Pax6-GFP Reporter Is An Efficient System To Derive Neural Progenitor Cells During Mouse Embryonic Stem Cells Differentiation

Degenerative diseases such as Alzheimer's disease,Parkinson's syndrome and Huntington's disease can be devastating for patients` exercise capacities and memories,putting a huge burden on relatives and society.However,there is no cure so far,mainly because of lacking suitable cells or animal models for drug therapy researches.Mouse embryonic stem cells(mESCs)are versatile in many disciplines due to their pluripotency to generate all somatic cell types.However,lineage specification of mESCs in vitro is heterogeneous and out of control.Such as mouse neuronal differentiation,there's no strict standard to guide mESCs to differentiate as predesigning.Neural progenitor cells(NPCs)are neural precursors that can differentiate into multiple neurons and in an intermediate state between mESCs and neurons.NPCs can be obtained through brain tissue separating from early embryo,or differentiation from mESCs in vitro,which has a significant application prospect in the field of clinical cell replacement therapy.During ESCs differentiation,there has been no systematic way to effectively indicate the differentiation process except to observe the changes in cell phenotype at all hours.Besides,the differentiation period is long,inefficient and the amount of NPCs obtained in vitro with current protocols are not sufficient enough for cell replacement therapy.The fundamental reason is that the molecular mechanism of neural differentiation is not clear.In order to promote the study of neural differentiation,cell replacement therapy and drug screening,an efficient and rigorous system to evaluate the mouse neural differentiation from mESCs is urgently needed.Here,we developed an endogenous fluorescence reporter system of mESCs by CRISPR/Cas9 technology.GFP was conjunct Pax6 with the same promoter to visualize the acquisition of neural identity.By fluorescence activated cell sorting(FACS)the GFP positive cells through a modified embryoid bodies differentiation process,we obtained a large amount of NPCs after 7 days` differentiation.In short,we`ve established an easier and quicker way to enrich mouse NPCs robustly with high purity and multiple potentials to derive functional neurons.This novel system can be an ideal tool for large-scale neural differentiation specific genes and medicines screening and further pave an avenue to the study of neural commitment.