Study On Directed Differentiation Of Cortical Glutamatergic Neurons From Mouse Embryonic Stem Cells In Vitro

Objective:Glutamatergic neurons, the most important excitatory neurons in the central nervous system, are involved in almost all cerebral function by regulating the release of excitatory neurotransmitter (glutamate) into synaptic cleft. The loss and abnormality of glutamatergic neurons can lead to glutamate abnormality, which induces a variety of neurological diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), schizophrenia, depression, epilepsy, and paroxysmal deafness. Embryonic stem cells (ESCs) are expected to be cell source for cell replacement therapy of neurological diseases with the characteristics of unlimited proliferation in vitro and pluripotency. Recently, many studies focus on inducing embryonic stem cells differentiating into neural cells in vitro, which not only has important implications for the treatment of neurodegenerative diseases or brain injuries, but also plays an important role for understanding developmental mechanism of central nervous system. Previous studies have concentrated on embryonic stem cells induced into neurons by external factors, which methods are too complex and induction efficiency is also low. So we aim to study an advanced induction method of mouse embryonic stem cells differentiating into our target neurons in this paper.Methods:1.BALB/c mice blastocyst inner cell mass were separated and placed on feeder cells to prepare mouse embryonic stem cells. After several passagings and identification, ESCs were inoculated into 96 well plates in low-density quantitative and cultured for 7 days.2. Then we detected the proportion of Bfl+and Emx+ telencephalon progenitors with flow cytometry.3. PCR and agarose gel electrophoresis tested the relative expression of marker genes (Oct3/4, Nestin, Tujl, GFAP) of the cells in different differentiation stages.4. Immunofluorescence staining was performed to observe and count the proportion of Tuj1+ and VGLUT1+ glutamatergic neurons.5. Cyclopamine treatment on telencephalic progenitors could induce them differentiating into VGLUT1+ glutamatergic neurons selectively.6. Patch clamp detection electrophysiological characteristics of VGLUT1+ neurons in vivo and in vitro.Results:1) Embryonic stem cells could be differentiated selectively into neural cells independently through this method with low concentration of external factors.2) This induction technology can make the mESCs efficiently differentiate into the Bf1+ telencephalon precursor cells (up to 70%), and most of which (89%) generated in this modified culture method co-expressed cortical marker Emxl.3) The method can make the mESCs differentiate effectively in an order of neurons-glia, which is consistent with the order of embryonic neural tissue development in vivo. 4) Cyclopamine can increase the percentage of VGLUT1+ neurons.5) The precursor cells induced by this method can differentiate into functional cortical glutamatergic neurons.Conclusions:By improving the technology of serum-free suspension culture, we succeeded in establishing a more effective induction method of mESCs differentiating selectively into cortical glutamatergic neurons, which not only provide a potential cell source for the treatment and research of neurodegenerative diseases or brain injuries, but also has certain scientific significance for understanding developmental mechanism of central nervous system.