Objective: Assessment of neurobehavioral function and motor recovery following thoracic spinal cord injury using standardized locomotor scoring with the Basso Mouse Scale
Materials & Equipment Checklist
6 items1 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment3
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Protocol Steps
View Abstract
Abstract Neural stem cells (NSCs) from embryonic or fetal/adult tissue sources have shown considerable promise in regenerative strategies for traumatic spinal cord injury (SCI). However, there are limitations with their use related to the availability, immunogenicity, and uncertainty of the mechanisms involved. To address these issues, definitive NSCs derived from induced pluripotent stem (iPS) cells generated using a nonviral, piggyBac transposon approach, were investigated. Committed NSCs were generated from iPS cells using a free-floating neurosphere methodology previously described by our laboratory. To delineate the mechanism of action, specifically the role of exogenous myelination, NSCs derived from wildtype (wt) and nonmyelinating Shiverer (shi) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation. Behavioral, histological, and electrophysiological outcomes were analyzed to assess the effectiveness of this treatment. The wt- and shi-iPS-NSCs were validated and shown to be equivalent except in myelination capacity. Both iPS-NSC lines successfully integrated into the injured spinal cord and predominantly differentiated to oligodendrocytes, but only the wt-iPS-NSC treatment resulted in a functional benefit. The wt-iPS-dNSCs, which exhibited the capacity for remyelination, significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk), histological outcomes, and electrophysiological measures of axonal function (sucrose gap analysis) compared with the nonmyelinating iPS-dNSCs and cell-free controls. In summary, we demonstrated that iPS cells can generate translationally relevant NSCs for applications in SCI. Although NSCs have a diverse range of functions in the injured spinal cord, remyelination is the predominant mechanism of recovery following thoracic SCI. Significance Gain-of-function/loss-of-function techniques were used to examine the mechanistic importance of graft-derived remyelination following thoracic spinal cord injury (SCI). The novel findings of this study include the first use of neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) derived using the clonal neurosphere expansion conditions, for the treatment of SCI, the first characterization and in vivo application of iPSCs from Shiverer mouse fibroblasts, and the first evidence of the importance of remyelination by pluripotent-sourced NSCs for SCI repair and regeneration.
1
Generate iPS cells using piggyBac transposon approach
Create induced pluripotent stem cells using nonviral piggyBac transposon methodology
Not specifiedNot specified
Note: This is the foundational step for generating neural stem cells
View evidence from paper
“Definitive NSCs derived from induced pluripotent stem (iPS) cells generated using a nonviral, piggyBac transposon approach”
2
Generate committed neural stem cells from iPS cells
Derive committed NSCs from iPS cells using free-floating neurosphere methodology
Not specifiedNot specified
Note: Two cell lines were used: wildtype (wt) and nonmyelinating Shiverer (shi) iPS cell lines
View evidence from paper
“Committed NSCs were generated from iPS cells using a free-floating neurosphere methodology previously described by our laboratory”
3
Validate iPS-NSC lines
Validate and characterize wt and shi iPS-NSC lines to confirm equivalence except in myelination capacity
Not specifiedNot specified
Note: Both cell lines must be validated before transplantation
View evidence from paper
“The wt- and shi- iPS-NSCs were validated and shown to be equivalent except in myelination capacity”
4
Perform thoracic spinal cord injury
Create thoracic spinal cord injury in mice
Not specifiedNot specified
Note: Injury location is thoracic region
View evidence from paper
“NSCs derived from wildtype ( wt ) and nonmyelinating Shiverer ( shi ) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation”
5
Perform subacute intraspinal transplantation
Transplant wt-iPS-NSCs, shi-iPS-NSCs, or perform cell-free control injection into injured spinal cord during subacute phase
Not specifiedNot specified
Note: Three treatment groups: wt-iPS-NSCs, shi-iPS-NSCs, and cell-free controls
View evidence from paper
“NSCs derived from wildtype ( wt ) and nonmyelinating Shiverer ( shi ) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation”
6
Assess neurobehavioral function using Basso Mouse Scale
Perform standardized locomotor scoring using the Basso Mouse Scale to measure motor recovery and neurobehavioral function
Not specifiedNot specified
Note: Primary behavioral outcome measure for assessing functional recovery
View evidence from paper
“significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk)”
7
Assess motor function using CatWalk
Perform gait analysis and motor function assessment using CatWalk system
