| BackgroundIn recent years,the incidence of End Stage Renal Disease(ESRD)has been increasing year by year.At present,there are 100 patients with End Stage Renal Disease in one million people in China.The main treatment methods are hemodialysis and kidney transplantation.The mortality rate of hemodialysis patients is still very high despite the great progress of related treatment technology in recent years.However,the shortage of kidney source in kidney transplantation is a current problem,and there is immune rejection in kidney transplantation,which requires lifelong treatment.In order to break through the limitations of renal replacement therapy,renal regeneration has become a research hotspot.With the development of stem cell technology and regenerative medicine,the application of stem cells in renal regeneration has brought new hope and treatment methods for patients with end-stage renal disease.Stem Cell(SC)is a kind of pluripotent cells with the ability of self-replication.Under certain conditions,it can differentiate into a variety of functional cells.Stem cells are widely used in many fields of medicine.At present,scientists have been able to identify,isolate,purify,amplify and culture human embryonic stem cells in vitro,and with such stem cells as "seeds",to produce some human tissues and organs.Embryonic stem cells(ESCs)and Induced pluripotent stem cells(iPSCs)can be directed to differentiate into most cell types and have the potential to repair damaged tissues.There are ethical problems with using ESCs,however,because the cells come from the inner cell mass of the early embryo,and the cell mass is damaged in the process.In contrast,IPSCs can be produced from differentiated cells by upregulating pluripotent factors,thus promising to provide a large amount of hard-to-obtain cell resources for regenerative medicine.Regenerative medicine research involving iPSCs is progressing significantly,and its treatments are already undergoing clinical trials.In addition,patient-derived iPSCs have the potential to generate disease-related specialized cells and associated organoids,helping researchers understand the underlying pathophysiology of the disease.Regenerative medicine has increasingly become the focus of scholars at home and abroad.At present,the constraction of kidney organoids has become a new research hotspot,and breakthrough research progress has been achieved.Due to the high complexity of kidney structure and function,there is still a big gap between kidney organoids and normal human kidneys.First of all,the vascularization obtained by renal organ transplantation in vivo is small blood vessels from the host,which cannot form a mature vascular network and lacks a functional vascular perfusion system.What'more,despite the constraction of kidney organoids can produce glomerular cells,but the glomerular filtration function,urine out cannot be achieved.On the basis of summarizing the protocol of constraction kidneyl organoids,and aiming at the above problems,this study will improve the maturity of induction of kidney organoids in vitro through the following research methods,promote the better differentiation of cells in the process of in vitro induction of kidney organoids,and obtain kidney organoids with more abundant structure.ObjectiveHuman Induced pluripotent stem cells(hiPSCs)due to its expansion and plasticity of growth signals,can be Induced to differentiate into kidney cells,so as to simulate the process of human genetic kidney disease.In order to accurately understand the process of kidney development and the pathogenesis of kidney disease,and ultimately provide a new therapeutic strategy for the treatment of End Stage Renal disease(ESRD),this study will use hiPSCs to constract kidney organoids in vitro,optimize the protocol of constraction kidney organoids,and then conduct in vivo transplantation experiment,so as to further promote the development and maturity of kidney organoids in vivo.Methods1.hiPSCs were induced into kidney organoids in vitro,specifically hiPSCs were implanted in T25 culture flask.After obtaining a certain cell density,CHIR99021,FGF9 and Heparin were successively added to induce differentiation.After 6 days of 2D induction in vitro,cell clusters were transferred into Transwell chamber for 12 days of 3D induction at the 7th day of in vitro culture,and kidney organoids were finally obtained.2.While constructing kidney organoids with the above method,kidney organoids were cultivated with nephron cells derived from hiPSCs.Based on the previous protocol,the in vitro culture scheme of kidney organoids was optimized,and the induction efficiency in vitro was relatively improved.hiPSCs-nepron cells were renal progenitor cells(NPCs)which were differentiated from hiPSCs.Then the renal progenitor cells were used for in vitro induction and differentiation,and finally obtained the kidney organoids.3.In order to further promote the maturity of kidney organoids,we digested kidney organoids into cell masses and injected them into the renal cortex of mice.From 3 weeks after transplantation,kidney organoids in vivo were removed for further identification.The purpose of this study is to research the influence of the microenvironment in renal cortex on kidney organoids,and whether kidney organoids can further differentiate and develop in vivo after digesting into cell masses,and to draw the corresponding conclusions.Results1.During the cultivation of kidney organoids by hiPSCs,according to the cell observation during the plane culture,the cell colonies began to disperse and grow with some changes in morphology after the addition of inducible factors.In the later period of 3D cultivation,the tubular structure began to appear on the third day of 3D transformation,and gradually increased and matured.After 18 days of cultivation,paraffin sectioning-HE staining of the kidney organoids confirmed the presence of a large number of tubular structures in the kidney organoids.2.At the same time,in order to improve the induction efficiency of kidneyl organoids in vitro and optimize the cultivation conditions of kidney organoids,we started the induction from hiPSCs-NPCs,and finally successfully cultivated kidney organoids.A large number of tubular structures were observed in the kidney organoids under the microscope.3.In order to promote the further differentiation and development of kidney organoids,the digested cells were injected into the renal cortex of mice in this study.After three weeks of in vivo culture,the kidneys were taken for identification.After paraffin section-HE staining and immunohistochemical staining identification,it was found that the kidney organoids were further mature in the renal cortex,which proved that the renal cortex of mice could be used for in vivo culture of kidney organoids.ConclusionAccording to the above two induction protocols,kidney organoids were successfully cultivated in vitro.After identification,it was found that the kidney organoids cultivated in vitro contained a large number of tubular structures,and it was speculated that the tubular structures were probably primary renal tubes and primary collecting tubes.The kidney organoids induced by hiPSCs-NPCs were injected into the renal cortex of mice after digestion.Three weeks after transplantation,kidney organoids were identified and further matured in the renal cortex of mice after digestion.There were antigen-specific tubular structures at the injection site,which proved that the renal cortex could promote the further maturation and differentiation of kidney organoids. |
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