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: To examine the mechanistic importance of graft-derived remyelination following thoracic spinal cord injury using iPS-derived neural stem cells, comparing wildtype myelinating versus nonmyelinating Shiverer cell lines to determine if remyelination is the predominant mechanism of functional recovery
This is a Spinal Cord Transplantation protocol using mouse as the model organism. The procedure involves 10 procedural steps, 3 equipment items, 4 materials. Extracted from a 2015 paper published in Stem Cells Translational Medicine.
Model and subjects
mouse • Wildtype and Shiverer (nonmyelinating) mouse lines • unknown • Not specified • Not specified
Study window
Estimated timing pending
Core workflow
Generate iPS cells from mouse fibroblasts • Differentiate iPS cells into committed neural stem cells • Validate iPS-NSC lines
Primary readouts
Key equipment and reagents
Verified items
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Create induced pluripotent stem cells from wildtype and Shiverer mouse fibroblasts using nonviral piggyBac transposon approach
Note: Two cell lines generated: wildtype (with myelination capacity) and Shiverer (nonmyelinating)
“Definitive NSCs derived from induced pluripotent stem (iPS) cells generated using a nonviral, piggyBac transposon approach”
Generate committed NSCs from iPS cells using free-floating neurosphere methodology
Note: Methodology previously described by the laboratory; produces both wt-iPS-NSCs and shi-iPS-NSCs
“Committed NSCs were generated from iPS cells using a free-floating neurosphere methodology previously described by our laboratory”
Characterize and validate both wildtype and Shiverer iPS-NSC lines to confirm equivalence except in myelination capacity
Note: Both lines validated and shown to be equivalent except in myelination capacity
“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 type and severity not specified in provided text
“NSCs derived from wildtype (wt) and nonmyelinating Shiverer (shi) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation”
Transplant iPS-derived neural stem cells (both wildtype and Shiverer lines) into the injured thoracic spinal cord at subacute timepoint
Note: Two treatment groups plus cell-free control group; transplantation timing and cell dosage not specified
“NSCs derived from wildtype (wt) and nonmyelinating Shiverer (shi) iPS cell lines were used following thoracic SCI with subacute intraspinal transplantation”
Measure neurobehavioral function and locomotor recovery using the Basso Mouse Scale (BMS)
Note: Timepoints for assessment not specified
“The wt-iPS-dNSCs, which exhibited the capacity for remyelination, significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk)”
Measure gait and locomotor function using CatWalk apparatus
Note: Timepoints for assessment not specified
“The wt-iPS-dNSCs, which exhibited the capacity for remyelination, significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk)”
Conduct histological examination of spinal cord tissue to assess graft integration and differentiation
Note: Both iPS-NSC lines successfully integrated into injured spinal cord and predominantly differentiated to oligodendrocytes
“Both iPS-NSC lines successfully integrated into the injured spinal cord and predominantly differentiated to oligodendrocytes”
Measure axonal function and conduction velocity using sucrose gap analysis
Note: Timepoints for measurement not specified
“Electrophysiological measures of axonal function (sucrose gap analysis) compared with the nonmyelinating iPS-dNSCs and cell-free controls”
Compare neurobehavioral, histological, and electrophysiological outcomes between wildtype iPS-NSC treatment, Shiverer iPS-NSC treatment, and cell-free control groups
Note: Wildtype treatment showed significant functional benefit compared to nonmyelinating and control groups
“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”
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.
To examine the mechanistic importance of graft-derived remyelination following thoracic spinal cord injury using iPS-derived neural stem cells, comparing wildtype myelinating versus nonmyelinating Shiverer cell lines to determine if remyelination is the predominant mechanism of functional recovery
Objective
To examine the mechanistic importance of graft-derived remyelination following thoracic spinal cord injury using iPS-derived neural stem cells, comparing wildtype myelinating versus nonmyelinating Shiverer cell lines to determine if remyelination is the predominant mechanism of functional recovery
Subjects
From papermouse • Wildtype and Shiverer (nonmyelinating) mouse lines • unknown • Not specified • Not specified
Cohort notes
From paperiPS cells generated from wildtype and Shiverer mouse fibroblasts using nonviral piggyBac transposon approach
Generate iPS cells from mouse fibroblasts (Not specified)
Differentiate iPS cells into committed neural stem cells (Not specified)
Validate iPS-NSC lines (Not specified)
Induce thoracic spinal cord injury (Not specified)
Neurobehavioral function (Basso Mouse Scale)
From paperBehavioral, histological, and electrophysiological outcomes were analyzed to assess treatment effectiveness; specific statistical methods not detailed 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
Gait and locomotor function (CatWalk)
From paperBehavioral, histological, and electrophysiological outcomes were analyzed to assess treatment effectiveness; specific statistical methods not detailed 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 integration and differentiation of transplanted cells
From paperBehavioral, histological, and electrophysiological outcomes were analyzed to assess treatment effectiveness; specific statistical methods not detailed 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
Oligodendrocyte differentiation
From paperBehavioral, histological, and electrophysiological outcomes were analyzed to assess treatment effectiveness; specific statistical methods not detailed 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 (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
Gait and locomotor function (CatWalk)
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 integration and differentiation of transplanted cells
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
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
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
Preprocessing / cleaning
Behavioral, histological, and electrophysiological outcomes were analyzed to assess treatment effectiveness; specific statistical methods not detailed in provided text
Scoring or quantification
Quantify the primary readouts for this experiment: Neurobehavioral function (Basso Mouse Scale); Gait and locomotor function (CatWalk); Histological integration and differentiation of transplanted cells; Oligodendrocyte differentiation.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Neurobehavioral function (Basso Mouse Scale), Gait and locomotor function (CatWalk), Histological integration and differentiation of transplanted cells, Oligodendrocyte differentiation.
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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