatomically.10-13 Cellular therapy is one of the only proposed treatments for SCI with the potential to reconstitute the cellular architecture of the damaged spinal cord.14 Safety with the use of HuCNS-SC® in clinical trials has been demonstrated in previously published data for experimental treatment with Neuronal Ceroid Lipofuscinoses, a liposomal storage disorder,15 Pelizaeus–Merzbacher disease 16 which is predominantly a disorder of myelination, and dry age-related macular degeneration.17 Human-derived NSCs also have demonstrated clinical safety in recent clinical trials for the treatment of amyotrophic lateralizing sclerosis by providing motoneuron replacement.18 The technique for the safe perilesional intramedullary injection of various cellular therapies has been previously described,19 and more recently, in the injection of NSCs into the cervical spinal cord for ALS.20 With the clinical groundwork for safe engraftment of NSCs into eloquent areas of the CNS, future potential applications of cellular therapy may further include more prevalent CNS disorders such as multiple sclerosis, potentially by oligodendrocyte replacement.21 The authors report 12-mo follow-up data for chronic cervical SCI treated with perilesional intramedullary transplantation with HuCNS-SC® (Stemcells, Inc). METHODS Institutional review board (IRB) approval was obtained. The Quorum IRB evaluated and managed all participating centers in a previous Phase I/II study of HuCNS-SC® (Stemcells, Inc) transplantation in thoracic SCI. This was filed under an Investigational New Drug application with the US Food and Drug Administration and Health Canada, then registered on ClinicalTrials.gov (NCT01321333). We report the 1 yr results of all subjects (n = 5) who were entered and randomized into the University of Miami Miller School of Medicine site. Following this study, subjects with traumatic, nonpenetrating SCI were enrolled in a phase II escalating dose safety and efficacy study of intramedullary injections of HuCNS-SC® (Stemcells, Inc) rostral and caudal to the C5-C7 injury level with American Spinal Injury Association Impairment Scale (AIS) grade A or B (NCT02163876). Cohort 1 consisted of an open label, dose-escalation protocol (15, 30, and 40 million cells), while cohort 2 was randomized, controlled, single blinded, with a treatment dose of 40 million cells. Injections were performed at least 4 mo after injury. Along with interim magnetic resonance imaging (MRI) evaluation of the cervical spine, preoperative motor and sensory evaluation instruments included the “Graded Redefined Assessment of Strength, Sensibility, and Prehension” (GRASSP), and the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) assessments as well as postintervention, and serially at 3, 6, 9, and 12 mo interim periods. All subjects received immunosuppression regimens prior to engraftment that were continued through 6 mo post-transplantation to prevent graft rejection and mitigate any potential clinically significant post-transplant inflammatory response. Patient consent was obtained for evaluation including screening and imaging. A separate consent was obtained if a subject was enrolled as a control or in the treatment group including transplantation surgery, blood draws, immunosuppressive medications, and all follow-up noninvasive data collection such as radiographic and functional outcomes data.FIGURE: A, Intraoperative photograph demonstrating sterile transfer of cellular therapy from vial to sterile syringe. HuCNS-SC® (Stemcells, Inc) were carefully transferred from vial to a syringe with an 18-gauge blunt needle. The cell suspension was aspirated up and down 3 times forming a solution of neural cell clusters, with care taken to avoid air formation. This was done by the senior surgeon (ADL) to ensure rigorous quality control during the trial. B, Intraoperative photograph, demonstrating the exposed cervical spinal cord at the lesion site with injection of HuCNS-SC®. Direct visualization of the spinal cord and ultrasound visualization allowed the operator to avoid vessel injury and hemorrhage with the needle at the depth of the pia and within the parenchyma below.Overview of Surgical Technique and HuCNS-SC Injection General anesthesia using total intravenous anesthesia to facilitate neurophysiologic monitoring (somatosensory and motor evoked potentials, spontaneous electromyography) did not deviate from the standard hospital protocol for cervical SCI. Using the previous midline cervical incision in most patients that underwent cervical decompression and stabilization, the lesions were localized on the exposed dura using ultrasound guidance (Hitachi HI Vision Ascendus, Hitachi Medical Systems Europe Holding AG, Switzerland/12 Mhz linear array transducer on an IU22 scanner (Hitachi Aloka Medical America, Inc, Wallingford, Connecticut)). After a midline dural opening, intramedullary injections of HuCNS-SC® (Stemcells, Inc) were performed by the senior surgeon (ADL) rostral and caudal to the lesion site with a 2-hand technique.22 Direct visualization of the spinal cord and ultrasound visualization allowed the operator to target “normal” white matter around the periphery of the spinal cord damage (Figure). Generally, injection sites were midway between the dorsal intermediate sulcus and the dorsal root entry zone at a 30° medial inclination to avoid unintentional transplant placement at or near the anterior motor tracts. After localizing the rostral and caudal extent of perilesional injection, the pia was opened with the tip of a #11 blade scalpel. A 30-gauge needle was used to make all injections at depths of 3 to 4 mm, with a rate of injection of 20 μL/s for a maximum injection time of 3:30 (70 μL) and a dwell time of 60 s prior to needle removal, which was monitored and recorded by an independent staff member in the operating room. Injection sites were visually inspected for bleeding and reflux of cellular material. A 1 mL vial of HuCNS-SC® was utilized in each patient in the open-label dose escalation cohort involving 3 doses (n = 2/dose): 15, 30, and 40 million cells. In the first 2 patients with 15 million cells, 2 microinjections delivered 10 million cells (140 μL) caudally, and 2 microinjections delivered 5 million cells (70 μL) rostrally. In the 30 million cell group, 4 microinjections totaling 20 million cells were injected caudally (280 μL) and 2 microinjections delivered 10 million cells rostrally (140 μL). In the 40 million cell group, 4 microinjections delivered 20 million cells caudally (280 μL) and 4 microinjections delivered 20 million cells rostrally (280 μL). In the randomized cohort, the 40 million cell dose injection paradigm was utilized. RESULTS A total of 17 patients were transplanted for the entire study. Five total subjects (2 subjects in in cohort 1, 3 subjects in cohort 2) with 1-yr follow-up are presented (1 with AIS grade A, 5 with AIS grade B injury) from the University of Miami enrollment site. Four subjects received HuCNS-SC® (Stemcells, Inc) injections (range 15-40 × 106 cells) and 1 was a control. The mean age was 24.3 yr (range 19-30, standard deviation = 3.78; Table 1). For the transplanted subjects, the mean GRASSP score at enrollment was 74.5 ± 14.5 (range 53-84), 76.8 ± 35.9 at 3 mo (range 23-97), 86.0 ± 21.0 at 6 mo (range 58-108), 90.5 ± 28.8 at 9 mo (range 50-113), and 89.3 ± 21.6 at 12 mo (range 57-103). The control subjects’ GRASSP score was 66 at enrollment, 73 at 3 mo, 64 at 6 mo, 64 at 9 mo, and 82 at 12 mo. The mean change in scores for all patients were 1.4 ± 2.51 for strength, 4.2 ± 4.76 for sensation, 1 ± 4 for prehension ability, and 8.4 ± 3.65 for prehension performance. The mean improvement from enrollment to month 12 in total GRASSP score was 14.8 ± 7.8 (range 4-21; Table 2) for the transplanted subjects and 16 for the control subject. Three subjects, 2 transplant and 1 control, with AIS B injury remained unchanged at the 12-mo period. One transplanted subject with AIS grade B declined to AIS grade A by 9 mo, persistent at 12 mo. One transplanted AIS grade A subject improved to AIS B at 12 mo. The mean improvement from enrollment to month 12 in ISNCSCI scoring was 2.3 ± 1.5 for upper extremity motor strength (–1 in control), 5.3 ± 3.8 for light touch sensation (5 in control), 9.8 ± 15.0 for pin prick sensation (8 in control), and the composite lower extremity motor score was zero was unchanged. The mean overall ISNCSCI score improved from 88.3 ± 43.1 at baseline (control = 101) to 105.5 ± 59.8 at 12 mo (control = 113), a mean improvement of 17.3 ± 16.8 points (Table 3). There were no serious adverse events attributed to spinal cord injection.TABLE 1: Baseline Characteristics of Cervical Study ParticipantsTABLE 2: Summary of GRASSP ScoringTABLE 3: Summary of ISNCSCI ScoringDISCUSSION HuCNS-SC® (Stemcells, Inc) transplantation has repeatedly demonstrated in animal models of contusive SCI to engraft, survive, migrate, and differentiate into neurons, astrocytes, and oligodendrocytes. This has been chiefly demonstrated with fetal-derived NSC/NPCs10-13,23,24 and more recently with human embryonic stem cell-derived NSCs.25 Human NSC transplantation in the chronic injury setting has been previously demonstrated to be safe and provide no additional morbidity to injection in the acute or subacute window.26 Moreover, NSC transplantation in the chronic time period may provide a more favorable, less-inflammatory environment for cell engraftment. On the other hand, the formation of glial scar in the form of chondroitin sulfate proteoglycans found in greater abundance in the chronic environment may provide an additional obstacle to axonal growth and reconnectivity. This concept is supported by enhanced migratory success observed with cotreatment of cell transplants with chondroitinase relative to control transplantation.27-30 To date, 29 SCI subjects (12 thoracic, 17 cervical) were enrolled across 2 clinical trials. The authors present the initial 12-mo follow-up of 5 cervical subjects transplanted with HuCNS-SC® (Stemcells, Inc), demonstrating that the cervical spinal cord can safely be treated with doses up to 40 million cells. All subjects demonstrated improvement in their total GRASSP score for all components with a mean improvement of 15 ± 6.78 points (range 4-21; Table 2). Also, ISNCSCI scores improved from 90.8 ± 37.7 points at baseline to 107 ± 51.9 points at 12 mo, a mean improvement of 16.2 points (Table 3). One control reached the 12-mo mark and was included for analysis (Table 1; subject 17-1007). Comparable improvements in ISNCSCI (Table 3) and GRASSP (Table 2) were demonstrated on follow-up between control and treatment subjects. One observation to note was that the control subject was the only case where a net decline in upper extremity motor strength was observed at 12-mo follow-up (19-18). This decline in function was not correlated on GRASSP scoring where a 16-point gain was observed on final follow-up. Moreover, the C6 injury level was unaffected in this patient and improvements in light touch and pin prick components were observed. However, beyond these observations, and until adequate follow-up is obtained from the remaining subjects, statements of relative efficacy cannot be made due to the limited enrollment numbers and variations of baseline GRASSP and ISNCSCI scoring can be observed, partly due to interrater reliability. The GRASSP scoring tool is 50% more sensitive than ISNSCI for evaluating sensory and motor function of the upper limb, with a high interrater reliability (0.84-0.96).31-33 No adverse events were attributed to manual perilesional spinal cord injection or the number of microinjections performed. Injections ranged from 4 to 8 microinjections administered bilaterally. One instance of paresthesia in the fifth metacarpal of the left hand shortly resolved after 2 wk. No cases of hyperalgesia or allodynia were observed. Serial MRI was obtained after injection, revealing dorsal column atrophy rostral the lesion cavity and to a less degree, lateral column atrophy caudal to the lesion cavity, which is a finding often indicative of Wallerian degeneration from chronic SCI.34 The rationale for the use of perilesional injection through the dorsal columns was supported by these MRI findings (data not shown) and the favorable safety profile encountered. Intraoperative ultrasonography (IOUSG) use throughout this study has been indispensable in localization of the SCI lesion. Also, confirmation of a sufficient rostral and caudal cervical laminectomy prior to durotomy was aided by IOUSG as well as identification of associated cyst cavities to further delineate the extent of exposure and subsequent durotomy.35 For intramedullary spinal cord surgery, IOUSG has been shown to be a timely and effective method of tumor localization, evaluation of resection extent, as well as confirmation of spinal cord decompression after tumor resection and even with ventral spinal cord compression after traumatic fracture.35-37 There were no instances of spinal cord hemorrhage on postoperative MRI, which potentially can be attributed to the effectiveness of IOUSG in avoidance of spinal cord vasculature.35 Postoperative cervical MRI was obtained after injection to confirm anatomic location of the cell graft and absence of spinal cord hemorrhage. Also, larger doses of 40 million cells and larger volumes of T2 hyperintensity were observed, changes most likely indicative of perilesional cell injectate rather than cord edema. These changes resolved over the course of the study. To limit the potential inflammatory effect from injectate or from spinal cord displacement by larger injection volumes, microinjections were divided into maximum volumes of 70 μL and injection rates of 20 μL per minute. CONCLUSION The transplantation of HuCNS-SC® (Stemcells, Inc) into the chronic cervical spinal cord can be safely performed, supported by 12-mo clinical follow-up. Improvements in overall mean functional outcomes measures at final follow-up were observed with perilesional injections of human CNS-derived NSC into the chronically injured cervical spinal cord. Further conclusions regarding clinical efficacy cannot be made without additional enrollment, follow-up including a rigorous comparison with a larger matched control group. Disclosures This study was funded by Stemcells, Inc and the respective Academic Institutions. Dr. Allan D. Levi receives teaching honoraria from Medtronic and grant support from the Department of Defense. Dr. Kim Anderson is a consultant for Vertex Inc. Acknowledgements We sincerely thank the multiple clinical care coordinators, rehabilitation, neuroradiology, immune suppression, anesthesia, back up and blinded physicians and nurses who participated in the study. In addition, we are grateful to the SCI patients around the world who participated in the current trials. We are greatly indebted to Linda Alberga for her editorial assistance.

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