Source Paper
Source Paper
Ryan P. Salewski, Robert A. Mitchell, Lijun Li, Carl Shen, Maria Milekovskaia et al.
Stem Cells Translational Medicine • 2015
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.
Objective: Assessment of neurobehavioral function and motor recovery following thoracic spinal cord injury using standardized locomotor scoring with the Basso Mouse Scale
This is a Basso Mouse Scale protocol using mouse as the model organism. The procedure involves 9 procedural steps, 3 equipment items, 2 materials. Extracted from a 2015 paper published in Stem Cells Translational Medicine.
Model and subjects
mouse • Not specified in provided text • unknown • Not specified in provided text • Not specified in provided text
Study window
Estimated timing pending
Core workflow
Generate iPS cells using piggyBac transposon approach • Generate committed neural stem cells from iPS cells • Validate iPS-NSC lines
Primary readouts
Key equipment and reagents
Verified items
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Create induced pluripotent stem cells using nonviral piggyBac transposon methodology
Note: This is the foundational step for generating neural stem cells
“Definitive NSCs derived from induced pluripotent stem (iPS) cells generated using a nonviral, piggyBac transposon approach”
Derive committed NSCs from iPS cells using free-floating neurosphere methodology
Note: Two cell lines were used: wildtype (wt) and nonmyelinating Shiverer (shi) iPS cell lines
“Committed NSCs were generated from iPS cells using a free-floating neurosphere methodology previously described by our laboratory”
Validate and characterize wt and shi iPS-NSC lines to confirm equivalence except in myelination capacity
Note: Both cell lines must be validated before transplantation
“The wt- and shi- iPS-NSCs were validated and shown to be equivalent except in myelination capacity”
Create thoracic spinal cord injury in mice
Note: Injury location is thoracic region
“NSCs derived from wildtype ( wt ) and nonmyelinating Shiverer ( shi ) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation”
Transplant wt-iPS-NSCs, shi-iPS-NSCs, or perform cell-free control injection into injured spinal cord during subacute phase
Note: Three treatment groups: wt-iPS-NSCs, shi-iPS-NSCs, and cell-free controls
“NSCs derived from wildtype ( wt ) and nonmyelinating Shiverer ( shi ) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation”
Perform standardized locomotor scoring using the Basso Mouse Scale to measure motor recovery and neurobehavioral function
Note: Primary behavioral outcome measure for assessing functional recovery
“significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk)”
Perform gait analysis and motor function assessment using CatWalk system
Note: Secondary behavioral outcome measure
“significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk)”
Conduct histological examination of spinal cord tissue to assess graft integration and differentiation
Note: Histological outcomes were analyzed as part of comprehensive assessment
“Both iPS-NSC lines successfully integrated into the injured spinal cord and predominantly differentiated to oligodendrocytes”
Conduct sucrose gap analysis to measure electrophysiological measures of axonal function
Note: Electrophysiological outcomes assess functional axonal integrity
“electrophysiological measures of axonal function (sucrose gap analysis)”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Assessment of neurobehavioral function and motor recovery following thoracic spinal cord injury using standardized locomotor scoring with the Basso Mouse Scale
Objective
Assessment of neurobehavioral function and motor recovery following thoracic spinal cord injury using standardized locomotor scoring with the Basso Mouse Scale
Subjects
From papermouse • Not specified in provided text • unknown • Not specified in provided text • Not specified in provided text
Cohort notes
From paperStudy used wildtype (wt) and nonmyelinating Shiverer (shi) iPS cell-derived neural stem cells
Generate iPS cells using piggyBac transposon approach (Not specified)
Generate committed neural stem cells from iPS cells (Not specified)
Validate iPS-NSC lines (Not specified)
Perform thoracic spinal cord injury (Not specified)
Neurobehavioral function assessed by Basso Mouse Scale
From paperNot specified in provided text
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
Motor function assessed by CatWalk gait analysis
From paperNot specified in provided text
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
Histological outcomes including graft integration and oligodendrocyte differentiation
From paperNot specified in provided text
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
Electrophysiological measures of axonal function via sucrose gap analysis
From paperNot specified in provided text
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
Neurobehavioral function assessed by Basso Mouse Scale
From paperRaw 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
Final reported form
Group comparisons of gait indices, stride metrics, or recovery curves
Motor function assessed by CatWalk gait analysis
From paperRaw 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
Final reported form
Group comparisons of gait indices, stride metrics, or recovery curves
Histological outcomes including graft integration and oligodendrocyte differentiation
From paperRaw 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
Final reported form
Group comparisons of gait indices, stride metrics, or recovery curves
Electrophysiological measures of axonal function via sucrose gap analysis
From paperRaw 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
Final reported form
Group comparisons of gait indices, stride metrics, or recovery curves
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
Preprocessing / cleaning
Not specified in provided text
Scoring or quantification
Quantify the primary readouts for this experiment: Neurobehavioral function assessed by Basso Mouse Scale; Motor function assessed by CatWalk gait analysis; Histological outcomes including graft integration and oligodendrocyte differentiation; Electrophysiological measures of axonal function via sucrose gap analysis.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Neurobehavioral function assessed by Basso Mouse Scale, Motor function assessed by CatWalk gait analysis, Histological outcomes including graft integration and oligodendrocyte differentiation, Electrophysiological measures of axonal function via sucrose gap analysis.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Ryan P. Salewski et al. (2015). Transplantation of Induced Pluripotent Stem Cell-Derived Neural Stem Cells Mediate Functional Recovery Following Thoracic Spinal Cord Injury Through Remyelination of Axons. Stem Cells Translational Medicine
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