Rapid Generation Of Forebrain Neurons From Human Embryonic Stem Cells Using Small Molecules

Forebrain is one of the most important and complex structures in the central nervous system of vertebrates,which includes cerebral cortex,basal ganglia,thalamus,hypothalamus,and etc.According to the secreted neurotransmitters,forebrain neurons can be classified intoγ-aminobutyric acid(GABA)neurons,glutamatergic neurons,cholinergic neurons,and etc.The integrity of the structure and function of forebrain neurons is essential for the maintenance of normal neurophysiology of human body.Defect and disfunction of forebrain neurons will lead to a variety of cognitive,memory,motor,emotional and other functional disorders,and even lead to neurodegenerative diseases including Alzheimer’s disease(AD)and Huntington’s disease(HD),which seriously affect the life quality of patients.But at present,there is no effective treatment for neurodegenerative diseases.In recent years,with the progress of stem cell research,cell replacement therapy has showed great perspective to alleviate or even cure these diseases.However,in order to obtain cells for the treatment of neurodegenerative diseases in vitro,several key bottleneck problems are needed to be solved.For example,how to induce stem cells to differentiate into specific types of neurons,how to obtain a large number of mature neurons for transplantation in vitro,and etc.But acquisition of forebrain neurons is not easy.In recent years,research on the molecular mechanism of forebrain neuron development and differentiation has directly promoted the generation of forebrain neurons via directed differentiation using pluripotent stem cells(PSCs)or somatic cell lineage reprogramming.However,the process mentioned above is complicated and time-consuming,and the neurons have problems of low-purification and immaturity.Therefore,researchers are now focusing on shortening the differentiation time from h PSCs to forebrain neurons,simplifying the differentiation process and improving the differentiation purity.There are two main methods to obtain neurons in vitro,gene modification and small molecule induction.Transfection and expression of the core transcription factors of differentiated target cells during PSCs differentiation can effectively promote the directional differentiation of stem cells and improve the purity of target cells.However,the complexity and safety of genetic manipulation limit the wide application of this strategy.Compared with the gene modification,small molecules have gradually shown great advantages in the field of directional differentiation of h PSCs due to its high efficiency,high safety and controllability of quantification.Our team has long been engaged in the study of directional differentiation from human embryonic stem cells(h ESCs)using small molecules.In our previous studies,we have successfully established primary self-renewing neural stem cells(NSCs)using small molecules in vitro,and these primary NSCs could be directed into dopaminergic neurons efficiently,which could rescue the symptoms when being transplanted into rats of Parkinson’s disease.And we also got self-renewing OLIG2~+neural progenitors from h ESCs for the first time.Besides,these self-renewing OLIG2~+neural progenitors had the potential to differentiate into both functional spinal motor neurons and oligodendrocytes within a short period.In this study,we followed the rules and signaling pathways of forebrain neurons development in vivo.We firstly inhibited Wnt(wingless-related integration site)signaling pathway by small molecules based on‘Dual-Smad’inhibition method under the condition of monolayer culture,and found that h ESCs could be efficiently and rapidly directed into forebrain neuroepithelial progenitors(f NEPs)in 1 week which expressed FOXG1(Forkhead box G1),SOX2(SRY-box transcription factor 2,N-Cadherin and Nestin.After that,compound SAG(activator of Smoothened)was used to activate Shh(sonic hedgehog)signaling pathway in order to ventralize these f NEPs.Within 20 days,a higher purity(>80%)NKX2.1~+GABAergic neurons derived from medial ganglionic eminence(MGE)area were obtained,and the somatostatin(SST)was detected,a subtype of GABAergic neurons.With the combined application of protein SHH and compound SAG,the cholinergic neurons expressing v ACh T(vascular acetylcholine transporter)were obtained in 20 days.The glutamate neurons expressing v Glu T2(vesicular glutamate transporter 2)were obtained in about 20 days when the Hedgehog pathway inhibitor GDC-0449 was added to the culture medium.In addition,the forebrain GABAergic neurons obtained showed electrophysiological activity in vitro,and could develop into mature neurons in about 6weeks after transplantation into immunodeficient mice.Spontaneous excitatory postsynaptic potentials(s EPSPs)and spontaneous inhibitory postsynaptic potentials(s IPSPs)of transplanted cells were detected at 3 months after transplantation.These results suggest that h ESCs-derived GABAergic neural progenitors could differentiate into functional neurons in mice.In vivo experiments show that SP8(specificity protein 8)plays an important role in the patterning of forebrain,especially in maintaining the characteristics of ventral forebrain cells.Specific knockout of SP8 in mouse forebrain inhibits the expression of NKX2.1 and leads to severe developmental defects of LGE(lateral ganglionic eminence)and MGE.Next,we hope to use our rapid differentiation induction system to study the effect of SP8 knockout on MGE and neural development in this region in vitro.We established a h ESCs cell line with SP8 gene deletion using clustered regularly interspaced short palindromicrepeats-Cas9(CRISPR-Cas9)system,and found that knocking out the SP8 gene in h ESCs had little effect on the induction of f NEPs,but significantly improved the purity of neurons in the late stage.Subsequently,we explored the effect of cyclins on the differentiation of forebrain neurons,and found that inhibition of CDK2 in cyclin-dependent kinases(CDK)had no significant effect on the differentiation speed of forebrain neurons in our study,while inhibition of CDK4/6 and Aurora kinase was not conducive to the differentiation of forebrain neurons in our culture system.It is still a challenge to obtain high-purity specific types of forebrain neurons quickly in vitro.In our study,a novel and well-defined induction culture system was constructed.In this culture system,we can quickly and efficiently induce h ESCs to differentiate into forebrain GABAergic neurons,glutamatergic neurons and cholinergic neurons under the condition of monolayer adherent culture.Therefore,our research will provide a suitable option for cell therapy,and will also contribute to the development of disease models and personalized drug.