Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal coRd Injury NOD- scid Mouse Model methods
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Methods and Findings
hCNS-SCns were transplanted into immunodeficient NOD- scid mice 30 days post spinal cord contusion injury. hCNS-SCns transplanted mice demonstrated significantly improved locomotor recovery compared to vehicle controls using open field locomotor testing and CatWalk gait analysis. Transplanted hCNS-SCns exhibited long-term engraftment, migration, limited proliferation, and differentiation predominantly to oligodendrocytes and neurons. Astrocytic differentiation was rare and mice did not exhibit mechanical allodynia. Furthermore, differentiated hCNS-SCns integrated with the host as demonstrated by co-localization of human cytoplasm with discrete staining for the paranodal marker contactin-associated protein.
Group design
Female mice were used in this study to avoid bladder complications and urolithiasis that frequently occur in male mice following SCI,. Animal surgeries were done in parallel over multiple days. Prior to surgery, NOD- scid mice (n = 48) were tested by the Basso Mouse Scale (BMS) to ensure normal locomotor function. One animal was excluded at this time because of abnormal gait. During the initial SCI surgery, one animal died and three additional animals were excluded because of surgical error. Pre-hoc criteria were established to exclude animals if any of the following conditions occurred: cord bruised or tearing of dura during laminectomy (n = 1); visual slippage of the mouse from the vertebral stabilization clamps (n = 0); abnormal time versus force curve indicating a bone hit or clamp slip (n = 1); or unilateral bruising of the cord (...
Spinal Cord Injury
Female NOD- scid mice (transferred from StemCells Inc, the Jackson Laboratory, stock # 001303), 8-10 weeks of age were anesthetized with tribromoethanol (312.5 mg/kg i.p. bolus) and vertebral T9 exposed by laminectomy as previously described. Mice received a 50-kDyne-contusion injury using the IH device (Precision Systems and Instrumentation LLC). After surgery, mice recovered overnight in cages with Alpha-Dri bedding (Newco Distributors Inc) placed on water-jacketed heating pads at 37°C. Mice were maintained on twice daily manual bladder expression for 2 weeks, followed by once daily manual bladder expression for the remaining survival period. Post-operative care included buprenorphine twice a day for 2 days, lactated ringers once a day for 4 days, and antibiotic daily for the duration of the study. All animals were maintained on rotating schedule of antibiotics; Baytril,...
Transplantation surgery
Isolation and culture of hCNS-SCns from fetal brain tissue (gestational 16-20 weeks) have been previously described,,. Briefly, FACS-sorted single cell suspensions were cultured in neurosphere initiation media consisting of Ex-Vivo 15 media with N2 supplement, FGF, EGF, LIF, neural survival factor-1, and NAC. Cells were propagated as neurospheres, fed weekly, and passaged every 2-3 weeks,,. Human fibroblasts derived from fetal liver were grown to confluence in Iscove's modified Dulbecco's medium/10% FBS, dissociated with trypsin, washed and concentrated to 50,000 cells per µl. Thirty days post-injury, mice were anesthetized with tribromoethanol (312.5 mg/kg i.p. bolus), the laminectomy site re-exposed and hCNS-SCns or hFbs were transplanted. Four injections of 250 nl each, bilateral from the midline, both rostral and caudal to the injury epicenter were performed...
BMS
Open-field locomotor recovery was assessed using the BMS locomotor rating scale prior to injury, 2 days after injury, weekly for 4 weeks until transplantation, then weekly following transplantation until sacrifice. Briefly, mice were observed in the open-field for 4 minutes each by two individuals blinded to the experimental groups,. Motor function of the hind limbs was rated, recorded, and converted to a score according to the published scale.
CatWalk Analysis
CatWalk video was recorded at 16 weeks post-transplantation. Individuals blinded to experimental groups analyzed video using CatWalk software version 6.13 for Windows.
Mechanical Allodynia
Von Frey hair testing of hind limbs was done at 15 weeks post-transplantation. Animals were tested for a withdrawal response to successively higher force filaments (Touch-Test Sensory Evaluators, North Coast Medical). The lowest filament from which an animal withdrew from was the threshold assigned for that animal. After a positive response was elicited, the previous filament was tested to confirm a lack of response and the next filament was tested to confirm a positive response.
Histological Assessment
Sixteen weeks post-transplantation, mice were anesthetized using pentobarbital (100 mg/kg) and transcardially perfused with 15 mls of PBS followed by 100 mls of 4% phosphate-buffered paraformaldehyde. A T6-T12 segment of the spinal cord was dissected based on counting the dorsal spinal roots for all mice to obtain an anatomically consistent region of the spinal cord for stereological analysis. Spinal roots were identified by visualization of the features of C6, C7 and C8 root brachial plexus and exposure of the distal thoracic roots. Dissected T6-T12 spinal cord segments were post-fixed in a 20% sucrose/4% paraformaldehyde solution for cryoprotection overnight at 4°C. Following cryoprotection, spinal cords were frozen in isopentane at -65°C, and serial sliding microtome sections were collected free floating for immunocytochemical staining. Parasagittal sections (n̴...
Measurement outputs
What raw and processed outputs should exist?
Gait recovery and locomotor function
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
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Acquisition
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Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
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Evidence quotes (8)
hCNS-SCns were transplanted into immunodeficient NOD- scid mice 30 days post spinal cord contusion injury. hCNS-SCns transplanted mice demonstrated significantly improved locomotor recovery compared to vehicle controls using open field locomotor testing and CatWalk gait analysis. Transplanted hCNS-SCns exhibited long-term engraftment, migration, limited proliferation, and differentiation predominantly to oligodendrocytes and neurons. Astrocytic differentiation was rare and mice did not exhibit mechanical allodynia. Furthermore, differentiated hCNS-SCns integrated with the host as demonstrated by co-localization of human cytoplasm with discrete staining for the paranodal marker contactin-associated protein.
Female mice were used in this study to avoid bladder complications and urolithiasis that frequently occur in male mice following SCI,. Animal surgeries were done in parallel over multiple days. Prior to surgery, NOD- scid mice (n = 48) were tested by the Basso Mouse Scale (BMS) to ensure normal locomotor function. One animal was excluded at this time because of abnormal gait. During the initial SCI surgery, one animal died and three additional animals were excluded because of surgical error. Pre-hoc criteria were established to exclude animals if any of the following conditions occurred: cord bruised or tearing of dura during laminectomy (n = 1); visual slippage of the mouse from the vertebral stabilization clamps (n = 0); abnormal time versus force curve indicating a bone hit or clamp slip (n = 1); or unilateral bruising of the cord (n = 1). Mice (n = 44) received BMS testing 2 dpi and weekly thereafter and were randomized into 3 balanced groups, to be transplanted with either vehicle control, hFbs, or hCNS-SCns, based upon their BMS scores at 28 dpi. To minimize the effect of variations in spinal cord da...
Female NOD- scid mice (transferred from StemCells Inc, the Jackson Laboratory, stock # 001303), 8-10 weeks of age were anesthetized with tribromoethanol (312.5 mg/kg i.p. bolus) and vertebral T9 exposed by laminectomy as previously described. Mice received a 50-kDyne-contusion injury using the IH device (Precision Systems and Instrumentation LLC). After surgery, mice recovered overnight in cages with Alpha-Dri bedding (Newco Distributors Inc) placed on water-jacketed heating pads at 37°C. Mice were maintained on twice daily manual bladder expression for 2 weeks, followed by once daily manual bladder expression for the remaining survival period. Post-operative care included buprenorphine twice a day for 2 days, lactated ringers once a day for 4 days, and antibiotic daily for the duration of the study. All animals were maintained on rotating schedule of antibiotics; Baytril, Amoxicillin, and Cipro were each given for 2 weeks, and then rotated to the next antibiotic.
Isolation and culture of hCNS-SCns from fetal brain tissue (gestational 16-20 weeks) have been previously described,,. Briefly, FACS-sorted single cell suspensions were cultured in neurosphere initiation media consisting of Ex-Vivo 15 media with N2 supplement, FGF, EGF, LIF, neural survival factor-1, and NAC. Cells were propagated as neurospheres, fed weekly, and passaged every 2-3 weeks,,. Human fibroblasts derived from fetal liver were grown to confluence in Iscove's modified Dulbecco's medium/10% FBS, dissociated with trypsin, washed and concentrated to 50,000 cells per µl. Thirty days post-injury, mice were anesthetized with tribromoethanol (312.5 mg/kg i.p. bolus), the laminectomy site re-exposed and hCNS-SCns or hFbs were transplanted. Four injections of 250 nl each, bilateral from the midline, both rostral and caudal to the injury epicenter were performed using a beveled glass (0.53 mm I.D, 1.14 mm O.D. Drummond Scientific Co.) micropipette (75-80 µm ID, 100-115 µm, 30° bevel Sutter Instrument Co.) and NanoInjector 2000 system (World Precision Instruments) delivering a total of 1 µl of hCNS-SCns at 75,000-cells/µ...
Open-field locomotor recovery was assessed using the BMS locomotor rating scale prior to injury, 2 days after injury, weekly for 4 weeks until transplantation, then weekly following transplantation until sacrifice. Briefly, mice were observed in the open-field for 4 minutes each by two individuals blinded to the experimental groups,. Motor function of the hind limbs was rated, recorded, and converted to a score according to the published scale.
CatWalk video was recorded at 16 weeks post-transplantation. Individuals blinded to experimental groups analyzed video using CatWalk software version 6.13 for Windows.
Von Frey hair testing of hind limbs was done at 15 weeks post-transplantation. Animals were tested for a withdrawal response to successively higher force filaments (Touch-Test Sensory Evaluators, North Coast Medical). The lowest filament from which an animal withdrew from was the threshold assigned for that animal. After a positive response was elicited, the previous filament was tested to confirm a lack of response and the next filament was tested to confirm a positive response.
Sixteen weeks post-transplantation, mice were anesthetized using pentobarbital (100 mg/kg) and transcardially perfused with 15 mls of PBS followed by 100 mls of 4% phosphate-buffered paraformaldehyde. A T6-T12 segment of the spinal cord was dissected based on counting the dorsal spinal roots for all mice to obtain an anatomically consistent region of the spinal cord for stereological analysis. Spinal roots were identified by visualization of the features of C6, C7 and C8 root brachial plexus and exposure of the distal thoracic roots. Dissected T6-T12 spinal cord segments were post-fixed in a 20% sucrose/4% paraformaldehyde solution for cryoprotection overnight at 4°C. Following cryoprotection, spinal cords were frozen in isopentane at -65°C, and serial sliding microtome sections were collected free floating for immunocytochemical staining. Parasagittal sections (n = 6 per group) were collected at 30 µm for human cytoplasm and paranodal protein (CASPR) double immunofluorescence and stereological analysis of hCNS-SCns and hFbs survival and migration, lesion volume, spared tissue volume, and glial scar area. Coronal sections (n = 3 per g...
Machine-readable layer
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