诱导多能干细胞向肾脏前体细胞定向分化及修复肾损伤的作用与机制
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摘要
研究背景及目的：
诱导多能干细胞（Induced pluripotent stem cells，iPS）是目前干细胞与组织再生研究领域的一项重大突破，因为其解决了胚胎干细胞来源有限和伦理学障碍的难题，在医学领域展示了广阔的应用前景。肾脏疾病发展到终末期都需要肾移植等替代治疗，干细胞移植是治疗肾脏疾病的理想治疗方式，但有关iPS细胞在肾脏再生领域的研究较少。本文旨在建立iPS细胞向肾脏前体细胞定向分化的体外诱导培养技术体系，观察iPS细胞来源的肾脏前体细胞在动物模型体内肾脏组织中的存活及分化能力，以及对大鼠肾缺血再灌注损伤的修复作用，探讨其作用机制，为iPS细胞在肾脏疾病方面的再生治疗提供实验依据。
方法：
1体外诱导iPS细胞向肾脏前体细胞分化
1) iPS细胞的培养：从胎鼠获得小鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEF），传至第3代，用γ射线辐照处理后冻存。MEF作为饲养层细胞，iPS细胞接种在饲养层细胞上培养，正常传代、扩增。
2)拟胚体（embryonic body，EB）的培养：iPS细胞用去除白血病抑制因子（leukemiainhibitory factor，LIF)的培养液悬浮培养EB。
3)诱导分化实验分组：EB培养两天后分组诱导培养。生长因子诱导组：EB培养液中添加维甲酸（retinoic acid，RA）、activin-A、骨形成蛋白7（Bone Morphogenetic Protein7，BMP7）三种生长因子诱导培养5天；对照组：EB培养液不加生长因子培养5天；生长因子+REGM诱导组：生长因子诱导组培养5天后继续以肾脏上皮细胞生长培养基（Renal Epithelial Cell Growth Medium，REGM）培养5天。
4)观察指标及检测方法：免疫荧光检测各组细胞中Pax2、Bry、WT1、E-cadherin蛋白表达情况；流式细胞技术检测Pax2、Bry、WT1、E-cadherin、CD24阳性细胞比例；realtime PCR检测间介中胚层标志物Pax2、Bry、Osr1，后肾间充质标志物WT1、Six2、Sall1，以及CD24、PDGFR、AQP1、E-cadherin基因表达情况。
2体内观察iPS细胞来源的肾脏前体细胞修复肾损伤的作用及机制
1)动物模型制作及实验分组：采取左肾动脉夹闭45分钟后再通+右肾切除的手术方式制作肾缺血再灌注急性肾损伤动物模型后，分2组进行实验，移植组：肾脏前体细胞+水凝胶移植治疗，每只大鼠造模成功后，立即将肾脏前体细胞与水凝胶混合，注射到肾缺血再灌注损伤模型大鼠肾脏实质内，约注射200μl细胞水凝胶悬液（细胞密度5×105个/ml）；对照组：使用无菌蔗糖溶液代替肾脏前体细胞，再加水凝胶移植，每只大鼠造模成功后，立即将无菌蔗糖溶液与水凝胶混合，注射到肾缺血再灌注损伤模型大鼠肾脏实质内，约注射200μl水凝胶悬液。
2)观察指标及检测方法：术后收集血清检测血Scr、BUN评价肾功能；术后第3、7、14、28、90天时间点各随机处死3只大鼠，收集标本；收集肾组织标本分别制作冰冻切片和石蜡切片，免疫荧光染色及免疫酶联组化染色检测绿色荧光蛋白（Green Fluorescent Protein，GFP)阳性细胞来确定移植的肾脏前体细胞在大鼠肾组织中的存活、分布和分化情况；HE染色对肾小管损害程度评分；TUNEL法检测肾组织细胞凋亡；免疫组化检测增殖细胞核抗原（ProliferatingCellNuclearAntigen，PCNA）观察肾组织细胞增殖情况。realtime PCR检测肾组织中抗炎症因子白细胞介素10(interleukin-10，IL-10)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)、转化生长因子-β1（transforming growth factor-β1,TGF-β1)和促肾小管修复因子表皮生长因子(Epidermal Growth Factor，EGF)、肝细胞生长因子(hepatocyte growth factor，HGF)、血小板衍生生长因子(Platelet derived growth factor，PDGF)基因表达情况。
结果：
1、体外诱导iPS细胞向肾脏前体细胞分化
1)细胞免疫荧光染色结果显示生长因子诱导组、生长因子+REGM诱导组细胞Pax2、WT1、E-cadherin蛋白的表达明显高于对照组；
2)流式细胞技术检测显示生长因子诱导组Pax2、Bry、WT1、E-cadherin和CD24阳性细胞比例较对照组增加，生长因子+REGM诱导组表达Pax2、E-cadherin和CD24的阳性细胞比例较生长因子诱导组进一步增加；
3) realtime PCR检测显示生长因子诱导组、生长因子+REGM诱导组间介中胚层标志物Pax2、Bry、Osr1，后肾间充质标志物WT1、Six2、Sall1，以及CD24、PDGFR、AQP1、E-cadherin的基因表达水平较对照组有不同程度升高（P<0.05或P<0.01），而生长因子+REGM诱导组成熟肾脏细胞标志因子AQP1、E-cadherin的基因表达水平较生长因子诱导组明显升高（P<0.05），有统计学意义。
2、iPS细胞来源的肾脏前体细胞对肾损伤的修复作用
1)肾功能检测结果显示：两组大鼠术后第1天血清Scr、BUN均较术前明显升高（P<0.01），并达到高峰，表明肾缺血再灌注大鼠动物模型制作成功。
2)移植组和对照组大鼠术后第二天开始血清Scr、BUN逐渐下降。移植组血清Scr、BUN较对照组均有不同程度降低，其中术后第一天移植组血清Scr、BUN较对照组相比明显降低（P<0.05），有统计学意义，表明肾脏前体细胞对肾损伤的有修复作用。
3) GFP免疫组化染色结果显示：移植组肾组织中可以观察到呈免疫荧光阳性或DAB阳性的移植细胞嵌合在肾小管上皮细胞之间。
4)肾脏前体细胞移植后安全性评估结果显示，移植组SD大鼠术后观察3个月，大体观察和肾组织标本病理检查均未发现异常增殖细胞群，表明无肿瘤组织形成。
5)肾脏病理检查显示，两组大鼠术后均出现明显的肾小管上皮细胞急性变性坏死，肾小管管腔扩张，管腔狭窄或阻塞，弥漫性间质水肿及炎症细胞浸润。移植组和对照组均显示在术后第3天肾小管损伤最严重，于第7天开始有所减轻，第14天、28天时肾小管损伤进一步好转。其中移植组在第3天、第7天、第14天时的肾小管损伤评分均低于对照组（P<0.05），有统计学意义。
6) TUNEL检测肾小管上皮细胞凋亡情况结果显示：移植组凋亡细胞数均较对照组减少，其中第7天、第14天、第28天移植组较对照组明显减少（P<0.01），有显著的统计学意义。
7)免疫组化PCNA检测肾小管上皮细胞增殖情况结果显示：移植组PCNA阳性细胞数均较对照组增加，其中第7天、第14天移植组较对照组PCNA阳性细胞计数明显增加（P<0.05），有统计学意义。
8)肾脏标本Realtime PCR检测结果显示，术后第3天、第7天、第14天、第28天移植组抗炎症因子IL-10、bFGF、TGF-β1及促肾小管修复因子EGF、HGF、PDGF基因表达水平较对照组均有不同程度的增加（P<0.05或P<0.01），有统计学意义。
结论：
1、本实验成功建立了iPS细胞向肾脏前体细胞定向分化的体外诱导培养技术体系。研究结果显示生长因子能诱导iPS细胞向肾脏前体细胞分化，联合肾脏上皮细胞生长培养基可进一步提高iPS细胞的诱导分化效率。
2、iPS细胞诱导来的肾脏前体细胞具有修复肾缺血再灌注损伤的功能，安全有效。能在大鼠动物模型体内成功存活，并嵌合到肾小管结构中。
3、iPS细胞来源的肾脏前体细胞具有修复肾缺血再灌注损伤的功能，分析其机制包括，肾脏前体细胞能抑制肾小管上皮细胞凋亡，促进肾小管上皮细胞增殖，从而改善肾小管损伤程度；同时可能与肾脏前体细胞促进抗炎症因子和促肾小管修复因子的表达有关。
Background and objective
The 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.
Methods
1. To induce iPS cells to differentiate toward renal progenitor cells in vitro
1) 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 methods
Pax2, 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 vivo
1) Animal models establishing and experimental grouping
Renal 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 methods
Blood 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.
Results
1. Induced iPS cells to differentiate toward renal progenitor cells in vitro
1) 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).
Conclusions
1.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.
诱导多能干细胞（Induced pluripotent stem cells，iPS）是目前干细胞与组织再生研究领域的一项重大突破，因为其解决了胚胎干细胞来源有限和伦理学障碍的难题，在医学领域展示了广阔的应用前景。肾脏疾病发展到终末期都需要肾移植等替代治疗，干细胞移植是治疗肾脏疾病的理想治疗方式，但有关iPS细胞在肾脏再生领域的研究较少。本文旨在建立iPS细胞向肾脏前体细胞定向分化的体外诱导培养技术体系，观察iPS细胞来源的肾脏前体细胞在动物模型体内肾脏组织中的存活及分化能力，以及对大鼠肾缺血再灌注损伤的修复作用，探讨其作用机制，为iPS细胞在肾脏疾病方面的再生治疗提供实验依据。
方法：
1体外诱导iPS细胞向肾脏前体细胞分化
1) iPS细胞的培养：从胎鼠获得小鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEF），传至第3代，用γ射线辐照处理后冻存。MEF作为饲养层细胞，iPS细胞接种在饲养层细胞上培养，正常传代、扩增。
2)拟胚体（embryonic body，EB）的培养：iPS细胞用去除白血病抑制因子（leukemiainhibitory factor，LIF)的培养液悬浮培养EB。
3)诱导分化实验分组：EB培养两天后分组诱导培养。生长因子诱导组：EB培养液中添加维甲酸（retinoic acid，RA）、activin-A、骨形成蛋白7（Bone Morphogenetic Protein7，BMP7）三种生长因子诱导培养5天；对照组：EB培养液不加生长因子培养5天；生长因子+REGM诱导组：生长因子诱导组培养5天后继续以肾脏上皮细胞生长培养基（Renal Epithelial Cell Growth Medium，REGM）培养5天。
4)观察指标及检测方法：免疫荧光检测各组细胞中Pax2、Bry、WT1、E-cadherin蛋白表达情况；流式细胞技术检测Pax2、Bry、WT1、E-cadherin、CD24阳性细胞比例；realtime PCR检测间介中胚层标志物Pax2、Bry、Osr1，后肾间充质标志物WT1、Six2、Sall1，以及CD24、PDGFR、AQP1、E-cadherin基因表达情况。
2体内观察iPS细胞来源的肾脏前体细胞修复肾损伤的作用及机制
1)动物模型制作及实验分组：采取左肾动脉夹闭45分钟后再通+右肾切除的手术方式制作肾缺血再灌注急性肾损伤动物模型后，分2组进行实验，移植组：肾脏前体细胞+水凝胶移植治疗，每只大鼠造模成功后，立即将肾脏前体细胞与水凝胶混合，注射到肾缺血再灌注损伤模型大鼠肾脏实质内，约注射200μl细胞水凝胶悬液（细胞密度5×105个/ml）；对照组：使用无菌蔗糖溶液代替肾脏前体细胞，再加水凝胶移植，每只大鼠造模成功后，立即将无菌蔗糖溶液与水凝胶混合，注射到肾缺血再灌注损伤模型大鼠肾脏实质内，约注射200μl水凝胶悬液。
2)观察指标及检测方法：术后收集血清检测血Scr、BUN评价肾功能；术后第3、7、14、28、90天时间点各随机处死3只大鼠，收集标本；收集肾组织标本分别制作冰冻切片和石蜡切片，免疫荧光染色及免疫酶联组化染色检测绿色荧光蛋白（Green Fluorescent Protein，GFP)阳性细胞来确定移植的肾脏前体细胞在大鼠肾组织中的存活、分布和分化情况；HE染色对肾小管损害程度评分；TUNEL法检测肾组织细胞凋亡；免疫组化检测增殖细胞核抗原（ProliferatingCellNuclearAntigen，PCNA）观察肾组织细胞增殖情况。realtime PCR检测肾组织中抗炎症因子白细胞介素10(interleukin-10，IL-10)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)、转化生长因子-β1（transforming growth factor-β1,TGF-β1)和促肾小管修复因子表皮生长因子(Epidermal Growth Factor，EGF)、肝细胞生长因子(hepatocyte growth factor，HGF)、血小板衍生生长因子(Platelet derived growth factor，PDGF)基因表达情况。
结果：
1、体外诱导iPS细胞向肾脏前体细胞分化
1)细胞免疫荧光染色结果显示生长因子诱导组、生长因子+REGM诱导组细胞Pax2、WT1、E-cadherin蛋白的表达明显高于对照组；
2)流式细胞技术检测显示生长因子诱导组Pax2、Bry、WT1、E-cadherin和CD24阳性细胞比例较对照组增加，生长因子+REGM诱导组表达Pax2、E-cadherin和CD24的阳性细胞比例较生长因子诱导组进一步增加；
3) realtime PCR检测显示生长因子诱导组、生长因子+REGM诱导组间介中胚层标志物Pax2、Bry、Osr1，后肾间充质标志物WT1、Six2、Sall1，以及CD24、PDGFR、AQP1、E-cadherin的基因表达水平较对照组有不同程度升高（P<0.05或P<0.01），而生长因子+REGM诱导组成熟肾脏细胞标志因子AQP1、E-cadherin的基因表达水平较生长因子诱导组明显升高（P<0.05），有统计学意义。
2、iPS细胞来源的肾脏前体细胞对肾损伤的修复作用
1)肾功能检测结果显示：两组大鼠术后第1天血清Scr、BUN均较术前明显升高（P<0.01），并达到高峰，表明肾缺血再灌注大鼠动物模型制作成功。
2)移植组和对照组大鼠术后第二天开始血清Scr、BUN逐渐下降。移植组血清Scr、BUN较对照组均有不同程度降低，其中术后第一天移植组血清Scr、BUN较对照组相比明显降低（P<0.05），有统计学意义，表明肾脏前体细胞对肾损伤的有修复作用。
3) GFP免疫组化染色结果显示：移植组肾组织中可以观察到呈免疫荧光阳性或DAB阳性的移植细胞嵌合在肾小管上皮细胞之间。
4)肾脏前体细胞移植后安全性评估结果显示，移植组SD大鼠术后观察3个月，大体观察和肾组织标本病理检查均未发现异常增殖细胞群，表明无肿瘤组织形成。
5)肾脏病理检查显示，两组大鼠术后均出现明显的肾小管上皮细胞急性变性坏死，肾小管管腔扩张，管腔狭窄或阻塞，弥漫性间质水肿及炎症细胞浸润。移植组和对照组均显示在术后第3天肾小管损伤最严重，于第7天开始有所减轻，第14天、28天时肾小管损伤进一步好转。其中移植组在第3天、第7天、第14天时的肾小管损伤评分均低于对照组（P<0.05），有统计学意义。
6) TUNEL检测肾小管上皮细胞凋亡情况结果显示：移植组凋亡细胞数均较对照组减少，其中第7天、第14天、第28天移植组较对照组明显减少（P<0.01），有显著的统计学意义。
7)免疫组化PCNA检测肾小管上皮细胞增殖情况结果显示：移植组PCNA阳性细胞数均较对照组增加，其中第7天、第14天移植组较对照组PCNA阳性细胞计数明显增加（P<0.05），有统计学意义。
8)肾脏标本Realtime PCR检测结果显示，术后第3天、第7天、第14天、第28天移植组抗炎症因子IL-10、bFGF、TGF-β1及促肾小管修复因子EGF、HGF、PDGF基因表达水平较对照组均有不同程度的增加（P<0.05或P<0.01），有统计学意义。
结论：
1、本实验成功建立了iPS细胞向肾脏前体细胞定向分化的体外诱导培养技术体系。研究结果显示生长因子能诱导iPS细胞向肾脏前体细胞分化，联合肾脏上皮细胞生长培养基可进一步提高iPS细胞的诱导分化效率。
2、iPS细胞诱导来的肾脏前体细胞具有修复肾缺血再灌注损伤的功能，安全有效。能在大鼠动物模型体内成功存活，并嵌合到肾小管结构中。
3、iPS细胞来源的肾脏前体细胞具有修复肾缺血再灌注损伤的功能，分析其机制包括，肾脏前体细胞能抑制肾小管上皮细胞凋亡，促进肾小管上皮细胞增殖，从而改善肾小管损伤程度；同时可能与肾脏前体细胞促进抗炎症因子和促肾小管修复因子的表达有关。
Background and objective
The 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.
Methods
1. To induce iPS cells to differentiate toward renal progenitor cells in vitro
1) 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 methods
Pax2, 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 vivo
1) Animal models establishing and experimental grouping
Renal 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 methods
Blood 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.
Results
1. Induced iPS cells to differentiate toward renal progenitor cells in vitro
1) 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).
Conclusions
1.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.
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70. Wu, D.-Q., T. Wang, B. Lu, et al., Fabrication of supramolecular hydrogels for drugdelivery and stem cell encapsulation. Langmuir,2008.24(18): p.10306-10312.
71. Bilousova, G., D.H. Jun, K.B. King, et al., Osteoblasts derived from inducedpluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo.Stem cells,2011.29(2): p.206-216.
72. Christman, K.L., H.H. Fok, R.E. Sievers, et al., Fibrin glue alone and skeletalmyoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction.Tissue engineering,2004.10(3-4): p.403-409.
73. Dai, W., L.E. Wold, J.S. Dow, et al., Thickening of the infarcted wall by collageninjection improves left ventricular function in rats: a novel approach to preservecardiac function after myocardial infarction. Journal of the American College ofCardiology,2005.46(4): p.714-719.
74. Davis, M.E., J.M. Motion, D.A. Narmoneva, et al., Injectable self-assembling peptidenanofibers create intramyocardial microenvironments for endothelial cells. Circulation,2005.111(4): p.442-450.
1. Sagrinati, C., G.S. Netti, B. Mazzinghi, et al., Isolation and Characterization ofMultipotent Progenitor Cells from the Bowman’s Capsule of Adult Human Kidneys.Journal of the American Society of Nephrology,2006.17(9): p.2443-2456.
2. Lazzeri, E., C. Crescioli, E. Ronconi, et al., Regenerative potential of embryonic renalmultipotent progenitors in acute renal failure. Journal of the American Society ofNephrology,2007.18(12): p.3128-3138.
3. Mazzinghi, B., E. Ronconi, E. Lazzeri, et al., Essential but differential role for CXCR4and CXCR7in the therapeutic homingof human renal progenitor cells. The Journal ofexperimental medicine,2008.205(2): p.479-490.
4. Lin, F., K. Cordes, L. Li, et al., Hematopoietic stem cells contribute to theregeneration of renal tubules after renal ischemia-reperfusion injury in mice. Journalof the American Society of Nephrology,2003.14(5): p.1188-1199.
5. Morigi, M., B. Imberti, C. Zoja, et al., Mesenchymal stem cells are renotropic, helpingto repair the kidney and improve function in acute renal failure. Journal of theAmerican Society of Nephrology,2004.15(7): p.1794-1804.
6. T gel, F., A. Cohen, P. Zhang, et al., Autologous and allogeneic marrow stromal cellsare safe and effective for the treatment of acute kidney injury. Stem cells anddevelopment,2009.18(3): p.475-486.
7. Thomson, J.A., J. Itskovitz-Eldor, S.S. Shapiro, et al., Embryonic stem cell linesderived from human blastocysts. science,1998.282(5391): p.1145.
8. Steenhard, B.M., K.S. Isom, P. Cazcarro, et al., Integration of embryonic stem cells inmetanephric kidney organ culture. Journal of the American Society of Nephrology,2005.16(6): p.1623-1631.
9. Kim, D. and G.R. Dressler, Nephrogenic factors promote differentiation of mouseembryonic stem cells into renal epithelia. Journal of the American Society ofNephrology,2005.16(12): p.3527-3534.
10. Vigneau, C., K. Polgar, G. Striker, et al., Mouse Embryonic Stem Cell–DerivedEmbryoid Bodies Generate Progenitors That Integrate Long Term into Renal ProximalTubules In Vivo. Journal of the American Society of Nephrology,2007.18(6): p.1709-1720.
11. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell,2006.126(4): p.663-676.
12. Morizane, R., T. Monkawa, and H. Itoh, Differentiation of murine embryonic stem andinduced pluripotent stem cells to renal lineage in vitro. Biochemical and BiophysicalResearch Communications,2009.390(4): p.1334-1339.
13. Song, B., A.M. Smink, C.V. Jones, et al., The directed differentiation of human iPScells into kidney podocytes. PLoS One,2012.7(9): p. e46453.
14. Lee, P., Y. Chien, G. Chiou, et al., Induced Pluripotent Stem Cells Without c-MycAttenuate Acute Kidney Injury via Down-Regulating the Signaling of Oxidative Stressand Inflammation in Ischemia-Reperfusion Rats. Cell transplantation,2012.
15. Li, L., R. Black, Z. Ma, et al., Use of mouse hematopoietic stem and progenitor cellsto treat acute kidney injury. American Journal of Physiology-Renal Physiology,2012.302(1): p. F9-F19.
16. Swetha, G., V. Chandra, S. Phadnis, et al., Glomerular parietal epithelial cells of adultmurine kidney undergo EMT to generate cells with traits of renal progenitors. Journalof cellular and molecular medicine,2011.15(2): p.396-413.
17. Prodromidi, E.I., R. Poulsom, R. Jeffery, et al., Bone Marrow‐Derived CellsContribute to Podocyte Regeneration and Amelioration of Renal Disease in a MouseModel of Alport Syndrome. Stem cells,2006.24(11): p.2448-2455.
18. Lee, R.H., M.J. Seo, R.L. Reger, et al., Multipotent stromal cells from human marrowhome to and promote repair of pancreatic islets and renal glomeruli in diabeticNOD/scid mice. Proceedings of the National Academy of Sciences,2006.103(46): p.17438-17443.
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1. Sagrinati, C., G.S. Netti, B. Mazzinghi, et al., Isolation and Characterization ofMultipotent Progenitor Cells from the Bowman’s Capsule of Adult Human Kidneys.Journal of the American Society of Nephrology,2006.17(9): p.2443-2456.
2. Lazzeri, E., C. Crescioli, E. Ronconi, et al., Regenerative potential of embryonic renalmultipotent progenitors in acute renal failure. Journal of the American Society ofNephrology,2007.18(12): p.3128-3138.
3. Mazzinghi, B., E. Ronconi, E. Lazzeri, et al., Essential but differential role for CXCR4and CXCR7in the therapeutic homingof human renal progenitor cells. The Journal ofexperimental medicine,2008.205(2): p.479-490.
4. Lin, F., K. Cordes, L. Li, et al., Hematopoietic stem cells contribute to theregeneration of renal tubules after renal ischemia-reperfusion injury in mice. Journalof the American Society of Nephrology,2003.14(5): p.1188-1199.
5. Morigi, M., B. Imberti, C. Zoja, et al., Mesenchymal stem cells are renotropic, helpingto repair the kidney and improve function in acute renal failure. Journal of theAmerican Society of Nephrology,2004.15(7): p.1794-1804.
6. T gel, F., A. Cohen, P. Zhang, et al., Autologous and allogeneic marrow stromal cellsare safe and effective for the treatment of acute kidney injury. Stem cells anddevelopment,2009.18(3): p.475-486.
7. Thomson, J.A., J. Itskovitz-Eldor, S.S. Shapiro, et al., Embryonic stem cell linesderived from human blastocysts. science,1998.282(5391): p.1145.
8. Steenhard, B.M., K.S. Isom, P. Cazcarro, et al., Integration of embryonic stem cells inmetanephric kidney organ culture. Journal of the American Society of Nephrology,2005.16(6): p.1623-1631.
9. Kim, D. and G.R. Dressler, Nephrogenic factors promote differentiation of mouseembryonic stem cells into renal epithelia. Journal of the American Society ofNephrology,2005.16(12): p.3527-3534.
10. Vigneau, C., K. Polgar, G. Striker, et al., Mouse Embryonic Stem Cell–DerivedEmbryoid Bodies Generate Progenitors That Integrate Long Term into Renal ProximalTubules In Vivo. Journal of the American Society of Nephrology,2007.18(6): p.1709-1720.
11. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell,2006.126(4): p.663-676.
12. Morizane, R., T. Monkawa, and H. Itoh, Differentiation of murine embryonic stem andinduced pluripotent stem cells to renal lineage in vitro. Biochemical and BiophysicalResearch Communications,2009.390(4): p.1334-1339.
13. Song, B., A.M. Smink, C.V. Jones, et al., The directed differentiation of human iPScells into kidney podocytes. PLoS One,2012.7(9): p. e46453.
14. Lee, P., Y. Chien, G. Chiou, et al., Induced Pluripotent Stem Cells Without c-MycAttenuate Acute Kidney Injury via Down-Regulating the Signaling of Oxidative Stressand Inflammation in Ischemia-Reperfusion Rats. Cell transplantation,2012.
15. Li, L., R. Black, Z. Ma, et al., Use of mouse hematopoietic stem and progenitor cellsto treat acute kidney injury. American Journal of Physiology-Renal Physiology,2012.302(1): p. F9-F19.
16. Swetha, G., V. Chandra, S. Phadnis, et al., Glomerular parietal epithelial cells of adultmurine kidney undergo EMT to generate cells with traits of renal progenitors. Journalof cellular and molecular medicine,2011.15(2): p.396-413.
17. Prodromidi, E.I., R. Poulsom, R. Jeffery, et al., Bone Marrow‐Derived CellsContribute to Podocyte Regeneration and Amelioration of Renal Disease in a MouseModel of Alport Syndrome. Stem cells,2006.24(11): p.2448-2455.
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