Construction Of Kidney Organoid With Human Induced Pluripotent Stem Cell

Research Background:Stem Cells(SC)are the progenitor cells for any kind of mature cell in living organisms.For the recent years,the application of SC in production of organoids in vitro is becoming more and more prevalent.Research of organoids refer to inducing stem cells to appointed kinds of mature cells with a sophisticated designed scheme,combining it with 3D culture environment,to produce an organ-like substance which may resemble the structure or even the function of mature organ.The ambition of organoids research is to replace the dysfunctional organ of human body.Function of kidney in advancing beings mainly concentrating on excretion and detoxication,regulating the homeostasis.Recently,the morbidity of End Stage Renal Diseases(ESRD)show an increasing tendency,while the dialysis and kidney transplantation can only delay the progression of ESRD,without curing it.If the clinical application of kidney organoids become true,it can probably be the best curing method for ESRD.However,because of the complication of kidney structure,the stem cell for kidney organoids must be highly pluripotent.The embryonic stem cell(ESC)possess the highest pluripotency among all kinds of stem cells.Although ESC can be acquired from human embryo or gonad,this technique has serious ethical issues,which hinders its application on organoid research.As for a substitution,some researchers invented human Induced Pluripotent Stem Cell(hIPSC)by introducing Oct4/Sox2/Klf4/c-Myc into human fibroblasts,which is also highly pluripotent,and has being prevalently applied in organoids research.Until now,there is no internationally consistent production method of kidney organoid.We summarized several schemes of kidney organoid researches,compiled a complete method for production of kidney organoid,and obtained kidney organoids which probably contained primary renal tubules and collecting ducts.Research Objective:Induce hIPSC toward kidney organoids in vitroResearch Method:hIPSC is acquired by the method described before,then cultured in T-25 bottle.After hIPSC reach certain density,perform a 6-day 2D induction by adding CHIR99021 and FGF9 to the cells.Digest and gather cells to acquire a cell mass,then transfer it onto Transwell membrane to perform a 12-day 3D induction,and finally obtain the kidney organoid.Pictures of inducing cells are taken on 1/3/6/11/18 day under microscope,protein on day 1/3/6/11/18 are gathered for the detection of markers for different stage.The final kidney organoids are dehydrated and fixed to paraffin section for further HE and immunohistochemistry identification.Research Result:Pictures under microscope showed hIPSC formed fewer colonies after the stimulating factors was added and grew separately.In the following 3D inducing process,several tubular-like structures were identified inside the kidney organoids.HE-staining of kidney organoid on day-18 also proved the occurrence of tubular-like structures.Further immunohistochemical identification indicated that the tubular-like structure in kidney organoid highly expressed PAX2,lowly expressed EC AD,and partially expressed GATA3.The Western Blot(WB)result for day 1/3/6 showed that,with the induction proceeding,the expression of hIPSC markers were down-regulated and the Nephronic Progenitor Cells(NPC)markers were up-regulated.WB result of day 1/3/6/11/18 provided further proof for expression of NPC markers,and discovered expression of several renal tubules and collecting duct markers,such as PAX2/ECAD/GATA3.Research Conclusion:With our precisely designed inducing scheme,hIPSC differentiated into kidney organoid which probably contain primary tubular structures resembling renal tubules and collecting ducts.