V3 interneurons are a major class of excitatory and commissural interneurons that are found in the spinal cord. These interneurons are essential for enabling a stable locomotor rhythm but they are notoriously difficult to derive from pluripotent stem cells. Given how crucial V3 interneurons are for movement, our lack of methods to derive these is an obstacle not only for in vitro spinal and motor disease modelling but for the development of new cell therapies for spinal cord disorders.
In new work from the Lieberam lab in collaboration with Elizabeth Bradbury at the Wolfson Centre for Age-Related Diseases, Berzanskyte et al. address this and present a new method for deriving V3 interneurons. In their paper, published in Scientific Reports, Ieva Berzanskyte (now at the LMB, University of Cambridge) and colleagues describe their protocol for the differentiation and isolation of V3 interneurons which combines extrinsic factor-mediated differentiation with magnetic-activated cell sorting. Through their work, the authors discovered that differentiation of V3 progenitor cells can be enhanced with a higher than usual concentration of Sonic Hedgehog agonist, in addition to culturing the cells in a 3D format.
Berzanskyte et al. developed a transgene reporter in their work which has part of the regulatory sequence of V3-specific gene Nkx2-2 to enable V3 progenitor purification from cultures with mixed differentiation. In human cells, NKX2-2 initially exhibited co-labelling with a motor neuron progenitor maker but as the differentiation culture progressed, V3 specificity became more apparent. In fact, in their study, Berzanskyte et al. were able to enrich V3 progenitors labelled with CD14 (a membrane marker expressed as a consequence of the transgene reporter) to approximately 95% purity and successfully mature the cells to postmitotic V3 interneurons.
This new research offers a valuable and reliable method for deriving V3 interneurons from human pluripotent stem cells. Berzanskyte et al.’s protocol can be applied to several different areas of research including the development of new cell therapies for spinal cord disorders.