Cerebral Organoids Repair Traumatic Brain Injury And The Study Of Nampt Gene Editing

PartⅠ:Cerebral organoids repair traumatic brain injuryTraumatic brain injury(TBI)is defined as the function disorder of brain tissue caused by violence.According to the damaged brain tssue weather communicated with exterior,TBI can be divided into closed brain injury and open brain injury.At present,TBI is the main reason of traumatic death and the disability of people under 40.Treatments for TBI include first aid in the early period,debridement of wounds and the rehabilitation treatment in the late.But presently there is no effective treatment to cure the neurological dysfunctions caused by TBI in clinic.Cerebral organoid is a 3D neural structure which induced from embryonic stem cell(ESC)or induced pluripotent stem cells.By using induced factors and 3D cell culture technology,ESCs can be induced into cerebral organoids which contain multiple types of neurons such as neural stem cell,dopaminergic neuron,glutamatergic neuron and have different brain regions includes dorsal cortex,choroid plexus,ventral forebrain.In addition,cerebral organoids recapitulate many key features of human brain in vivo including the recapitulation the zone of putative outer radial glia cells.In brief,compared to the conventional nerve cell line,cerebral organoid has more kinds of neurons,various distinct brain regions and certain neurological functions.Stem cell transplantation therapy of TBI has proven to be effective in many animal models.However,there is no report about the cerebral organoid transplantation therapy of TBI.Therefore,in this study we built special TBI model of rat and transplanted cerebral organoid with different culture time to study the therapeutic effect of CO transplantation on TBI.In addition,we also preliminarily explored the mechanism of CO transplantation in TBI treatment.Methods 1.Culture COs according to the references.Perform COs in different culture time to immunofluorescence staining and cell count,compare and analyse the differences of cell numbers and cell composition between different 55d-COs and 85d-COs.2.Train the Sprague-Dawley rats on the balance beam till the rats can through the balance beam quickly and smoothly,the week before the experiment.After that divide rats into Sham group(only went craniotomy without brain injury),TBI group(went brain injury without COs transplantation),55d-CO transplantation group(went brain injury and transplanted with COs that were cultured of 55 days),85d-CO transplantation group(went brain injury and transplanted with COs that were cultured of 85 days)randomly.Open a small window on the skull of rats,then using biopsy punch to cause damage in the cerebral cortex,making rats appearing neurological dysfunction.After damage the cerebral cortex,transplanting COs in the lesions.In the 2,5,7,11,14,21,28,35,42 days after surgery,evaluate the behavioral dysfunction of the rats.m NSS(modified neurological severity scores)and Balance beam experiment were used to evaluate the behavioral dysfunction of the rats.3.In the 7,14,28 and 56 days after surgery,collect the brain tissue of rats in each group.Then perform these brain tissue were to immunofluorescence staining including Brd U/Nestin、Brd U/DCX、Brd U/Neu N.Then count the Brd U+/Nestin+,Brd U+/DCX+,Brd U+/Neu N+ cell numbers of lesions,subgranular zone(SGZ)of the hippocampal dentate gyrus and the subventricular zone(SVZ)of the lateral ventricles.Perform the brain tissue to immuohistochemical staining including IL-1α(Interleukin)、TNF-α(Tumor necrosis factor)and ICAM(Intercellular cell adhesion molecule)and test the ICAM level of serum to detect the level of inflammation of lesions and serum.In addition,collecting the ipsilateral hippocampus of rat brain tissues and extracting the protein of hippocampus.Then perform protein to Western blot,analyze the expression level of postsynaptic density protein 95(PSD95),synaptophysin(SYN)and brain derived neurotrophic factor(BDNF)and explore the mechanism of the protective effect of CO transplantation on TBI.4.In the 7,14,28,56 days after surgery,collect the brain tissue of rats in 55d-CO transplantation group and perform brain tissue to immunofluorescence staining including Olig2、Chat、v Glut1、SATB2、STEM121.Then observe the Survival,differentiation and neural migration of CO within the host.Meanwhile detect the vascular connection between CO and rat brain by immunofluorescence staining and immuohistochemical staining.Results 1.By cell counting and immunofluorescence staining of COs,we found that with the increasing of culture time,the number of neural stem cells within CO decreased,while the number of mature neurons increased.When cultured about 80 days,CO showed different brain regions.In addition,the number of CO cells increased along the culture time,and the number of 85d-CO cells was almost double that of 55d-CO cells.2.By counting the numbers of Brd U+/DCX+,Brd U+/Neu N+ cells in the lesions,SGZ and SVZ,we found that the numbers newborn neurons of CO transplantation group was significantly more than the TBI group and Sham group.Moreover the numbers newborn neurons of 55d-CO transplantation group were more than 85d-CO transplantation group.This suggested that CO transplantation could promote nerve regeneration of TBI rats and 55d-CO transplantation had better effect.In addition,through the m NSS and balance beam experiment in the 2,5,7,11,14,21,28,35 and 42 day after transplantation,we found that CO transplantation could significantly improve the neurological function of TBI rats.3.Through the immunofluorescence staining of Olig2,Chat,v Glut1,SATB2,STEM121 and other neural markers of CO,we discovered that CO could survive for a long time in the rat brain and differentiate into a variety of neurons of the motor cortex,Meanwhile,CO cells also migrated extensively in the rat brain.In addition,immunofluorescence and immunohistochemical staining of CD31 and CD105 proved that there was vascular connection between CO and rat cerebral cortex.We detected that the expression levels of PSD95,SYN and BDNF in the ipsilateral hippocampus by Western blot,and found that neural connection proteins of CO transplantation group was higher than TBI group and Sham group.Conclusion 1.There are differences of cell numbers and composition between COs with different culture time.Compared to 55d-CO,85d-CO has more neurons and cell numbers and less neural stem cells.2.CO transplantation can promote the nerve regeneration of TBI rats and improve the neural function.Moreover 55d-CO transplantation has a higher survival rate of transplanted cells and shows better neuroprotective effect.3.Transplanted CO can survive in the rat cortex and differentiate into a variety of neurons of the motor cortex.Meantime cells of CO can migrate extensively in the rat brain and form vascularconnection between CO and rat cerebral cortex.4.CO transplantation can increase the expression of neural connection proteins and neurotrophic factors of TBI rat.PartⅡ:The effect of Nampt gene editing in the growth of human embryonic stem cellsNicotinamide phosphoribosyl-transferase(Nampt)is the biosynthetic rate-limiting enzymes of the nicotinamide adenine dinucleotide(NAD)which determines the level and speed of generation of NAD and plays an important role in energy metabolism of cells.Meanwhile,Studies have shown that Nampt also plays an important role in promoting cell proliferation and differentiation and regulating apoptosis.Van der Veer et al.confirmed that Nampt could promote the maturation and differentiation of vessel smooth muscle cells and prolong the life of vessel smooth muscle cells.Currently,the effect of Nampt on proliferation and differentiation of human embryonic stem cells has been rarely reported.In this study,we usecrispr-cas9 technology to knockout Nampt gene of embryonic stem cells and study the effect of Nampt gene knockout on the survival and growth of human embryonic stem cells.Methods 1.Design gRNA sequences according to the design principles of gRNA target genes and construct CRISPR/Cas9 vector.2.Culture human embryonic stem cells,digest cells when the cell fusion rate reaches about 70%,perform electroporation transfection by using a torsion apparatus.3.On the first day after electroconvulsion,perform drug resistance screening by m Te SR1 medium containing 0.3 g/ml puromycin.Select drug-resistant clones and verify Gene sequence drug-resistant clones with PCR sequencing.4.Select a batch of embryonic stem cells in good growth state,when the cell fusion rate reaches about 70%,add Nampt inhibitor FK866 to the medium in different concentrations(0.3 nmol/L,1 nmol/L,3 nmol/L,10 nmol/L).Use cck-8 kit to detect cell viability after 48 hours of administrationResults 1.After electroporation transfection of human embryonic stem cells,performe 0.3 g/ml puromycin to drug resistance screening for 5 days.Then select cells and extract DNA for sanger sequencing,the result showed that the Nampt gene was not edited.2.To ensure rapid cell proliferation,reduce the concentration of puromycin to 0.1 g/ml.5 days after drug resistance screening,select cells and extract DNA for sanger sequencing,the result showed that the Nampt gene was also not edited.3.Add ROCK inhibitor Y27632(10μ Μ mol/L)on the first day after electroconvulsion,at the same time perform 0.3 g/ml puromycin to drug resistance screening for 3 days.Extract DNA of the hybrid cell line and perform to sanger sequencing.the result showed that three bases of the Nampt gene were deleted.Extract DNA of the hybrid cell line 7 days after drug resistance screening,and perform to sanger sequencing.the result showed that the Nampt gene was not edited.4.The growth inhibition rates of 0.3 nmol/L,1 nmol/L,3 nmol/L and 10 nmol/L Nampt inhibitor FK866 on embryonic stem cells were 2.95% ± 0.01、13.75% ± 0.01、90.05% ± 0.01 和 95.61% ± 0.02,respectively.All the dose groups showed statistical difference.Conclusion Nampt gene plays an indispensable role in maintaining the growth of human embryonic stem cells,deletion of the Nampt gene causes cell death.