Inducing human induced pluripotent stem cell differentiation through embryoid bodies:A practical and stable approach

Human induced pluripotent stem cells(hiPSCs)are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use.They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research.However,significant limitations exist when using conventional flat culturing methods especially concerning cell expansion,differentiation efficiency,stability maintenance and multicellular 3D structure establishment,differentiation prediction.Embryoid bodies(EBs),the multicellular aggregates spontaneously generated from iPSCs in the suspension system,might help to address these issues.Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve,EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing,differentiation efficiency enhancement,ex vivo simulation,and organoid establishment.EBs can potentially also be used in early prediction of iPSC differentiation capability.To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs,critical factors including iPSC pluripotency maintenance,generation of uniform morphology using micro-pattern 3D culture systems,proper cellular density inoculation,and EB size control are discussed on the basis of both published data and our own laboratory experiences.Collectively,the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.

Embryoid body formation from embryonic and induced pluripotent stem cells:Benefits of bioreactors

Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening.Embryoid body (EB)formation from ES cells is a common method for producing different cell lineages for further applications. However,conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation.For standardized mass EB production,a well defined scale-up platform is necessary.Recently,novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems(spinner flasks),rotating cell culture system and rotary orbital culture have allowed large-scale EB formation.Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods.This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems.Furthermore,an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently,new insights in induced pluripotent stem(iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research.These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity.Hence,culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells,although direct evidence of their use for iPS cells is still limited.

日本落叶松胚状体干化处理对萌发的影响

[目的]提高日本落叶松胚状体的萌发能力,优化日本落叶松良种繁育技术。[方法]本研究根据子叶数量和胚轴形态,对胚状体进行分类,并分别统计其萌发率;利用“滤纸容器法”对类型Ⅰ胚状体进行干化处理,统计其萌发情况。[结果]胚状体分为10种类型。类型Ⅰ胚状体萌发率为4.48%,其余类型胚状体难以萌发。干化处理14 d后,类型Ⅰ胚状体萌发率提高到69.43%,且具有干化响应的胚状体更易萌发。[结论]本研究表明日本落叶松胚状体干化处理后萌发率明显提高,为该物种良种繁育提供依据。

丝瓜子房培养类胚体发生途径诱导研究

[目的]以丝瓜(Luffa ssp.)品种“徐绿一号”的未授粉子房为试验材料,分析不同因素对类胚体形成的影响。[方法]设置L 16(215)正交试验分析子房发育阶段、4℃预冷处理、35℃黑暗热激处理、TDZ、6-BA、ABA、NAA、2,4-D对丝瓜离体子房培养获得类胚体的影响。[结果]不同因素对子房片的发育过程具有不同的效果,其中采集当天开放的雌花、4℃预冷处理、在MS培养基中添加0.04 mg/L TDZ对子房片的类胚体形成具有促进作用,添加0.04 mg/L 2,4-D对丝瓜子房类胚体形成具有抑制作用,但可以促进子房片生根;另外预冷处理与TDZ、TDZ与雌花采集时间、4℃预冷和ABA的两两交互作用对丝瓜类胚体的形成也具有显著影响。[结论]有效形成丝瓜子房类胚体途径:当天采集子房+4℃预冷处理+0.04 mg/L TDZ。

利用人诱导多能干细胞构建心脏类器官方法的研究

目的:探索人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)分化为心脏类器官的方法。方法:首先通过免疫荧光染色验证hiPSCs多能性标志物的表达;之后将hiPSCs培养为拟胚体(embryoid bodies,EBs),利用双向调控WNT(wingless and Int-1,WNT)信号通路法诱导EBs向心脏类器官分化,记录分化过程中的形态变化。最后,通过免疫荧光染色、实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)和微电极阵列(multi-electrode array,MEA)技术检测心脏类器官的细胞成分、基因表达谱、电生理特性和收缩舒张功能,验证心脏类器官分化方案的有效性。结果:免疫荧光染色显示hiPSCs中OCT4和SSEA4高表达。利用双向调控WNT信号的方法成功诱导hiPSCs产生跳动的心脏类器官。免疫荧光染色表明心脏类器官中cTNT阳性心肌细胞比例为(59.3±12.8)%,非心肌细胞比例为(40.7±12.8)%。qRT-PCR结果表明其中非心肌细胞包括平滑肌细胞、成纤维细胞及内皮细胞。与分化15D相比,分化30D的类器官中心肌成熟相关基因和心脏各项功能相关基因表达水平更高。MEA检测结果显示,随分化时间增加,类器官逐渐具备类似在体心脏的电生理特征,跳动频率减小[分化15d时跳动(58±5)vs.30d(38±5)次/min],收缩幅度增加15d时收缩幅度[(8.57±3.7)vs.30d(16.4±5.9)%]。结论:通过该方法能使hiPSCs分化出成熟且有功能的心脏类器官。

人胚胎早期发育与干细胞

人胚胎早期发育包括三个重要阶段:①从受精卵到晚期囊胚的着床前阶段;②从晚期囊胚到原肠运动前的围着床阶段;③从原肠运动到早期器官发生的原肠后阶段。后两个阶段统称为着床后早期发育阶段。妊娠过程中,不育(胚胎着床失败或流产)和胎儿出生缺陷,很大程度上是胚胎的着床后早期发育出现异常所致。人着床后早期胚胎,由于位于母体子宫,且尺寸较小,不易对其观察和研究,因此,这一阶段的胚胎发育过程长期处于“黑匣子”状态。近年来,随着单细胞组学技术和胚胎体外延长培养系统的建立,以及胚胎和胚外干细胞、类器官和类胚胎领域的快速发展,使得人胚胎着床后早期发育的神秘面纱被慢慢揭开。本文从人胚胎早期发育、胚胎和胚外干细胞、类胚胎和类器官研究的视角,结合细胞通信、谱系互作、信号梯度、黏附分子、生物力学和细胞外基质等因素对细胞分选、迁移重排和自我组织的影响,概述了人胚胎早期发育过程中的发育原理,当前胚胎和胚外干细胞的研究进展以及用其模拟人胚胎早期发育的研究现状、存在问题和发展方向,以期能够帮助理解人胚胎早期发育的奥秘。

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES cells differentiation is the growth into embryoid bodies （EBs）,which are utilized widely as the trigger of in vitro differentiation.In this study,a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established.When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds,they grew into aggregates gradually and formed simple EBs with circular structures.After 7 days＇ culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers.Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types;they were also able to form into tissue-like structures.Moreover,with introduction of ascorbic acid,ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19.The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

人诱导多能干细胞成骨分化的研究进展

骨骼疾病如骨质疏松、骨关节炎等已成为亟待解决的人类健康问题,细胞治疗及组织工程技术被认为是理想的治疗方法之一。人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)具备体外长期自我更新和分化所有三胚层来源体细胞的独特功能,已成为目前最有前景的成骨细胞来源。因此,需要构建成分明确的hiPSCs体外成骨向诱导分化体系,获得符合临床应用要求的成骨样细胞。许多团队在促进hiPSCs向成骨分化成熟的直接路径和经间充质干细胞的间接路径方面取得了实质性的进展,本文针对这两类成骨分化路径及其应用现状进行综述,以期为骨再生技术提供参考。现有研究借助拟胚体法和单层诱导法,基于生物材料,构建可支持hiPSCs体外培养和成骨向诱导分化体系。然而,目前的研究主要存在成分不明确,分化效率低等局限,基于特定化合物严格调控的分阶段式和三维定向诱导体系是未来的主要研究方向。

高渗环境调控钙黏蛋白表达并影响拟胚体分化

目的:渗透压对拟胚体(embryoid body,EB)分化的确切影响尚不明确,本研究旨在探讨增加渗透压对EB分化的影响,及该过程与钙黏蛋白之间潜在的联系。方法:本研究使用聚乙二醇300来增加培养液的渗透压,在高渗透压和常规培养液下培养EB。实验设计包括对照组、实验组(高渗组)、不同浓度的聚乙二醇300处理组和高渗+抑制剂组,采用Western blot、RT-qPCR、AM/PI染色、CCK-8、免疫细胞化学染色等检测方法,对EB的细胞活力及上皮钙黏蛋白(E-cadherin,CDH1)和神经钙黏蛋白(N-cadherin,CDH2)、三胚层标志物以及多能性标志物的表达进行了分析。结果:高渗不影响EB的细胞活力。在实验组和对照组的EB中,钙黏蛋白CDH1和CDH2的表达显示出显著差异,但无明显的上皮间质转化转变趋势。实验组中CDH2的表达显著下调,并且与渗透压变化呈现明显相关性。此外,在高渗组中,相较于对照组,EB的多能性标志物在第2~5天表达显著提高。同时,高渗组中胚层标志物的表达量明显增加,而外胚层标志物呈现下调趋势。本研究使用SB431542特异性抑制TGF-β信号上调CDH2表达,结果显示高渗组EB的中胚层和外胚层标志物的表达趋势发生了逆转。结论:当渗透压较高时,会促进EB中胚层的分化效率,这一过程可能与渗透压引起的CDH1和CDH2变化相关。

LMO4在小鼠胚胎干细胞分化为血管内皮细胞和血管生成中的作用

目的探讨转录因子LIM结构域蛋白4(LMO4)在小鼠胚胎干细胞(mESC)分化为血管内皮细胞及新生血管中的作用。方法使用逆转录聚合酶链反应(RT-PCR)方法从小鼠红系白血病细胞系MEL)中克隆小鼠Lmo4的cDNA,并亚克隆到含有小鼠胎肝激酶-1(Flk-1)启动子驱动表达Gfp的载体(pFG),构建成血管细胞特异性表达的LMO4的载体pFLG。将表达载体转染mESC,通过遗传霉素(G418)筛选,获得mESC/pFG和mESC/pFLG的细胞株;将这些mESC在体外进行自我分化,以形成4 d和10 d的胚胎体(EB),并进行成血管细胞的集落细胞形成实验(BL-CFC);以10 d-EB进行新生血管出芽实验,观察和分析出芽长短、数目;运用蛋白免疫印迹(Western blot)或定量RT-PCR方法对目的基因的表达进行检测。结果PCR结果证实成功构建了成血管细胞特异性表达的LMO4的表达载体pFLG。通过G418筛选获得mESC/pFG和mESC/pFLG的细胞株。这些mESC通过自我分化形成4 d-EB和10 d-EB,荧光显微镜下观察到EB内均可见绿色荧光标记的细胞。Western blot检测显示:与mESC相比,4 d-EB和10 d-EB的LMO4的表达显著增加。过量表达LMO4的mESC/pFLG产生BL-CFC效率为(7.70%±1.27%),而mESC/pFG细胞产生BL-CFC效率为(1.15%±0.48%),二者差异有统计学意义(P=0.021)。定量RT-PCR结果显示,Flk-1、C-kit、Tie-2、Ve-cad基因在10 d-EB/pFLG中的表达,均较10 d-EB/pFG中表达增加2倍以上。新生血管出芽实验结果显示,10 d-EB/pFLG的新生血管数量和长度均较10 d-EB/pFG增加(P<0.05)。结论过量表达LMO4促进mESC形成成血管细胞,并有利于血管内皮细胞的分化和血管的新生。

Human embryoid bodies to hepatocyte-like clusters:Preparing for translation

End-stage liver disease and acute liver failure are some of the most common causes of death worldwide,affecting over 40,000 patients in the United States,for most of whom liver transplantation is the only treatment.Transplantable livers are obtained primarily from deceased donors in the west and living donors in the east,with demand outstripping supply,leading to thousands of deaths each year for those on the transplant waiting lists.As a bridge to liver transplantation,human primary hepatocytes have been transplanted with low success,owing to their inability to grow and expand in vitro,their high sensitivity to cold storage-induced damage,and their dedifferentiation following two-dimensional culture.In the past decade,human induced pluripotent stem cells(hiPSCs)have been studied as a potential alternative to liver and primary hepatocyte transplantation through their differentiation into hepatocyte-like cells(HLCs).Differentiation of hiPSCs into HLCs is limited by the low percentage of differentiated cells that reach a mature hepatic phenotype,poor reproducibility of existing differentiation protocols,and inadequate long-term viability and function in vitro and in vivo.In this review,we will discuss the mechanisms of the various techniques that aim to improve the hepatic differentiation of hiPSCs into mature and genotypically stable HLCs for use in drug studies,as a potential bridge for liver transplantation after liver failure or as therapy for liver regeneration and replacement.

外源激素嘧啶醇与NAA对芦笋胚状体再生植株生长的影响

以芦笋花粉粒培养获得的胚状体为试材,在其形成的不定芽中,以MS+蔗糖30 g·L^(-1)+琼脂粉8 g·L^(-1)为基本培养基,添加不同质量浓度嘧啶醇(0、0.05、0.10、0.50、1.00、2.00 mg·L^(-1))和NAA(0、0.05、0.10、0.20、0.50、1.00 mg·L^(-1)),研究了嘧啶醇和NAA浓度对不同基因型再生植株生长的影响,以期优化和提高芦笋植株体外扩繁效率。结果表明:培养基添加0.50 mg·L^(-1)嘧啶醇处理的再生植株净生长高度、茎数分别增加1.05 cm和1.0根,根系变长,侧根增多;MS培养基添加0.50 mg·L^(-1)嘧啶醇和0.20 mg·L^(-1) NAA后再生植株净生长高度、茎数分别增加1.60 cm和2.0根,根系生长旺盛;并且0.50 mg·L^(-1)嘧啶醇和0.20 mg·L^(-1) NAA对芦笋胚状体再生植株的促进作用无基因型的显著差异。综上,在MS培养基中添加0.50 mg·L^(-1)嘧啶醇和0.20 mg·L^(-1) NAA,可以提高芦笋胚状体再生植株繁殖效率。

Differential Effects of Cold and Heat Shock on Embryogenic Induction and Green Plant Regeneration from Wheat (Triticum aestivum L.) Microspores

Albinism is a common problem encountered by researchers in anther/microspore cultures of cereal crops. The present study investigates the effects of temperature variations on embryogenesis of wheat (Triticum aestivum L.) microspores. Following a cold (4°C - 13°C) vs. heat (33°C) shock to wheat tillers, microspores were isolated and cultured in a liquid medium to obtain embryoids. Data on embryogenic microspore%, embryoid yield, plant regeneration% and green plant% were collected and analyzed. Cold pretreatment of 4°C or 10°C for a period of 6 or 10 days were more effective than other cold temperature regimes in inducing microspore embryogenesis. The heat shock of 33°C yielded the highest numbers of embryogenic microspores and embryoids. The albino-prone genotypes produced significantly higher green plant% following optimal cold shock, as compared to the standard 33°C heat shock. Results from present study suggest that cold shock may be a desirable alternative for germplasm that produce lower green plant% using heat shock. Lowered incubation temperature during embryoid development did not result in higher green plant.

油棕病毒诱导的基因沉默体系的建立及优化

为开展油棕油脂代谢调控相关基因鉴定研究,以油棕八氢番茄红素脱氢酶基因(phytoene desaturase gene,PDS)作为报告基因,探索以病毒载体TRV为载体在油棕胚状体上应用病毒诱导基因沉默(virus-induced gene silencing,VIGS)的可能性,并对油棕胚状体VIGS体系的相关参数进行优化。结果显示:以EHA105为菌种、侵染菌液OD_(600)=0.5、侵染时间5 min、乙酰丁香酮(AS)质量浓度20 mg/L、共培养48 h、侵染后培养时间为12 d能取得最佳的基因沉默效果。在此基础上,利用优化后的VIGS体系对油棕二酰甘油酰基转移酶基因(diacylglycerol acyltransferase gene,DGAT)进行沉默,取得了预期的基因沉默效果。

烟草未授粉子房离体培养诱导胚状体的影响因素研究

源于未授粉子房的雌性单倍体在烟草育种利用价值上优于源于花药或小孢子的雄性单倍体,开展烟草未授粉子房的离体培养对烟草单倍体育种具有重要意义。为了建立未授粉子房离体培养技术体系,分别以不同的杂种F1代为试材,采用HW培养基探讨了消毒方法、接种方式、接种密度、无机盐浓度、外源激素配比和琼脂浓度6个关键因素对胚状体诱导的影响。结果表明:花蕾不需剥开花瓣而直接放入0.1%升汞中浸泡8 min,剥开子房壁并横切子房后接种,每个125 mL三角瓶中接种2块子房,培养基中无机盐浓度额外增加60%、附加IAA 0.5 mg/L和6-BA 2 mg/L或者附加KT 2.0 mg/L和2,4-D 0.5 mg/L、琼脂浓度9 g/L有助于对胚状体的诱导。据此进一步完善了烟草未授粉子房离体培养过程中的技术参数。

黑麦胚性愈伤组织和体细胞胚胎形成过程中内源IAA和Zt变化的初步研究

黑麦胚性愈伤组织和体细胞胚胎形成过程中内源ＩＡＡ和Ｚｔ变化的初步研究邢登辉吴琴生刘大钧（首都师范大学农学系北京１０００３７）（南京农业大学农学系南京２１００９５）体细胞胚胎发生已经成为许多植物细胞全能性得以实现的主要途径，黑麦也不例外。许多报道就...

Optimization of Culture Techniques for DH Line in Brassica napus L.

[Objective] The aim of this study is to reveal the disinfectants and disinfection methods,medium components,and embryoid culture method on dissociative microspore culture.[Method] B5 as basic medium appended with different concentrations of sucrose,agar and different hormone combinations was used to optimize the culture technique for DH line in Brassica napus L.[Result] Both the 15 min disinfection of NaClO containing 5% Cl-and 10 min disinfection of 0.1% HgCl2 performed well in disinfection and subsequent embryo production;in the extraction process of dissociative microspores,B5 medium containing 2% sucrose could achieve a good embryo production effect;under dark condition microspores were firstly incubated at 32 ℃ 5-7 d,then at 25 ℃ 12-15 d,and finally transferred to 25 ℃ oscillator(60-65 r/min)for 3-7d,when the embryoid would become full ripeness;1/2MS medium appended with 1.2% agar,0.02% NAA,2.0 mg/L 6-BA,3.4 mg/L AgNO3 and 2% sucrose was helpful for embryoid differentiation and plantlet generation,presenting low degree of browning and slight vitrification.[Conclusion] The results may facilitate DH Line in rape production in large scale and high efficient transformation system.

Influences of Carbon Sources and Plant Growth Regulators on Anther Culture Efficiency of Pepper

[Objective]In order to increase anther culture efficiency of pepper.[Method]MS culture media and Bolajiaohong were used in this experiment to study the influences of carbon sources and concentrations on anther callus induction of pepper.Jiayu was taken as a material to study influences of plant growth regulators and concentrations on anther callus induction of pepper according to L16（4^5） orthogonal design.[Result]The average callus and embryoid induction rates of maltose at all concentrations were higher than these of sucrose but the difference was not significant.Taking maltose or sucrose as a carbon source,3% to 6% concentration was good for increasing induction frequencies of calli and embryoids.However,If the concentration was over 6%,the induction rates were declined dramatically with the increase of sugar concentration.The influences of growth regulators on induction rate of calli were listed as 2,4-D﹥ZT﹥NAA﹥KT﹥6-BA;the influences on induction rates of embryoids were listed as 2,4-D﹥NAA﹥ZT﹥KT﹥6-BA.The 2,4-D,ZT,NAA and KT had signficant or extremely significant influences on induction rates of calli and embryoids.2,4-D,ZT at 1.0 mg/L and NNA,KT at 0.5 mg/L had the best effects.The influences of ZT on calli and embryoids were better than those of KT and 6-BA.1.0 mg/L 2,4-D ＋1.0 mg/L ZT ＋0.5 mg/L KT ＋0.5 mg/L 6-BA was the best regulator combination for induction culture of Jiayu anther.[Conclusion]The experiment provided research basis for anther culture of pepper.

玉米幼胚的组织培养及其植株再生的研究

以成单 14 ,15 ,18三种基因型玉米为材料 ,对幼胚愈伤组织的诱导和继代培养、植株再生、再生苗的培养和移栽等方面进行了系统的研究。三种基因型玉米幼胚经诱导均可产生愈伤组织 ,但只有成单 15 ,18两种基因型玉米幼胚产生的愈伤组织可继代培养下去 .对愈伤组织进行了分类 ,并将能继代的愈伤组织转入分化培养基 ,观察到愈伤组织通过胚状体萌发和直接分化成苗两种途径再生出完整植株 .可能由于气温太低的缘故 ,再生植株经壮苗、移栽后只有少数可成活 .

锦丰梨花粉植株的诱导

采用花粉发育单核期的‘锦丰’梨花药,接种在1/2 MS附加IAA 0.2mg/L、BA1～2mg/L的培养基上,经过120天产生胚状体。胚状体转入MS附加GA3 0.1mg/L、IBA 0.2mg/L、BA 1mg/L的分化培养基上,经过85天分化幼梢。无根植株转入1/2 MS附加IAA 1.5mg/L的培养基上,经过14天诱导生根,移栽和嫩枝嫁接成活情况良好。