Source Paper
Transplantation of Induced Pluripotent Stem Cell-Derived Neural Stem Cells Mediate Functional Recovery Following Thoracic Spinal Cord Injury Through Remyelination of Axons
Ryan P. Salewski, et al.
Materials & Equipment Checklist
8 items1 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment3
Materials4
Generated in-house using piggyBac transposon approach • Not specified • Not specified • Not specified
Generated in-house using piggyBac transposon approach • Not specified • Not specified • Not specified
Generated in-house • Not specified • Not specified • Not specified
Generated in-house • Not specified • Not specified • Not specified
Software1
Not specified • Not specified
1 item available from ConductScience
Estimated: $2,610.00
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Protocol Steps
1
Generate iPS cells from mouse fibroblasts
Create induced pluripotent stem cells from wildtype and Shiverer mouse fibroblasts using nonviral piggyBac transposon approach
Not specifiedNot specified
Note: Two cell lines generated: wildtype (with myelination capacity) and Shiverer (nonmyelinating)
View evidence from paper
“Definitive NSCs derived from induced pluripotent stem (iPS) cells generated using a nonviral, piggyBac transposon approach”
2
Differentiate iPS cells into committed neural stem cells
Generate committed NSCs from iPS cells using free-floating neurosphere methodology
Not specifiedNot specified
Note: Methodology previously described by the laboratory; produces both wt-iPS-NSCs and shi-iPS-NSCs
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
Characterize and validate both wildtype and Shiverer iPS-NSC lines to confirm equivalence except in myelination capacity
Not specifiedNot specified
Note: Both lines validated and shown to be equivalent except in myelination capacity
View evidence from paper
“The wt- and shi- iPS-NSCs were validated and shown to be equivalent except in myelination capacity”
4
Induce thoracic spinal cord injury
Create thoracic spinal cord injury in mice
Not specifiedNot specified
Note: Injury type and severity not specified in provided text
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 iPS-derived neural stem cells (both wildtype and Shiverer lines) into the injured thoracic spinal cord at subacute timepoint
Subacute timing (not precisely defined)Not specified
Note: Two treatment groups plus cell-free control group; transplantation timing and cell dosage not specified
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 behavioral outcomes using Basso Mouse Scale
Measure neurobehavioral function and locomotor recovery using the Basso Mouse Scale (BMS)
Not specifiedNot specified
Note: Timepoints for assessment not specified
View evidence from paper
“The wt-iPS-dNSCs, which exhibited the capacity for remyelination, significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk)”
7
Assess behavioral outcomes using CatWalk
Measure gait and locomotor function using CatWalk apparatus
Not specifiedNot specified
Note: Timepoints for assessment not specified
View evidence from paper
“The wt-iPS-dNSCs, which exhibited the capacity for remyelination, significantly improved neurobehavioral function (Basso Mouse Scale and CatWalk)”
8
Perform histological analysis
Conduct histological examination of spinal cord tissue to assess graft integration and differentiation
Not specifiedNot specified
Note: Both iPS-NSC lines successfully integrated into injured spinal cord and predominantly differentiated to oligodendrocytes
View evidence from paper
“Both iPS-NSC lines successfully integrated into the injured spinal cord and predominantly differentiated to oligodendrocytes”
9
Perform electrophysiological analysis using sucrose gap method
Measure axonal function and conduction velocity using sucrose gap analysis
Not specifiedNot specified
Note: Timepoints for measurement not specified
View evidence from paper
“Electrophysiological measures of axonal function (sucrose gap analysis) compared with the nonmyelinating iPS-dNSCs and cell-free controls”
10
Compare outcomes between treatment groups
Compare neurobehavioral, histological, and electrophysiological outcomes between wildtype iPS-NSC treatment, Shiverer iPS-NSC treatment, and cell-free control groups
Not specifiedNot specified
Note: Wildtype treatment showed significant functional benefit compared to nonmyelinating and control groups
View evidence from paper
“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”
Subjects / Specimens
- Species
- mouse
- Strain
- Wildtype and Shiverer (nonmyelinating) mouse lines
- Age
- Not specified
- Sex
- unknown
- Weight
- Not specified
iPS cells generated from wildtype and Shiverer mouse fibroblasts using nonviral piggyBac transposon approach