Reprogramming of astrocytes into motoneuron-like cells by a Ascl1-Myt1l-Pou3f2-Isl1 cocktail

Reprogramming of spinal astrocytes into motor neurons holds great promise for spinal cord injury in regeneration medicine. Here we identified a set of four transcription factors-achaete-scute complex homolog-like 1, myelin transcription factor 1 like, POU class 3 homeobox 2, and ISL LIM homeobox 1-collectively referred to as the 4 F cocktail. The 4 F cocktail reprograms rat and human reactive astrocytes into motoneuron-like cells. The reprogrammed cells display neuronal morphology and stain positive for microtubule-associated protein 2 (MAP2), a neuronal-specific marker, and choline acetyltransferase, which is a known motor neuron-specific marker. Early in the process of astrocytic reprogramming mediated by the 4 F cocktail, quantitative real-time reverse transcription polymerase chain reaction revealed that the expression of the astrocytic gene glial fibrillary acidic protein was inhibited. Additionally, the expression of the neural progenitor cell markers SRY-box transcription factor 2 and neural cell adhesion molecule 1 was upregulated within 5 days, while the motor neuron progenitor marker oligodendrocyte transcription factor 2 was activated within 7 days. Furthermore, the expression of the neural genes Map2, synapsin I, tubulin β-3, and neurofilament heavy, as well as the motor neural genes ISL LIM homeobox 1, motor neuron and pancreas homeobox 1, LIM homeobox 1, LIM homeobox 3, and NK6 homeobox 1, and survival of motor neuron 1 increased in the reprogrammed cells after induction with the 4 F cocktail. More importantly, 4 F cocktail-reprogrammed motoneuron-like cells can release acetylcholine and exhibited vulnerability to glutamate-induced excitotoxicity. Our findings demonstrate that the 4 F cocktail first reprograms spinal astrocytes into a neural progenitor-like and motor neuron progenitor-like intermediate plastic state, which then gives rise to motoneuron-like cells.