Induced Adipose-derived Stem Cells Differentiation In Vitro To Construct Engineered Kidney

BackgroundChronic kidney disease is a kind of diseases that threatens human health,coupled with increased morbidity.According to statistics,there are more than one million patients with end-stage renal disease(ESRD)in China,which has become a serious social problem.Adult kidneys have a fixed number of nephrons,thus it is difficult to regenerate once damaged.Currently,there are two main ways to treat end-stage renal disease:dialysis therapy and kidney transplantation.Dialysis brings great suffering to patients,seriously affecting the quality of life and makeing families bear a huge financial burden.It will also cause many complications such as cardiovascular disease,bone disease and so on.Kidney transplantation is the most effective treatment for ESRD at present,but the shortage of donors and severe immune rejection limit its wide application.Therefore,there is an urgent need for clinicians to find a new treatment for ESRD.Kidney regeneration aims to establish an artificial kidney with renal structure and function.At present,there are six strategies for kidney regeneration:decellularized kidney scaffold(DKS),metanephros transplantation,kidney organoid,blastocyst complementation,adult kidney stem cells and bioartificial kidney(BAK).Blastocyst complementation is the process of injecting exogenous stem cells into the blastocysts of mice without Sall-1 gene to make stem cells develop into chimeras together with blastocysts,and then to obtain kidneys derived from exogenous stem cells.This method is of controllable cell sources and able to obtain complete kidneys.However,chimeras produced by exogenous stem cells and blastocysts has serious ethical problems,thus it cannot be used as an ideal method for kidney regeneration.Induced pluripotent stem cells differentiate into various cell types of kidney in vitro,and the latter could self-assemble into tissue organs.Adding growth factors to the culture medium can induce pluripotent stem cells to differentiate into kidney cells in vitro.After that,kidney organs with three-dimensional structure can be obtained by 3D culture.Metanephros transplantation can relieve renal failure by transplanting embryonic metanephros for renal failure animal.Metanephros cells are at the stage of renal progenitor cells,so they have developmental pluripotency and low immunogenicity,however,metanephors transplantation is limited by the ethical considerations.Kidney stem cells are the latest kind of stem cell that can be found in the niches of kidney theoretically.And it can be cultured in vitro to differentiate into kidney cells for the study of kidney regeneration.Renal stem cells are derived from the ureteral bud-induced metanephric mesenchyma,but the isolation of renal adult stem cells remains to be further studied.Stem cells combined with kidney acellular scaffolds is a new method of kidney regeneration,which is expected to play a role in renal replacement therapy.Therefore,it has become one of the hot topics in regenerative medicine in recent years.Renal acellular scaffold is a kind of tissue engineering scaffold constructed by removing autogenous cells derived from the original kidney by chemical and physical methods to form non-immunogenic or low-immunogenic materials.Then the acellular scaffolds were perfused with adult cells or stem cells,and tissue engineering kidneys with certain function were obtained after a period of culture.The acellular scaffolds were significantly reduced in immunogenicity due to the elution of the original donor cells,remaining normal renal structure and extracellular factors,which could contribute to the growth and differentiation of seed cells in the scaffold.For the choice of seed cells,the types of cells in normal kidney are more complex,it is difficult to select one or several adult cells for kidney regeneration.In recent years,stem cells have been studied more and more deeply.With the characteristics of multidirectional differentiation and infinite proliferation,pluripotent stem cells can differentiate into different cell types under different environmental conditions,so they are the ideal seed cells.In previous studies,embryonic stem cell(ESCs),)umbilical vein endothelial cells(HUVECs),)and renal interstitial cells(RMSCs)of rats were used in the study of acellular scaffolds,but the further application in clinical practice of the cells mentioned above was limited by the ethical problems.Adipose mesenchymal stem cells(ADSCs)that have multiple differentiation potential are easily obtained and cultured.Adipose mesenchymal stem cells derived from mesoderm can differentiate across the embryo layer under the influence of specific growth factors and environment.Mesoderm(IM)is a part of mesoderm stage,the main differentiation direction of which is kidney and gonad.Therefore,it became the source of renal parenchyma structure.ObjectiveIn this study,adipose mesenchymal stem cells were induced to differentiate into mesoderm cells in vitro for recellular renal acellular scaffolds so as to obtain engineered kidneys with renal structure and function.Methods1.Isolation and Culture of Primary ADSCs.The primary ADSCs were cultured to P4 for subsequent experiments.The morphology of cells was observed under light microscope.Adipogenic differentiation was induced in vitro for 14 days.After 21 days of osteogenic differentiation,oil red O staining was performed to identify the lipid properties.Alizarin red staining was used to identify its osteogenic.After 21 days of chondrogenic differentiation,Alcian blue staining was performed in chondrogenic pellets.Flow cytometry was used to detect the expression of CD29,CD34,CD45,CD90 and CD105.2.Induction of ADSCs differentiating into Intermediate Mesoderm Cells(IM).CHIR99021 and FGF9 factors were added into ADSCs medium for 7 days.The expression of OSR1 and PAX2,the IM specific markers,was identified by immunofluorescence.Western blot was used to verify the expression of OSR1 and PAX2 in the cells after induction.The mouse embryos on day 9.5 were used as positive control.3.Preparation of decellularized scaffold.After 10%chloral hydrate anesthesia,abdominal median incision was taken to expose abdominal aorta,and sterile PBS water was infused with 24G cannula needle to flush out renal blood.After kidney removal,24 G and 26 G cannula needles were placed through renal artery and ureter,respectively.The kidneys were continuously perfused with 0.5%sodium dodecyl sulphate(SDS)for 6 hours,and then the residual SDS was rinsed by PBS.4.Comparison of the Histological Changes before and after Acellular Treatment.The morphology and structure of normal kidney and decellularized scaffolds were observed by HE staining,and the expression of collagen in normal kidney and decellularized scaffolds was observed by Masson staining.The difference of microstructure between kidney and scaffolds was compared by scanning electron microscope.5.Measurement of DNA,Collagen and Cytokines in Renal Tissue and Decellularized scaffolds.The contents of DNA and collagen in renal tissue and decellularized scaffolds were detected by DNA and collagen assay kit,and the difference of HGFV EGF-? in renal tissue and stents was compared by ELISA,and the statistical analysis was carried out.6.Recellularization of the Decellularized Scaffolds.The induced IM cells were injected through the cannula of renal artery and ureter,coupled with continuous supplying with oxygen and infused in medium.And then,the recellularized scaffolds were obtained after 10-day co-culture.The adhesions of cells in the scaffolds were observed by HE staining.Immunofluorescence was used to identify the expression of kidney tubular marker E-CAD-GATA3 and podocyte marker WT1.ADSCs perfusion scaffolds,ADSCs and IM cells cultured in vitro were used as controls.Results1.ADSCs appeared to be spindle-like under a microscope and no obvious differences were observed between cells at passages 1,3,and 5.After adipogenic induction.for 14 days,Oil Red O testing showed red lipid droplets.After osteogenic induction for 21 days,the cells were positively stained for Alizarin Red.And after 21 days of chondrogenic induction,the chondrogenic pellets were strong with Alcian Blue staining.These tests demonstrated that the ADSCs possessed the ability of differentiating into adipocytes osteoblasts and chondrocytes.Flow cytometry analysis of cell phenotype indicated that the percentage of CD29,CD90,CD105,CD34,and CD45 was 99.9%,100%,97.2%,1.1%,and 2.4%,respectively.The identified cells were used for the subsequent experiments.2.After 7-day intermediate mesoderm differentiation,the morphological changes of cells could be observed.Immunofluorescence showed that the expression of OSR1 and PAX2 in IM cells was significantly higher than that in ADSCs.Western blot showed positive expression of OSR1 and PAX in IM cells.3.After decellularized treatment with 0.5%SDS for 6 hours,the kidney gradually became transparent,which indicated that the cells in the scaffold were completely eluted from the original kidney structure.4.HE staining showed that there were no cells in the decellularized scaffolds,indicating that the decellularized scaffolds were complete.Masson staining showed that collagen content did not decrease significantly in the decellularized process.Scanning electron microscopy showed that the microstructure of decellularized scaffold was not significantly damaged compared with that of normal kidney tissue.5.DNA content in decellularized scaffolds was less than 5%of that in normal kidney,and collagen content was not significantly different from normal group,HGF,VEGF and TGF-? were not significantly changed compared with normal group.6.HE staining showed that ADSCs were distributed in blood vessels,glomeruli and renal tubules of decellularized scaffolds,which were also observed in IM.The expression of E-CAD,GATA3 and WT1 was detected by immunofluorescence assay.The differentiation efficiency of IM in the scaffold was higher than that of ADSCs.The expression of these markers was not observed in ADSCs and IM cultured in vitro.ConclusionWe found that induced IM cells in decellularized scaffold could have higher differentiation efficiency than original stem cells.IM cells grow and differentiate toward tubular-like cells and podocyte-like cells.Inducing the differentiation of stem cells in scaffolds is a feasible strategy for renal regeneration.And DC scaffolds have the ability to induce stem cells differentiation,which could be used as an ideal biomaterial for kidney regeneration.