Differentiation Of IPS Cells Into Renal Precursor Cells And Mechanism For Repair Of Kidney Injury

Background and objectiveThe induced pluripotent stem (iPS) cells technology is a major breakthrough in thefield of stem cells and tissue regeneration research with wide application prospects.However the research of iPS cells in the field of kidney regeneration is limited. This paperaims to establish the technical system of directional differentiation of iPS cells towardrenal progenitor cells in vitro, observing the ability of iPS cells-derived renal progenitorcells to survive and differentiate in renal tissues of animal models, as well as the repaireffects towards renal ischemia reperfusion injury in rats, and analyzing the possiblemechanism of its repair effects and providing experimental evidence for regenerativetherapy of kidney disease.Methods1. To induce iPS cells to differentiate toward renal progenitor cells in vitro1) Culture of iPS cells: The mouse embryonic fibroblast(MEF) cells were prepared frommouse embryos and generally cultured to passage3. MEF cells were cryopreserved aftertreatment with gamma ray irradiation. iPS cells were normally passaged and explandedwith MEFs as a feeder layer.2) Culture of embryonic body (EB): iPS cells were cultured in the medium withoutleukemia inhibitory factor (LIF) to induce EB formation.3) Induced differentiation experiment grouping:EB was cultured for2days and dividedinto groups.Growth factors-treated group: The EB were grown in the presence of the following growthfactors: RA, activin-A, Bmp7for5days. Control group: The EB were grown without growth factors for5days.Growth factors plus REGM-treated group:After cultured for5days. Growth factors-treatedgroup continued to be cultured in renal epithelial cell growth medium for another5days.4) Observation indexes and detection methodsPax2, Bry, WT1and E-cadherin protein were detected by immunofluorescence staining.Flow Cytometry technology analyzed the proportion of Pax2+, Bry+, WT1+, E-cadherin+and CD24+cells.Real time PCR analyzed gene expressions of intermediate mesoderm markers Pax2, Bry,Osr1, metanephric mesenchyme markers WT1, Six2, Sall1, and maturing kidney markersAQP1, CD24, E-cadherin, PDGFR.2. To observe the repair effects and mechanism of iPS cells-derived renal progenitor cellstowards acute kidney injury in vivo1) Animal models establishing and experimental groupingRenal ischemia reperfusion injury animal models were built with occlusion of the left renalartery for45minutes followed by reperfusion plus right kidney removal. The models weredivided into2groups.Treated group: transplanted with renal progenitor cells and hydrogel, after each of the ratssucceeded in modeling, instantly mixed the renal progenitor cells with hydrogel, theninjected about200μl hydrogel/cell suspensions (cell concentration5×105/ml) into renalparenchyma of renal ischemia reperfusion injury rat models.Control group: replaced sterile sucrose solution with renal progenitor cells, after each ofthe rats succeeded in modeling, instantly mixed the sterile sucrose solution with hydrogel,then injected about200μl hydrogel suspensions into renal parenchyma of renal ischemiareperfusion injury rat models.2) Observation indexes and detection methodsBlood samples were collected for Scr and BUN to assess renal function.Three rats were killed randomly at five time points of the3rdday,7thday,14thday,28thday,90thday after surgery, and collected the samples. Renal tissue specimens were collected for making frozen sections and paraffin sections,GFP positive cells detected by immunofluorescence staining and ELISAimmunohistochemistry were to determine the survival, distribution and differentiation ofthe transplanted renal progenitor cells in SD rats.Renal tubular injury was graded by HE staining.TUNEL method was used to detect apoptosis of renal cells.PCNA expressions were detected by immunohistochemistry, in order to observeproliferation of renal cells.Realtime PCR analyzed gene expressions of anti-inflammatory factors:IL-10, bFGF,TGF-β1, and cell factors promoting renal tubular cells repair:EGF, HGF, PDGF.Results1. Induced iPS cells to differentiate toward renal progenitor cells in vitro1) Cell immunofluorescence staining results showed that the expressions of Pax2, WT1and E-cadherin protein were higher in growth factors-treated group and growth factors plusREGM-treated group compared with control group.2) Flow Cytometry technology results detected that the proportion of Pax2+, Bry+, WT1+,E-cadherin+and CD24+cells in growth factors-treated group was higher than that incontrol group, and the proportion of Pax2+,E-cadherin+and CD24+cells in Growthfactors plus REGM-treated group was significantly higher than that in Growthfactors-treated group.3) Realtime PCR results showed that the gene expressions of intermediate mesodermmarkers Pax2, Bry, Osr1, metanephric mesenchyme mrakers WT1, Six2, Sall1andCD24,PDGFR,AQP1,E-cadherin were elevated to varying degrees in Growthfactors-treated group and Growth factors plus REGM-treated group compared with controlgroup(P<0.05or P<0.01), while the maturing kidney markers AQP1, E-cadherin geneswere significantly increased in growth factors plus REGM-treated group than that ingrowth factors-treated group, and the differences had statistical significance(P<0.05).2. The repair effects of iPS cells-derived renal progenitor cells towards kidney injury. 1) Detection of Renal function showed that Scr and BUN were significantly elevated onthe1stday after surgery compared with before surgery (P<0.01), and peaked, whichdemonstrated that the rat models of renal ischemia-reperfusion injury succeeded.2) Scr and BUN of treated group and control group gradually declined from the2nddayafter surgery. Scr and BUN of treated group declined to varying degrees compared withcontrol group, and significantly declined on the1stday after surgery compared with controlgroup with statistical significance(P<0.05), which demonstrated that renal progenitor cellshad repair effects towards kidney injury.3) GFP immunohistochemistry staining results showed that transplanted cells presentedwith positive immunofluorescence or DAB staining positive could be observedembedded in the renal tubular epithelial cells in treated group.4) After transplantation of renal progenitor cells, safety assessment results showed thatno abnormal proliferative cell groups were found in3months after surgery in SD rats bygross observation and renal tissue sample pathological examination in treated group, whichdemonstrated that no tumor formed.5) Kidney pathology examination: Rats in two groups both showed obvious renal tubularepithelial cells necrosis,distension of the lumen of renal tubular,lumen narrowing andblocking, diffusing interstitial edema, inflammatory cells infiltration.In both groups kidney lesions reached the peak on the3rdday and began to lessen on the7thday, and further better on the14thday and on the28thday. Tubular injury scores in treatedgroup were lower than control group on the3rdday, on the7thday and on the14thday, andthe differences had statistical significance(P<0.05).6) TUNEL detect showed the counts of renal epithelial cells apoptosis in treated groupwere lower than control group, especially lower on the7thday,on the14thday, and on the28thday, and the differences had statistical significance(P<0.01).7) Immunohistochemistry PCNA staining showed proliferation of renal tubular epithelialcells in treated group was more obvious than control group, especially higher on the7thday,on the14thday, and the differences had statistical significance.(P<0.05). 8) Realtime PCR results showed that gene expressions of anti-inflammatory factors:IL-10,bFGF, TGF-β1, and cell factors promoting renal tubular cells repair: EGF, HGF, PDGF oftreated group on3rdday, on the7thday, on the14thday and on the28thday after surgerywere elevated to varying degrees compared with control group, and the differences hadstatistical significance(P<0.05or P<0.01).Conclusions1.The research succeeded in proposing the technical system of directional differentiation ofiPS cells toward renal progenitor cells in vitro and the results showed that growth factorscould induce iPS cells to differentiate toward renal progenitor cells, as well as furtherimprove differentiation efficiency of iPS cells combined with renal epithelial cell culturemedium.2. iPS cells-derived renal progenitor cells had the repair effects towards renal ischemiareperfusion injury safely and effectively. And they could successfully survive in rat modelsand embedded in renal tubular epithelial cells.3. iPS cells-derived renal progenitor cells could repair renal ischemia reperfusion injury.The mechanism could be possibly related to inhibiting renal tubular epithelial cellsapoptosis, promoting renal tubular epithelial cells proliferation, improving renal tubularinjury, and enhancing the expressions of anti-inflammatory factors and cell factorspromoting renal tubular cells repair.