Study On Transdifferentiation Of Adult Cells From Rhesus Monkey Into Neurons

The patients with neurological disorders are increasingly with our aging society.These diseases severely treat the people's health and life quality.For these degenerative or injured neurological diseases,the traditional medicine is unable to treat them.The therapy by stem cell transplantation to replace lost cells in vivo provides one possibility to treat them.However,the lack of appropriate methods to obtain sufficient patient-specific functional neurons or neural stem cells limits its study and application in clinics.Although iPSCs technology can provide us with personalized cell neurons,the efficiency of iPSC production is low.Importantly,there is a potential safety risks,such as possible oncogenes activation and tuMor formation in vivo,which imply that it is not the ideal method to obtain neuronal cells.In contrast,cell transdifferentiation directly induce one cell type into another cell fate bypassing pluripotent state.Furthermore,the higher efficiency of cell transdifferentiation,shorter duration and relatively lower safety risks make the method become the focus in the field.Neural stem cells undergo two different developmental stages in the developing brain:neuroepithelial stem cells(NESCs)and radial glial progenitor cells(RGPCs).NESCs are from neural stem cells(NSCs)in the neural tube.NESCs have strong proliferation ability and differentiation potentials into nerve cells of the whole brain,and produce neurons with high purity(>80%).The NESCs are transiently maintained in vivo,and their abnormal proliferation and differentiation will results in neural tube defects and,subsequently,defective brain development.Therefore,NESCs have important scientific and application value.In contrast,RGPCs are high instability,high heterogeneity and low efficiency of neuron differentiation,which limit its basic research and clinical needs.Therefore,the direct conversion of fibroblasts into NESCs with long-term self-renewal is not only beneficial to explore the development of the brain,neural circuits and signal transduction and other related scientific issues,but also provides ideal donor cell source to treat neurological diseases.In this study,we first used nonh?Man primates as a model to study NESC transdifferentiation.The retroviruses carrying three transcription factors of Sox2,Oct4 and KIf4 were firstly used to infect fetal monkey fibroblasts,and then induced these infected fibroblasts into NESCs by using the optimized culture conditions.The identities of purified NESCs were confirmed by morphologies,long-term passaging and differentiation potential,respectively.These transdifferentiated NESCs not only express NSC markers,Nestin and PAX6,but also express N-cadherin,which is specific for polarized NESCs.These NESCs can maintain stable proliferation ability in the long-term culture,and the differentiation potential to three lineages in the never system.On the other hand,during the course of neruonal damage such as spinal cord injury,a large npMber of glial cells are activated,undergo large proliferation,and form glial scars.Glial scar is one of the main barriers for axonal regeneration of central system neurons.Removal of glial scar is the key to the regeneration of neurons after neuronal injury.Unfortunately,there is still lack of efficient methods.The issue becomes an important unopened question of nerve regeneration.From the perspective of regenerative medicine,if we can directly convert activated astrocytes into neurons,two benefits are obtained.First,the over-activated astrocytes are removed from the glial barrier,which overcome the scar barrier and promote nerve regeneration and reconstruction of neural function by improving the brain's microenvironment.Second,the transformed neurons can replace the lost neurons and rebuild functional circuits.Previously,the traditional method of transdifferentiation was the use of viral methods with security risks.The small molecule-induced approach is undoubtedly the key for solving the problem.In this study,we initially use small chemical molecules to directly induce the astrocytes into neurons.After a large n?Mber of molecular screenings,our experiments found that the combination of four small molecule compounds(CHIR99021,LDN193189,Purmorphamine,Repsox)can efficiently convert astrocytes from mouse cortexs into neurons.Further experiments showed that the four small molecule compounds are required for the conversion.In conclusion,we first establish a new procedure for rapid acquisition of monkey neuroepithelial stem cells from fibroblasts.The conveted NESCs have unlimited proliferative abilities in vitro,is easy to expand in vitro and facilitate gene repair and other operations.NESCs can be differentiated into a variety of neurons and glial cells,which will provide an ideal cell model for recapitulating neurological disease pathogenesis and for cell replacement therapy and other related studies.At the same time,the study of small molecules to induce astrocytes into neurons will provide a new strategy for nerve repair of neural injury.