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Method Article
Randolph Ashton
Ashton Lab, University of Wisconsin–Madison
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
This chemically defined protocol facilitates the differentiation of human pluripotent stem cells (hPSCs) to highly pure neural stem cells (NSCs) characteristic of diverse anatomical regions throughout the posterior central nervous system (CNS). Whereas previous protocols generated NSC cultures of predominantly cervical spinal cord or heterogeneous regional identity, this protocol can generate NSCs with highly uniform (60-90% purity), distinct positional identity from discrete regions spanning the rostrocaudal neuraxis of the hindbrain thru cervical, thoracic, and lumbosacral spinal cord. The process relies on inducing colinear HOX activation in a pre-NSC culture by modulating Wnt/β-catenin, fibroblast growth factor (FGF), and growth differentiation factor (GDF) signaling. Then, the application of retinoic acid (RA) halts HOX activation and transitions the cultures to PAX6+ NSCs expressing HOX factors characteristic of specific hindbrain and spinal cord locations. Region-specific NSCs, which may have clinical utility, can be generated within 6-14 days depending on their rostrocaudal location.
Keywords
Hox transcription factors, Neural Tube, Neuroepithelium, Neuroectoderm
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Table 2 Troubleshooting advice.
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Table 1 Antibodies used for immunocytochemistry (ICC) and flow cytometry (FC).
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Associated Publications
Deterministic HOX Patterning in Human Pluripotent Stem Cell-Derived Neuroectoderm
by Ethan S. Lippmann, Clay E. Williams, David A. Ruhl, +4
Stem Cell Reports (18 August, 2015)
Method Article
Randolph Ashton
Ashton Lab, University of Wisconsin–Madison
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 03 Sep, 2015
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
This chemically defined protocol facilitates the differentiation of human pluripotent stem cells (hPSCs) to highly pure neural stem cells (NSCs) characteristic of diverse anatomical regions throughout the posterior central nervous system (CNS). Whereas previous protocols generated NSC cultures of predominantly cervical spinal cord or heterogeneous regional identity, this protocol can generate NSCs with highly uniform (60-90% purity), distinct positional identity from discrete regions spanning the rostrocaudal neuraxis of the hindbrain thru cervical, thoracic, and lumbosacral spinal cord. The process relies on inducing colinear HOX activation in a pre-NSC culture by modulating Wnt/β-catenin, fibroblast growth factor (FGF), and growth differentiation factor (GDF) signaling. Then, the application of retinoic acid (RA) halts HOX activation and transitions the cultures to PAX6+ NSCs expressing HOX factors characteristic of specific hindbrain and spinal cord locations. Region-specific NSCs, which may have clinical utility, can be generated within 6-14 days depending on their rostrocaudal location.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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Associated Publications
Deterministic HOX Patterning in Human Pluripotent Stem Cell-Derived Neuroectoderm
by Ethan S. Lippmann, Clay E. Williams, David A. Ruhl, +4
Stem Cell Reports (18 August, 2015)