Small Molecules Induce Direct Conversion Of Somatic Cells Into Motor Neurons

Objective: Motor neuron loss or degeneration is the typical pathological phenotype of motor neuron diseases or spinal cord injury.Transplantation of motor neurons is a promising strategy for improving the functional recovery of the spinal cord.In addition,acquisition of motor neuron disease patient-specific motor neurons can provide a disease model for studying the pathogenesis of motor neuron diseases and developing and evaluating novel therapeutic drugs.To date,there is lack of an efficient and safe way to generate motor neurons.This study aims to screen small molecules that can directly convert somatic cells into motor neurons.Methods: With the knowledge of neuronal development,we chose a pool of candidate small molecules and made different combinations of these small molecules to induce human astrocytes HA1800.On the basis of morphological changes,immunofluorescence staining,gene expression analysis and electrophysiological activities,we determined that the induced cells whether possessed the neuronal morphology,the phenotype of motor neurons,and electrophysiological properties of neurons and identified the effective small molecule cocktail.Further studies explored whether the same small molecule cocktail could induce direct conversion of mouse spinal cord astrocytes,human and mouse fibroblasts,and human bone marrow mesenchymal stem cells toward motor neurons.Finally,whether the small molecules could induce motor neuron generation in vivo were investigated.After transplantation of small molecule-soaked beads in the hypodermis of mouse back skins or near the rat sciatic nerves,immunostaining assay analyzed whether small molecules could induce nearby cells into motor neurons.Results: Five small molecules were identified to induce direct conversion of HA800 astrocytes into motor neurons efficiently and rapidly,without involving a proliferative neural progenitor stage.The induced cells showed a typical morphology of neuron,expressed general neuron markers such as TUJ1 and motor neuron-specific markers HB9,ISL1,and CHAT.Gene expression analyses showed that the neuron-associated genes were activated and the astrocyte-associated genes were suppressed.The induced neurons possessed electrophysiological properties and could form neuromuscular junctions with myotubes and control myotube contractions when co-culturing with myotubes.The same five small molecules were capable of converting mouse spinal cord astrocytes into motor neurons.When the same five small molecules were applied to human embryonic lung fibroblasts,neonatal skin fibroblasts,adult skin fibroblasts,mouse embryonic fibroblasts,and human mesenchymal stem cells,the small molecules also could convert them into motor neurons.Furthermore,small molecule-soaked beads were able to induce expression of motor neuron markers in cells within the hypodermis of mouse back skins as well as in cells near rat sciatic nerves,suggesting that the small molecules might produce motor neurons in vivo.Conclusions: We used small molecules to induce direct conversion of human and mouse astrocytes into motor neurons,without the involvement of a neural progenitor stage.The induced motor neurons displayed electrophysiological properties and control myotube contractions.The same five small molecules also induce other cells such as fibroblasts and mesenchymal stem cells into motor neurons and induce in vivo expression of motor neuron markers in cells surrounding the hypodermis of mouse back skins and rat sciatic nerves.