Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors

Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitryin vivo, or for optimally informative disease modeling and/or therapeutic screeningin vitro, it is important to reproduce the type or subtype of neurons involved. No such appropriatein vitromodels exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factorSox6is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and thatSox6suppresses a latent neurogenic program by repressing inappropriate proneuralNeurog2expression by progenitors. We FACS-purify these genetically accessible progenitors from postnatal mouse cortex and establish a pure culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activatingNeurog2, Fezf2, while antagonizingOlig2withVP16:Olig2). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors, and find that these neurons differentiate with remarkable fidelity compared with corticospinal neuronsin vivo. They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employedNeurog2-driven differentiation. Neurons induced byNeurog2instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from genetically accessible cortical progenitors sets a precedent and foundation forin vitromechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair.