The Study Of Zidovudine On The Proliferation And Differentiation Of Neurons Derived From Human Pluripotent Stem Cells

Neurological disease is a progressive degenerative disease of the brain,spinal cord or other nerves.Examples of nervous system diseases include Parkinson's disease(PD),Alzheimer's disease(AD),stroke and brain damage.According to a survey of Ministry of Health in 1982,about 5.4%Chinese peoplc suffering from various types of neurological diseases.We know that most neurons can't regenerate after injure in normal conditions.So there is still no medicine for curing neurological diseases.These diseases impact patients' quality of life seriously,and increases the burden on society.Human pluripotent stem cells are undifferentialed cells that can differentiate into specialized cells and have self-renewal ability.By using suitable transcription factors in vitro,stem cells can differentiation into all the specialized cells-neurons,osteoblasts,skin fibroblasts and so on.Human pluripotent stem cells act as a repair system for the body,and have immeasurable value for regenerative medicine.Neurons differentiated from human induced stem cells or embryonic stem cells can replace of dying nerve tissues,and provide a new direction of treating neurodegenerative diseases.In animal experiments,it has been proved that by grafting stem cell derived neurons,nervous function injury can be restored.However,undifferentiated cells have the capacity to proliferation,that means they may have risks of tumor formation.How to exclude undifferentiated cells from neural populations is the problem that need urgent solution.Telomerase activity controls telomere length and plays a pivotal role in cell aging.In somatic cells there is no telomerase expression,so telomere length is progressively shortened with each cell division.While in stem cells and tumor cells,telomerase has been shown to be specifically expressed.AZT(azidothymidine,also called zidovudin)is an antiretroviral medication used to prevent and treat HIV/AIDS.It works by selectively inhibiting HIV's reverse transcriptase,the enzyme that the virus uses to make a DNA copy of its RNA.Nowadays,researches demonstrated that AZT binds preferentially to telomeres,inhibits telomerase and induces tumor cell senescence.Therefore,we hypothesized AZT could inhibits telomerase of neuron stem cells at differentiation stage,increase neuron maturation.In this research,we used AZT to inhibit telomerase activity in hIPSC-derived neural progenitors.Thereby we hoped to reduce cell division ability of immature progenitor cells,and to promote neuron maturation.By in vitro experiment,we demonstrate the proliferation ability of cells treated with AZT reduced significantly.Cell proliferation marker K167&PH3 were significantly decreased.And the expressions of neural progenitor maker SOX2 and cortical progenitor maker PAX6 were dramatically decreased after AZT treatment.Furthermore,the cortical layer markers TBRI,CTIP2 and SATB2 were signifi cantly increased under the AZT treatment,which means AZT could accelerate the process of neuron differentiation.AZT also increased the length of the longest neurite and numbers of primary branches,which indicated the neurons showed mature morphology.Besides in vitro studies,we also did cell transplantation experiment.We treated hPSC-derived neural progenitors with AZT before transplantation to new born SCID mice,and measured the cell proliferation 4 weeks post injection.In control group,neural progenitors still had a strong ability to proliferate,in which KI67 and PH3 positive cell percentage was high.While in AZT treatment group,KI67 positive cells dramatically decreased,and hippocampus dentate gyrus cell marker PROX1 positive cells were increased,which indicated more grafted cells became postmitotic upon AZT treatment.10 weeks after transplantation,TBR1 and CTIP2,were significantly increased in AZT pre-treated group,which suggested that AZT promoted the differentiation of cortical neurons post transplantation.In this study,we identified that the telomerase inhibitor AZT suppressed the proliferation of hPSC-derived neural progenitors,promoted the differentiation of hPSC-derived cortical neurons and enhanced the maturation of hPSC-derived neurons.AZT would be a very useful tool to sculpture the hPSC derived neural differentiation process and improve the safety of hPSC transplantation therapy.