Directed Differentiation of V3 Interneurons from Mouse Embryonic Stem Cells

STEM CELLS AND DEVELOPMENT, 2015 · DOI: 10.1089/scd.2015.0122 · Published: July 13, 2015

Spinal Cord Injury Regenerative Medicine Neurology

Simple Explanation

This study focuses on creating V3 interneurons (INs) from mouse embryonic stem cells. V3 INs are important for controlling movement and are found in the spinal cord. The scientists tested different amounts of a chemical (SAG) that mimics a natural signal (Shh) and another chemical (RA) to see how they affect the development of these stem cells into V3 INs. They found that using less RA and exposing the cells to SAG for a longer time helped the stem cells turn into V3 INs. This method could be useful for studying how these cells help with movement and spinal cord injury recovery.

Study Duration

Not specified

Participants

Mouse Embryonic Stem Cells

Evidence Level

Not specified

Key Findings

1
Lower concentrations of retinoic acid (RA) and a longer duration of smoothened agonist (SAG) exposure led to increased levels of p3 and V3 marker expression.
2
Prolonged exposure to Shh signaling, rather than increasing its concentration, was more effective for inducing V3 interneurons.
3
Decreasing RA concentration increased Nkx2.2 and Sim1 gene expression and decreased Hb9 expression, indicating further ventralization of the culture.

Research Summary

The study established a novel protocol to generate V3 interneurons (INs) from mouse embryonic stem cells (ESCs) by assessing the effects of increasing Shh signaling, prolonging the duration of Shh exposure, and decreasing RA concentration. The findings indicated that a longer duration of Shh exposure and lower concentrations of RA significantly improved the differentiation of ESCs into V3 INs, as evidenced by increased expression of V3 IN markers and decreased expression of motoneuron markers. The generated V3 INs were confirmed to be non-hypothalamic and expressed the p3 progenitor marker Nkx2.2, making this protocol a potentially scalable method to obtain V3 INs for studying central pattern generators (CPGs) and spinal cord injury (SCI) therapy.

Practical Implications

Cell-Based Therapies

The protocol provides a method for generating V3 INs, which could be used for cell replacement therapies in spinal cord injury.

Understanding Locomotor Pathways

The availability of V3 INs allows for enhanced understanding of their role in central pattern generators and locomotor coordination.

Drug Discovery

The ability to generate V3 INs in vitro can facilitate drug screening and development for spinal cord injury treatment.

Study Limitations

1
Sim1 is not uniquely expressed in V3 INs, necessitating additional steps to ensure the induced cells are indeed V3 INs and not other Sim1+ neural populations.
2
The study relies on population-averaged data from qRT-PCR experiments, which may not fully capture the heterogeneity of the induced cell population.
3
The induction protocol results in approximately 8% of the induced cells being V3 INs, requiring further optimization to increase the efficiency of differentiation.

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