Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Ciacci ( jciacci@ucsd.edu ). Cell source requests should be directed to and will be fulfilled by Karl Johe, Ph.D. (Neuralstem Inc., kjohe@neuralstem.com ).

(F) Position of the injection floating cannula after placement of the injection tip into the spinal cord tissue. A stop-cock ring and floating portion of the cannula can now be seen (white arrow). The guiding stainless-steel tubing was retracted to permit the flotation of the injection cannula.

(E) Spinal cell injections were performed using a free-floating cannula attached to an XYZ manipulator. The XYZ manipulator is mounted on a self-supporting platform attached to patient vertebral column (vertebral laminae) above and below the level of laminectomy using four stainless steel posts and spinal screws.

Each subject received total of 6 intraspinal injections (2 × 10cells/injection delivered in 10μL of hibernation buffer). The injections were placed bilaterally into the remaining tissue lateral to the injury site and within the medial white matter-appearing tracts of approximately one segment below the injury site, as verified by intra-operative fluoroscopy imaging Injections were made using a customized stereotactic cell injection device ( Figure 5 E), ().

DNA of NSI-566 was subjected to high resolution sequencing analysis of major HLA loci: Class I locus A, B, and C; and Class II locus DRB1, DRB3/4/5, DPB1, DQA1, and DQB1. Based on the genotypes, followed antibodies in treated subjects were monitored: HLA-A2; HLA-A68; HLA-B62; HLA-B71; HLA-Cw7; HLA-DR9; HLA-DR12; HLA-DR52; HLA-DR53; HLA-DQ7, HLADQ9 (and the DQ alpha specificities 03 and 06); HLA-DP2 and HLA-DP6.

The clinical lot of NSI-566 had undergone extensive preclinical safety and efficacy studies in various small and large animal studies, which had been reviewed by US FDA under an IND (Investigational New Drug) application (#014413). Potential of NSI-566 to form tumor cells was evaluated in 3 different animal models in 5 studies. In an accepted mouse model of tumorigenicity, there was no evidence of tumorigenicity 1 or 3 months following subcutaneous injection with 1x10 7 cells. Four studies were conducted in which possible tumor formation was evaluated after injection of maximal feasible dose (0.45 – 0.6 × 10 6 cells) into the clinically intended target site, the spinal cord: 2 non-GLP 9-month survival studies, 1 GLP 6-month survival study, and 1 GLP 9-month survival study. No evidence of tumorigenicity due to NSI-566 was observed in any of these studies, as assessed either by mitotic activity or histopathology. In some of the rodent intraspinal injection studies, a 2-5-fold increase in the cell number was observed post transplantation. This increase is believed to represent a normal pattern of division of the spinal cord-derived human neural stem cells and their glial progenies, which gradually decline over time. The cell dose used in this study included such anticipated in vivo increase in graft size.

Before proceeding with cell administration, the cells suspension was inspected for cell viability of at least 70% using the method of trypan blue exclusion in order to proceed with the implantation. The viability ranged from 87%–92% and there were no failed deliveries or rejections due to out-of-range release specifications. Sterility and endotoxin level of each cell batch were verified by post hoc testing of retention sample kept at the manufacturing site, and the test result was notified to the study investigators within 14 days of surgery. There was no incidence of nonconformance in regard to sterility or endotoxin level.

For cell administration, NSI-566 was provided as a live-cell suspension, ready-to-inject, formulation, requiring no further manipulation. Briefly, NSI-566 cell suspension was prepared one day prior to each scheduled surgery at a cGMP facility. One or more vials of the cryopreserved CCB were thawed at once, washed of the freezing medium by repeated centrifugation in a hibernation medium (HM), and concentrated to a final concentration of 2x10cells/mL of HM. This target concentration had been established for being safe and adequate for intraspinal injections by series of preclinical () and clinical studies ((). The HM is a sterile, buffer solution free of preservatives and antibiotics developed for intraparenchymal injections into CNS, which was provided by Neuralstem, Inc. The cell suspension was then packaged in a custom-designed insulated shipping container that maintained the cell vials at 2°C–8°C, and shipped to the surgery site for overnight delivery by a commercial package courier (Federal Express). Prior shipping stability studies had validated > 70% cell viability up to 60 hours under these conditions. The expiry window for this study was 48 hours from the time of preparation.

NSI-566, is a human spinal cord-derived neural stem cell line. Neural stem cells are the precursor cells present in the neuro-epithelium along the neuraxis during mammalian fetal development. NSI-566 was derived from a single post-mortem spinal cord of an 8-week gestational age fetus. This tissue was obtained in compliance with the National Institutes of Health (NIH) and Food and Drug Administration (FDA) Good Tissue Practice Guidelines, and under a protocol approved by an outside independent review board. Neural stem cells were isolated by dissociating a single piece of spinal cord tissue of lower cervical/upper thoracic region and expanding it as a single line. Three-tiered cell banks, consisting of a master cell bank (MCB), a working cell bank (WCB), and a clinical cell bank (CCB), were established and cryopreserved under cGMP. The current MCB is > 99% nestin-positive, adherent neural stem cells established at passage 6. A WCB at passage 9 was manufactured from this MCB, and a CCB at passage 12 was manufactured from the WCB. All cell doses used in this study were prepared from the passage 12 CCB. Karyology of the clinical lot cells showed normal 44, X, and Y chromosomes. Greater details of the cGMP generation of clinical material was previously described ().

Inclusion and exclusion criteria are listed in Methods S1 . This was a Phase I safety study of human spinal cord-derived neural stem cell transplantation for the treatment of chronic spinal cord injury, defined as at least one year but no more than 2 years after traumatic SCI. A sample of 4 subjects with chronic SCI classified as AISA-A, a motor and sensory complete SCI, levels T2-T12, who met eligibility criteria were enrolled. No control group was included. Inclusion and exclusion criteria are listed in Methods S1 . All subjects received spinal cord injections of human spinal cord derived neural stem cells (NSI-566). The trial was registered in ClinicalTrials.gov as NCT 01772810. IRB approval granted by UCSD Health Center, Human Research Protections Program (HRPP), 9452 Medical Center Drive, La Jolla, CA 92037

Animal housing: Animals were single-housed in cages with irradiated bedding. Acclimation times prior to the experiment varied but were at least 8 days. Environmental controls were set to maintain temperatures from 64° to 79°F (18° to 26°C), with fluorescent lighting on a 12 hour/12 hour on/off inverted cycle. Animals were fed irradiated Harlan Teklad Global 18% Protein Rodent Diet T.2918.15 (Harlan Laboratories, Placentia, CA) ad libitum. In addition, animals that were selected for addition behavioral testing (see below) also received Fruit Crunchies (BioServ product # S05798-1) and HydroGel (ClearH2O®, ME 04101). Animals were provided with purified water (Mountain Dairy, Riverside, CA, USA) ad libitum.

The study design and animal usage were reviewed and approved by the Institutional Animal Care and Use Committee (MC 0071, UCSD, 9500 Gilman Dr., La Jolla, CA). Animal welfare for this study was in compliance with the U.S. Department of Agriculture’s (USDA) Animal Welfare Act (CFR 9 - Parts 1, 2, and 3), and the Guide for the Care and Use of Laboratory Animals. Animals: Rats, Athymic Nude (rnu-/rnu-) - Hsd: RH-Foxn1rnu (Harlan, Madison, WI, USA) were used. A total of 90 male rats were enrolled, of which 8 were used for descending motor tracts labeling ( Figure 1 A). Animal ages at injury ranged from 10 to 13 weeks, and body weights varied between 309 to 345 g.

Method Details

Induction of spinal injury (SCI): The day prior to surgeries the back of the animals was shaved. On SCI surgery days, each animal received lactated ringer’s (5 mL, s.c.). Animals were anesthetized with isoflurane (5% for induction, ∼2% for maintenance, in air). The surgery site was wiped with alcohol and chlorhexidine diacetate solution (2%–4%). Rat body temperature was maintained by a water blanket heating system. The skin over the vertebral column was opened, paravertebral muscles were dissected away, and the animal was mounted onto a Stereotaxic frame (Stoelting Lab Standard Stereotaxic - Single, Cat# 51600 Lab Standard) with Spine Adaptors (Stoelting, Cat# 51695 Rat Spinal Adaptor). A thoracic spinal segment (Th10) was then exposed using a dental drill. A moderate injury was made by letting the rod of a MASCIS (NYU) Impactor dropped from a height of 12.5 mm onto the exposed surface of the spinal cord. Next, the impactor was removed immediately, the animal was detached from the frame, the surgical site was irrigated with sterile saline, layers were closed with absorbable Vicryl suture, and Bacitracin/Neomycin/Polymyxin (triple antibiotic) ointment was applied to the incision site. Cefazolin (10mg/kg, s.c.) was also given at the day of surgery.

Dosing surgery: One week following SCI surgeries, prior to cell grafting, animals were allocated to groups by stratified randomization based on body weights. Animals were assigned to either the vehicle group (n = 45) or the cell-graft group (n = 45). There were no exclusion criteria for dosing surgery other than appearing healthy enough. Moribund animals or animals found dead were replaced (up to 30 days post injury). The day prior to grafting the animals were anesthetized to remove any remaining skin sutures and to shave the back of the animals. The spinal cord was re-exposed. The dorsal aspect of the vertebra immediately caudal to the existing laminectomy was removed using the dental drill. Special care was taken to keep the dura intact. For the current safety assessment, a maximum feasible cell dose was estimated by several preliminary dose-range finding studies using subcutaneous and intraspinal delivery. The cell suspension (Group A) or vehicle/hibernation buffer-only (Group B) was injected as follows: 1) 20 injections peripheral from the injury epicenter (1 μL each; 2 μL/min) were made bilaterally into about 2 segments above and/or below the injury epicenter at 1mm intervals.

2) 5 injections (5 μL each; 2 μL/min) were injected around the borders of injury epicenter. One week following SCI surgeries, prior to cell grafting, animals were allocated to groups by stratified randomization based on body weights. Animals were assigned to either the vehicle group (n = 45) or the cell-graft group (n = 45). There were no exclusion criteria for dosing surgery other than appearing healthy enough. Moribund animals or animals found dead were replaced (up to 30 days post injury). The day prior to grafting the animals were anesthetized to remove any remaining skin sutures and to shave the back of the animals. The spinal cord was re-exposed. The dorsal aspect of the vertebra immediately caudal to the existing laminectomy was removed using the dental drill. Special care was taken to keep the dura intact. For the current safety assessment, a maximum feasible cell dose was estimated by several preliminary dose-range finding studies using subcutaneous and intraspinal delivery. The cell suspension (Group A) or vehicle/hibernation buffer-only (Group B) was injected as follows:

A total of 25 injections were made in each spinal cord, resulting in a total of 45 μL (cells suspended at 104 cells/μL, resulting in a total of ∼4.5 ⨯ 105 cells).

The required volume of dosing for completing all injections of one animal was drawn into a 100 μL Nanofil syringe (World Precision Instruments) with a 33-gauge needle (World Precision Instruments, Cat# NF33BV). The syringe was mounted onto a manually controlled syringe holder/injector (David Kopf, Model 5000+5001) attached to the stereotaxic frame. The needle tip was lowered to a depth of 1-1.5 mm from the pial surface at the Dorsal Root Entry Zone (DREZ) into the spinal parenchyma, and the injection was made. After a thirty second pause, the needle was gently drawn out of the spinal cord. The syringe/needle ensemble was cleaned by repeatedly rinsing with and immersion in 70% isopropanol/water solution for a minimum of 15 minutes before and after each animal.

GFP labeling of descending motor tracts: A small group of SCI animals (n = 8; 4 with cell treatment and 4 vehicle-injected), was used to study the innervation of grafted cells by descending motor axons of the host. To label descending motor tracts animals were anesthetized and received four brain injections of recombinant Adeno-Associated Virus (AAV) engineered to express Green Fluorescent Protein under the human synaptophysin promoter (AAV.synGFP; neuronal specific expression). Bilaterally injected vectors were targeted into motor cortex and nucleus ruber. Stereotaxic coordinates used for the injections were: motor cortex: bregma −2.0mm, lateral 3.0mm, depth 1.5 mm, red nucleus: bregma −5.8, lateral 0.8mm, depth 7.1 mm. Each injection consisted of 5 μL of the virus suspension at 1013 gc/mL and was injected over 5 min period using 32G stainless steel needle. Vector was injected 4-6 weeks before sacrifice.

Post-surgical care: All animals received Sulfamethoxazole and Trimethoprim oral suspension, (USP 200 mg/40 mg per 5 mL) in the water (5 mL per 250 mL) for 1-2 days before, up to 30 days after initial contusion, and as needed in case of infections. Animals were checked for bladder retention at least two times daily for the duration of the study. If full, bladders were emptied by manual expression. Consecutively, the surgical area was checked. Ketoprofen (4.0 mg/kg, s.c.) was given approximately one hour prior to surgery and approximately every 24 hours for up to 48 hours. Animals found ill were given up to 5 mL (s.c., ≤ 2 daily) Lactated Ringer’s solution until improvement was observed. Hexa-Caine Spray and/or Bacitracin/Neomycin/Polymyxin triple antibiotic ointment was used to treat scabs or wounds.

Cells: The cells, named “NSI-566RSC,” were produced by Neuralstem Inc. (Rockville, MD, USA), as described before ( Johe et al., 1996 Johe K.K.

Hazel T.G.

Muller T.

Dugich-Djordjevic M.M.

McKay R.D. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. 4 cells/μL. Each batch (and vehicle) was prepared at Neuralstem Inc. (Rockville, MD, USA) one day before surgery and shipped by overnight service in temperature monitored containers. The cells, named “NSI-566RSC,” were produced by Neuralstem Inc. (Rockville, MD, USA), as described before (). The grafted cells were a clinical lot (Lot No. CRL471925-3, passage 12), a human neural stem cell line derived from fetal spinal cord of 8 gestational weeks. They were suspended in hibernation buffer at 10cells/μL. Each batch (and vehicle) was prepared at Neuralstem Inc. (Rockville, MD, USA) one day before surgery and shipped by overnight service in temperature monitored containers.

Post injury health observations, measurements, and specimens: General Health Observations (GHOs) were performed on the dosing day, pre-dose, post-dose, and weekly thereafter. GHOs included weight, general appearance, stool appearance, toxicity symptoms and other appearing additional health issues. In addition, tumor presence was assessed by full body palpation. Any palpable cell mass was measured using a caliper. All animals were observed daily to ensure no loss of animals for necropsy.

Behavioral testing: Behavioral testing was performed under red light or dark conditions. Open field locomotor rating: Locomotor recovery after spinal cord contusion injury was monitored using the Basso, Beattie, and Bresnahan (BBB) open field locomotor rating scale. In the present study, the BBB score was obtained weekly until eight weeks post-injury, biweekly up until week 20, and every four weeks thereafter. Each examination was conducted by two experienced examiners and during five minutes. The animals selected for BBB testing comprised of the last cohorts of 20 animals in both therapeutic groups (n = 40 total).

Additional tests, performed only once near the end of the study: Additional behavioral testing was conducted on animals showing weight support during the last BBB assessments. Behavioral testing was conducted approximately 1.5 months prior to sacrifice. For training/testing, animals were transferred to the testing room in their original housing and allowed to habituate to the room for 30 minutes, during which white noise was generated. The following behavioral tests were conducted:

Activity assessment in an open field: One week prior to the beginning of the test trials, animals were adapted to the apparatus/arenas (76 × 152 × 50 cm; W × L × H) for five minutes on each day. After habituation, the animals were placed in the center of a clean arena and tracked by a video tracking system under dark conditions (night/active portion of their diurnal rhythm), over a period of three hours. The distance traveled and a number of various movement variables was assessed (EthoVision, Noldus Technology, the Netherlands). Three separate observation periods were evaluated. Each observation period was separated by at least 24 hours. During testing, animals had access to Hydrogel and food pellets.

Beam walk: Rats were trained to traverse elevated (1 m) narrow beams (3, 2.5, and 2 × 200 inches; W × L) toward a darkened goal box (10 × 10 × 10 inches). Only motor impaired animals would produce foot faults (slips from beam), which number than represent a motor deficit. Bright light and white noise were produced near the start to promote beam crossing. Rats were allowed to fall from the beam (onto a container with padding). For training, each rat was placed in goal box for two minutes and afterward placed on the balance beam for 1 minute per trial. The rat was considered trained when it was able to remain on the beam for three consecutive trials. Mean scores out of two trials for each beam were used (i.e., right and left hind paw foot faults and falls). The animal was allowed to take ∼5 min to complete a crossing. In between runs an animal was placed in their home cage for ≥ 15 s. If an animal was able to cross a beam for at least two thirds, it was also tested on the narrower beam (i.e., 2.5 or 2 inch).

Catwalk gait analysis: The CatWalk apparatus (CatWalk 7.1, Noldus Technology, the Netherlands) was used to quantify gait parameters by footprint analysis during walkway crossings. Animals walk down a horizontal glass walkway (109 × 15 × 0.6 cm; L × W × H), of which the glass is illuminated along the long edge. The light only enters the (side of the) glass and reflects merely internally (when the glass is bordered by air). As an animal crosses the walkway, light reflects off of the animal’s paws, producing a series of bright footprints when viewed through the glass, which are recorded by a video camera. Hence, testing was performed in a darkened room. Walkway crossing was stimulated by rewarding the animal with little Fruit Crunchies, placed at the end of the walkway. In conjunction, animals were further motivated by food restriction prior to training/testing. Hence, food was removed from their cages approximately 18 hours before testing (and returned after training/testing). In addition, the following criteria concerning walkway crossings needed to be met: (1) the animal needed to walk uninterrupted across the walkway, at a constant pace, and (2) a minimum of three such crossings per animal were required. Data from three proper crossings was averaged for statistical analysis.

Sensory function assessment (mechanical and thermal): In this test, pain thresholds for a supraspinal response (vocalization or escape behavior) to a below-level evoked mechanical or thermal stimulus were assessed. The animals were habituated to the set-up, test room, and investigator for one week (no responses were elicited), twice daily for five minutes, prior to testing. The investigator held the animal in an upright position, fixed the tested hindpaw, and then the hindpaw was stimulated. When a response was elicited, the stimulus was stopped immediately and the maximal pressure elicited was recorded. Both hind paws were tested for four times while alternating between paws, with at least a 1-hour interval between trials. The first scores of each paw were removed.

The mechanical stimulus was created using a rigid tip mounted on a pressure transducer (IITC life sciences Electronic von Frey Anesthesiometer, Cat# 2393, Woodland Hills, CA), which was operated by a second investigator (SvG). The stimulus was applied on the dorsal side of a hind paw. Compressing the paw of the animal with the tip on the pressure transducer at the distal metatarsal or metacarpal area in a gradual incremental fashion (paw rests on table surface).

For eliciting the thermal stimulus an infrared beam apparatus (Ugo Basile, Cat# 37360, Collegeville, PA, USA) was used. For testing, the investigator held the animal’s plantar side of a hind paw over the infrared light beam mounted in the apparatus.

Necropsy: The post-grafting survival period varied between 272-274 days (∼10 months). Animals were sacrificed by 2 mg pentobarbital and 0.25mg phenytoin (0.5mL of Beuthanasia-D, Intervet/Schering-Plough Animal Health Corp., Union, NJ, USA) followed by transcardial perfusion of saline, trailed by 4% paraformaldehyde (phosphate buffered). The central nervous system tissue was dissected out and preserved in 4% paraformaldehyde. The following tissues were also taken out and placed in 10% formalin: skeletal muscle (thigh), skin, adrenals, aorta, rectum, cecum, colon, duodenum, esophagus, epididymides, eyes with optic nerves, femur, Harderian gland, heart, ileum, jejunum, stomach, kidneys, spleen, liver, lung with bronchi, mesenteric lymph node, mammary gland, pancreas, thymus, thyroid with parathyroid glands, trachea, pituitary, prostate, mandibular salivary glands, tongue, sciatic nerve, testes, seminal vesicles, and bladder.

Animals found clinically ill and moribund were euthanized, per protocol. On these animals, and all animals found dead, a gross necropsy was conducted (i.e., external surfaces and orifices, musculoskeletal system, cranial cavity (and brain), neck with associated tissues, and thoracic, abdominal and pelvic cavities (incl. associated organs and tissues).

At necropsy, any tissue with discoloration, lesion, necrosis, distension, malformation, and/or suspected tumor/mass were sent for external histopathological evaluations by a board-certified veterinary pathologist (J.E. Sagartz DVM PhD DACVP, Seventh Wave Laboratories LLC, Chesterfield, MO), as were the histological sections of the central nervous system. Evaluation was performed in a manner blinded to treatment nature. Severity grades for pathology diagnoses were based on a 4-point scale as follows: Grade 1 = minimal, Grade 2 = moderate, Grade 3 = marked and Grade 4 = severe. After completion of pathology analysis, the pathologist was unblinded and the final conclusion was made.

Central nervous system histology: Each cord was photographed against a ruler (cm) along with its identification number. The cords were bisected into approximately equal rostral and caudal pieces and embedded horizontally into a gelatin block (6.5 × 4.5 × 2.5 cm L × W × H). Spinal cords from 16 - 20 animals were embedded in 4 horizontal layers in a block 1. The rostral pieces were placed in the first and third layers and the caudal pieces were in the second and fourth layers, with the ventral side facing down. The layers were separated by ± 0.4 cm of gelatine. The cords (columns) were separated by ± 0.2 cm from each other. After solidification of the gelatin block, it was placed in 30% sucrose in 4% formaldehyde for 48 hours. Then, it was frozen in cold 2-methylbutane using dry ice, cryostat sectioned horizontally (40μm thick), and mounted with intervals of 12 sections. Whole brains were also embedded into gelatin blocks, cryostat sectioned coronally at 40 μm thickness. All stained slides were sent to a Board-certified veterinary pathologist for evaluation (J.E. Sagartz DVM PhD DACVP, Seventh Wave Laboratories LLC, Chesterfield, MO). For histopathological stainings every 24th section was used. The spinal cords used to assess the neuronal integration (see below) were not gelatin embedded but were frozen directly (after 48h in 30% sucrose) using isopentane (−80°C), mounted in OCT compound (Tissue-Tek®) and cryostat sectioned at 30 μm in the horizontal, sagittal, or transverse plane, and collected for free-floating immunofluorescence staining.

Stainings used entailed: hematoxylin & eosin (H&E), human axonal neurofilament antibody (h.HO14; rat 1:100; human specific axonal marker; gift from Dr. Virginia Lee, University of Pennsylvania, PA, USA), Glial Fibrillary Acidic Protein antibody (h.GFAP; rabbit 1:500; human specific astroglial marker; Origene, Rockville, MD, USA), and Green Fluorescent Protein antibody (GFP; chicken 1:2000; Aves, Tigard, OR, USA), Neuron-Specific Enolase antibody (h.NSE; mouse 1:500; human specific neuronal marker; Vector Labs, Burlingame, CA, USA) Synaptophysin antibody (h.Syn; mouse 1:500; human specific synaptic marker; Millipore, Billerica, MA, USA), Human Nucleus antibody (h.HuMA; mouse 1:200; Millipore, Billerica, MA, USA), Glial Fibrillary Acidic Protein antibody (GFAP; mouse 1:500; Sigma-Aldrich; St. Louis, MO, USA), Oligodendrocyte lineage transcription factor 2 antibody (Olig2; rabbit 1:1000; Abcam ab81093; Cambridge, MA, USA), Ki67 antibody (proliferation marker; Abcam ab16667; 1:100; rabbit), and DAPI (In Prolong®; Life Technologies, Carlsbad, CA, USA). Immunostainings were finished using fluorescent-conjugated secondary donkey antibodies (Alexa® Fluor 488 & 647; Jackson Immuno Research, West Grove, PA, USA; & Alexa® Fluor 555; Invitrogen; 1:500).

Surgical and Cell-Grafting Procedure Tadesse et al., 2014 Tadesse T.

Gearing M.

Senitzer D.

Saxe D.

Brat D.J.

Bray R.

Gebel H.

Hill C.

Boulis N.

Riley J.

et al. Analysis of graft survival in a trial of stem cell transplant in ALS. The intervention included placing an anesthetized subject in the prone position and sterile processing of the associated surgical trial materials. An approximately 10 - 15 cm incision was performed in the dorsal midline and a bilateral laminectomy performed over the injured spinal cord segments. All prior fusion hardware was removed during the surgical procedure to allow for optimum serial magnetic resonance imaging (MRI). Following laminectomy an incision of approximately 2-4 cm was made in the dura, which was then tacked up, allowing exposure of the spinal cord. The stereotaxic injection platform was then attached to 4 percutaneous posts through an approximately 1cm skin incision immediately above and below the laminectomy site ( Figure 5 E), (). The injection device consisted of a Z-drive holding a 30-gauge beveled needle in perpendicular position over the exposed spinal cord. The top end of the needle was attached to tubing which was attached to a microprocessor-controlled syringe pump. The syringe is back-filled with mineral oil to eliminate air and to create an immiscible barrier against aqueous solution in the syringe. The syringe plunger is inserted into the syringe, pushed toward the end, and attached to the drive spindle of the injection pump. Separately, the injection cannula is manually filled with sterile injectable saline in order to eliminate air. The saline-filled cannula is attached to the Hamilton syringe. Using the injection pump in reverse, a small air space is created at the cannula tip to prevent mixing of the cell suspension with the saline. Using the injection pump in reverse, the cannula is loaded with the required volume of the cell suspension. The capacity of the cannula is at least 500 μL so that the injection volume never touches the tip of the syringe. Prior to initiating spinal cord injections, a 5 μL bolus (1 injection) is ejected under direct vision of the surgeon to ensure that the system is open and unobstructed. After the needle is inserted into the spinal cord, the guide sheath is retracted, converting the cannula into a “floating cannula.” This feature allows for accuracy of delivery without suspension of respiration and other facets of homeostasis, including blood pressure and other vital signs. This setup is different as compared to other injection devices that are fixed to the operating room bed and require suspension of ventilation to ensure proper injection. Bilateral injection positions were determined by preoperative MRI and target approximately 1mm lateral to the rim of the remaining tissue bordering the injury site. The needle was lowered into the spinal cord to the depth of approximately 4 mm from the pial surface ( Figures 5 D and 5F). The cell suspension was then injected using the syringe pump at flow rate of 5.0 μL per min for a period of 2 minutes. The needle was left in place for 1 min after injection and then slowly pulled out of the cord, advanced to the next position along the cord avoiding visible blood vessels and the injection procedure was repeated. At this time once all injections had been completed the dura was then closed in a watertight fashion, and the posterior spinal fascia and skin closed in meticulous layers. Subjects were then extubated and recovered in a post-anesthesia care unit, followed by recovering in an intermediate level care unit of the acute care hospital.

Immunosuppression Glass et al., 2012 Glass J.D.

Boulis N.M.

Johe K.

Rutkove S.B.

Federici T.

Polak M.

Kelly C.

Feldman E.L. Lumbar intraspinal injection of neural stem cells in patients with amyotrophic lateral sclerosis: results of a phase I trial in 12 patients. Tadesse et al., 2014 Tadesse T.

Gearing M.

Senitzer D.

Saxe D.

Brat D.J.

Bray R.

Gebel H.

Hill C.

Boulis N.

Riley J.

et al. Analysis of graft survival in a trial of stem cell transplant in ALS. All 4 subjects were initiated and maintained for 12 weeks on a combination cocktail of immunosuppressive (IS) regimen that had previously been used successfully in ALS trials with the same cell line (). This included the use of three separate medications. Basiliximab (Simulect) 20 mg intravenous (IV) administered within 2 hours prior to transplantation surgery. A second dose of 20 mg was given on postoperative Day 3 or 4. No additional doses of Basiliximab were used. Tacrolimus was started on post-transplant Day 1. It was initially dosed at 0.1 mg/kg/day divided every 12 hours by mouth (PO). Trough levels were measured while the subjects were hospitalized and the dose of tacrolimus was adjusted as necessary to ensure maintenance of a trough serum level of 4-8 ng/ml. Following discharge, trough levels were measured at the 2-week post-operative visit and at scheduled visits thereafter. Mycophenolate mofetil was started on post-transplant Day 1 at 500 mg twice a day, on post-transplant Day 8 increased to 500mg in the morning and 1 g at night, and on post-transplant Day 15 increased to 1 g twice a day. In all 4 subjects Tacrolimus and mycophenolate mofetil were withdrawn after 12 weeks post-transplantation. The dose of both medications was reduced by half at Week 13 and by another half at Week 14, followed by complete cessation at Week 15. Presence of antibodies against donor HLAs were monitored during this period and at scheduled intervals thereafter. Because assessment of rejection is an efficacy metric rather than a safety metric, more comprehensive immunological workup thoroughly assessed in the Phase 2 study, including complement and interleukin levels. Changes in MRI intensity at the cell transplant area were also monitored before and after the IS withdrawal. All subjects tolerated the immunosuppressive regimen well. Subjects were monitored on a nearly weekly basis per our study protocol. See Methods S2

Outcome Measures Lee et al., 2004 Lee D.C.

Lim H.K.

McKay W.B.

Priebe M.M.

Holmes S.A.

Sherwood A.M. Toward an objective interpretation of surface EMG patterns: a voluntary response index (VRI). Sherwood et al., 1996 Sherwood A.M.

McKay W.B.

Dimitrijević M.R. Motor control after spinal cord injury: assessment using surface EMG. Subjects were assessed for adverse events including pain and infection, motor function, and quality of life. Additional secondary outcome assessments were made to measure any postoperative changes from baseline in neurologic deficits, neurophysiology, imaging studies, bladder and bowel function, allodynia and neuropathic pain. ISNC-SCI (International Standards for the Neurological Classification of Spinal Cord Injury) examination was used to monitor neurologic deficits. Neurophysiological changes were monitored when feasible by needle electromyography (EMG) and/or surface poly-electromyography (Brain Motor Control Assessment (BMCA) (). Imaging studies were done by standard 1.5T MRI for safety monitoring. Diffusion tensor imaging (DTI) imaging of the spinal cord was performed for longitudinal experimental studies (1.5T, TR 2500-5000ms, TE 64-95ms, slice thickness 3.5-4 mm, FA 90, DFOV 190-340, NSA 1-8). Bladder and Bowel Function and Pain and Allodynia questionnaires were administered. Quality of life was assessed by Functional Independence Measure (FIM) and Spinal Cord Independence Measure (SCIM) questionnaires. Subjects were followed postoperatively at 2 weeks, monthly for 6 months and at every 6 months thereafter in post-study safety are planned to be followed up for total 60 months post stem cell treatment. Patients did not receive any additional rehabilitation beyond their routine outpatient physical and occupational therapy