CN112574944A - 基于体外诱导eps发育形成类囊胚结构的方法 - Google Patents
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Abstract
本发明涉及生命科学技术领域,具体涉及一种基于体外诱导EPS发育形成类囊胚结构的方法,包括如下步骤:A.利用BMP4诱导EPS细胞分化为滋养外胚层样细胞;B.将滋养外胚层样细胞和EPS细胞按比例使用N2B27‑LCDM培养基或/和IVCI培养基的混合培养为类胚囊结构。本发明的方法可行性强,可重复,可以成功利用人EPS体外诱导培养形成的人工类囊胚结构。
Description
技术领域
本发明涉及生命科学技术领域,具体涉及一种基于体外诱导EPS发育形成类囊胚结构的方法。
背景技术
胚胎发育是人类个体形成过程中的起始环节,人类受精卵的发育始于一系列卵裂和形态发生重排,形成囊胚。晚期囊胚包括三个不同的细胞谱系(epiblast,EPI)和两个胚外组织(primitive endoderm,PE;trophoectoderm,TE),在植入子宫后,前者产生三胚层细胞以及整个胎儿,而后两者分别产生卵黄膜和胎盘。对于囊胚前期人胚胎的发育,已经开展了广泛的研究,取得了重要的进展(可参见:Yan L,Yang M,Guo H,Yang L,Wu J,Li R,LiuP,Lian Y,Zheng X,Yan J et al:Single-cell RNA-Seq profiling of humanpreimplantation embryos and embryonic stem cells.Nature structural&molecularbiology 2013,20(9):1131-1139.;Deglincerti A,Croft GF,Pietila LN,Zernicka-Goetz M,Siggia ED,Brivanlou AH:Self-organization of the in vitro attachedhuman embryo.Nature2016,533(7602):251-254.)。然而在发育到第7天,人胚胎需要植入母亲的子宫内膜中才能继续发育。这个阶段的胚胎在子宫内发生的变化以及导致这些变化的关键细胞和分子事件,由于材料的无法获得以及缺乏相应的体外研究体系导致这些问题仍不清楚(可参见:Nakamura T,Okamoto I,Sasaki K,Yabuta Y,Iwatani C,Tsuchiya H,Seita Y,Nakamura S,Yamamoto T,Saitou M:A developmental coordinate ofpluripotency among mice,monkeys and humans.Nature 2016,537(7618):57-62.;Rossant J,Tam PPL:New Insights into Early Human Development:Lessons for StemCell Derivation and Differentiation.Cell stem cell 2017,20(1):18-28.)。着床后人胚胎发育是生命发生的重要研究领域,胚胎无法植入以及在此阶段的发育异常是导致早期妊娠流产的主要原因(可参见:Rossant J:Human embryology:Implantation barrierovercome.Nature 2016,533(7602):182-183.)。着床后胚胎发育关键事件的解析对组织器官再生研究和早期胚胎发育疾病的预防与治疗具有重要的科学意义(可参见:Bedzhov I,Zernicka-Goetz M:Self-organizing properties of mouse pluripotent cellsinitiate morphogenesis upon implantation.Cell 2014,156(5):1032-1044.)。
由于取材及伦理的限制,人胚胎着床后的发育研究几乎不可能开展,该领域的进一步发展亟待创新的技术突破。2016年英国剑桥大学和美国洛克菲勒大学科学家Magdalena Zernicka-Goetz和AliH.Brivanlou共同建立胚胎体外延迟培养技术,突破了着床后胚胎发育的研究瓶颈,他们在2维(2D)培养皿中成功将人囊胚(E5-E6)延迟培养到12-13天(可参见:Shahbazi MN,Jedrusik A,Vuoristo S,Recher G,Hupalowska A,Bolton V,Fogarty NNM,Campbell A,Devito L,Ilic D et al:Self-organization of the humanembryo in the absence of maternal tissues.Nature cell biology 2016,18(6):700-708.;Deglincerti A,Croft GF,Pietila LN,Zernicka-Goetz M,Siggia ED,BrivanlouAH:Self-organization of the in vitro attached human embryo.Nature 2016,533(7602):251-254.)。这些2D培养的胚胎初步显示出体内胚胎发育的一些简单结构,扩展了对人类早期胚胎发育的认识,Science杂志将其评为2016年全球十大科技进展之首。2019年北京大学汤富酬教授和北京大学第三医院的乔杰教授团队运用这一技术,结合单细胞多组学技术,揭示了人类早期胚胎着床后发育的基因表达调控网络和DNA甲基化动态变化规律(可参见:Zhou F,Wang R,Yuan P,Ren Y,Mao Y,Li R,Lian Y,Li J,Wen L,Yan L et al:Reconstituting the transcriptome and DNA methylome landscapes of humanimplantation.Nature 2019,572(7771):660-664.)。然而在培养皿中进行2D培养的胚胎存在一些重要的缺陷,比如二维培养的胚胎是扁平的,与体内3D胚胎有明显的差异;2D培养的胚胎其细胞之间的拓扑关系和连接与体内胚胎存在明显的差异;虽然2D培养的胚胎的滋养外胚层12天后仍在继续存活,但是整个胚胎结构发生了坍塌和出现了发育的紊乱,导致很多细胞类型(羊毛上皮)、腔(羊膜腔、卵黄囊腔)和结构(基底膜、前后轴、原条)无法在2D培养的胚胎中清楚地观测到(可参见:Martyn I,Kanno TY,Ruzo A,Siggia ED,BrivanlouAH:Self-organization of a human organizer by combined Wnt and Nodalsignalling.Nature 2018,558(7708):132-135.)。因此,这些缺陷使得该体系很难真正模拟体内胚胎的发育。2020年昆明理工大学季维智教授和李天晴教授团队首次建立人胚胎三维培养系统,绘制了人原肠胚前期胚胎的发育全景图,这些3D培养条件下的胚胎能高度地模仿体内胚胎的发育,经历不同形态的发育并自发组装成2D条件下无法产生的3D结构,包括胚胎双层胚盘、羊膜(amnion)、基底膜(basal membrane)、初级和灵长类独特的次级卵黄膜囊、前后轴以及原条(可参见:Xiang L,Yin Y,Zheng Y,Ma Y,Li Y,Zhao Z,Guo J,Ai Z,Niu Y,Duan K et al:A developmental landscape of 3D-cultured human pre-gastrulation embryos.Nature 2020,577(7791):537-542.)。这项研究成果成为人着床后的早期胚胎发育建立了重要的研究基础。
由于人胚胎体外培养不能超过14天规则的限制,上述工作停止于受精后的14天,因此诸如三胚层分化、细胞间互作、原始生殖细胞特化等许多科学问题尚未回答,而这些问题又是业界关心的重要基础科学问题。2019年中科院动物所王红梅研究员、李磊研究员、昆明动物所郑萍研究员合作团队和昆明理工大学季维智教授、谭韬教授团队分别利用灵长类动物模型实现了食蟹猴/猕猴胚胎连续培养20天的体外培养,成功模拟了胚胎着床和原肠运动过程(可参见:Ma H,Zhai J,Wan H,Jiang X,Wang X,Wang L,Xiang Y,He X,Zhao ZA,Zhao B et al:In vitro culture of cynomolgus monkey embryos beyond earlygastrulation.Science 2019,366(6467).;Niu Y,Sun N,Li C,Lei Y,Huang Z,Wu J,SiC,Dai X,Liu C,Wei J et al:Dissecting primate early post-implantationdevelopment using long-term in vitro embryo culture.Science 2019,366(6467).)。研究者们结合食蟹猴/猕猴胚胎体外延迟培养系统、形态学鉴定以及单细胞组学测序等分析,证实了体外和体内发育胚胎在形态学与基因表达特征上高度相似,且均能在体外重现体内胚胎的多个重要生物学事件,研究结果同时揭示了灵长类着床后胚胎发育中不同细胞谱系间的相互作用,为进一步理解着床后胚胎发育微环境(niche)的重要作用提供了新的思路,这项研究结果有助于加深人们对早期着床后胚胎发育的理解,对器官再生研究也有重要指导意义。
发明内容
基于现有技术,本发明的目的在于提供基于体外诱导EPS发育形成类囊胚结构的方法。
本发明采用如下技术方案:
本发明提供一种基于体外诱导EPS(人潜能扩展的多能干细胞)发育形成类囊胚结构的方法,包括如下步骤:
A.利用BMP4(骨形态发生蛋白4:Gibco,Catalog#PHC9531,使用浓度25ng/mL)诱导EPS细胞分化为滋养外胚层样细胞;
B.将滋养外胚层样细胞和EPS细胞按比例使用N2B27-LCDM培养基和IVCI培养基的混合培养基培养为类胚囊结构。
进一步地,步骤A中,所述滋养外胚层样细胞的分化过程如下:
1)将EPS细胞使用BMP4诱导培养,经清洗、破膜、封闭处理后,置于分化一抗中过夜孵育;
2)次日清洗后,置于分化二抗中室温孵育,孵育完成后,清洗;
3)清洗完成后的干细胞置于CK7直染抗体(Anti-Cytokeratin7antibody[EPR1619Y]-Cytoskeleton Marker(Alexa647),购买于Abcam,Catalog#ab192077,使用比例1:200)和DAPI(ProLong Gold Antifade Mountant with DAPI,购买于Invitrogen,Catalog#P36941)混合溶液中室温孵育,清洗。DAPI,即4',6-二脒基-2-苯基吲哚。
进一步地,步骤1)中,将EPS细胞使用25ng/ml的BMP4诱导培养基培养3-5天,每天更换培养基。
进一步地,步骤1)中,所述清洗、破膜、封闭处理的过程如下:将培养完成的细胞用PBS清洗后,加入4%甲醛,室温固定30分钟,PBS清洗后加入0.3%Triton-x100(Sigma-Aldrich,Catalog#X100-100mL),室温条件下破膜1小时;PBS清洗后加入2%BSA(Sigma-Aldrich,Catalog#V900933-100G),室温封闭30分钟。BSA一般指牛血清白蛋白。
进一步地,所述分化一抗为:OCT4(Human Oct-4A mouse mAb,小鼠来源,购买于R&D,Catalog#MAB17591,使用比例1:200)和GATA3(Anti-GATA3antibody[EPR16651],兔来源,购买于Abcam,Catalog#ab199428,使用比例1:200)一抗。
进一步地,所述分化二抗为:anti-mouse 555(Anti-mouse IgG Fab2 AlexaFluor(R)555Molecular Probes,购买于Thermo Fisher Scientific,Catalog#4409S,使用比例1:500)、anti-rabbit 488(Goat anti-Rabbit IgG(H+L)Highly Cross-AdsorbedSecondary Antibody,Alexa Fluor Plus 488,购买于Thermo Fisher Scientific,Catalog#A32731,使用比例1:500)。
进一步地,步骤B中,所述类囊胚结构的培养过程如下:将滋养外胚层样细胞和EPS细胞按比例用消化酶消化成单细胞,经两次培养后,挑选收集类囊胚结构。
进一步地,所述滋养外胚层样细胞和EPS细胞按比例用Tryple消化酶消化成单细胞,其中滋养外胚层样细胞和EPS细胞以50万:10万/孔的细胞量混合,1300g离心5分钟,使细胞聚集于小槽底部,置于37℃5%O2培养箱中培养。
进一步地,所述两次培养过程如下:第一天使用N2B27-LCDM培养基进行第一次培养;第二天更换重组胚胎培养基进行培养,连续培养五天。
进一步地,所述重组胚胎培养基为按1.5:1比例混合N2B27-LCDM培养基和IVCI培养基制成重组胚胎培养基。
另一方面,本发明提供一种EPS细胞分化为滋养外胚层样细胞的的方法,其分化过程如下:
a)将EPS细胞使用BMP4诱导培养,经清洗、破膜、封闭处理后,置于分化一抗中过夜孵育;
b)次日清洗后,置于分化二抗中室温孵育,孵育完成后,清洗;
c)清洗完成后的干细胞置于CK7直染抗体和DAPI混合溶液中室温孵育,清洗。
进一步地,步骤a)中,将EPS细胞使用25ng/ml的BMP4诱导培养基培养3-5天,每天更换培养基。
进一步地,步骤a)中,所述清洗、破膜、封闭处理的过程如下:将培养完成的细胞用PBS清洗后,加入4%甲醛,室温固定30分钟,PBS清洗后加入0.3%Triton-x100(Sigma-Aldrich,Catalog#X100-100mL),室温条件下破膜1小时;PBS清洗后加入2%BSA(Sigma-Aldrich,Catalog#V900933-100G),室温封闭30分钟。
进一步地,所述分化一抗为:OCT4(Human Oct-4A mouse mAb,小鼠来源,购买于R&D,Catalog#MAB17591,使用比例1:200)和GATA3(Anti-GATA3antibody[EPR16651],兔来源,购买于Abcam,Catalog#ab199428,使用比例1:200)一抗
进一步地,所述分化二抗为:anti-mouse 555(Anti-mouse IgG Fab2 AlexaFluor(R)555Molecular Probes,购买于Thermo Fisher Scientific,Catalog#4409S,使用比例1:500)、anti-rabbit 488(Goat anti-Rabbit IgG(H+L)Highly Cross-AdsorbedSecondary Antibody,Alexa Fluor Plus 488,购买于Thermo Fisher Scientific,Catalog#A32731,使用比例1:500)。
进一步地,所述直染抗体和DAPI为:CK7直染抗体(Anti-Cytokeratin7antibody[EPR1619Y]-Cytoskeleton Marker(Alexa647),购买于Abcam,Catalog#ab192077,使用比例1:200),DAPI(ProLong Gold Antifade Mountant with DAPI,购买于Invitrogen,Catalog#P36941)。
与现有技术相比,本发明具有如下的有益效果:
本发明通过添加BMP4诱导EPS细胞分化为滋养外胚层样细胞,滋养外胚层样细胞和EPS细胞混合消化为单细胞后再经N2B27-LCDM培养基和/或IVCI培养基的混合培养为类胚囊结构,类囊胚结构可经过进一步地培养为人造胚胎,为器官再生研究提供新的途径。本发明的方法可行性强,可重复,可以成功利用人EPS体外诱导培养形成的人工类囊胚结构。
本发明通过采用BMP4诱导EPS细胞分化,大大缩短了滋养外胚层样细胞的获得时间,分化时间为72小时左右,所得细胞数量也高;
本发明提高了滋养外胚层样细胞培养为类囊胚结构的效率,经过N2B27-LCDM培养基培养EPS和滋养外胚层样细胞的混合细胞,经过6天即完成培养,得到类囊胚结构,为研究早期胚胎发育模式和早期妊娠失败的发生发展机制提供技术支持。
本发明是根据目前所使用的研究所用的人胚胎的不足而提出的,经过反复模式和调试所总结的新的技术方法,类囊胚结构与人正常囊胚结构相比,不仅在结构形态上高度相似,而且各分子标记物表达和胚层发育特点高度吻合,同时回避了医学伦理学中人类胚胎体外培养14天的限制,为以类囊胚结构为模型研究胚胎早期发育模式和早期妊娠发育失败的分子机制提供技术保障和理论基础。
附图说明
图1将人EPS细胞体外经过72小时诱导分化成滋养外胚层样细胞;图中,(A)BMP4诱导第1天,细胞由高亮的克隆变扁,且细胞增大;(B)BMP4诱导第2天,细胞继续增大变扁;(C)BMP4诱导第3天,上皮样细胞逐渐增多并融合;(D)BMP4诱导第1天,细胞仍然高表达OCT4,仅仅少量细胞表达滋养层细胞标记物GATA3,而不表达CK7;(E)BMP4诱导第2天,细胞表达OCT4降低,表达滋养层细胞标记物GATA3的细胞增多,开始出现CK7表达的细胞;(F)BMP4诱导第3天,细胞表达OCT4降低,表达滋养层细胞标记物GATA3的细胞增多,开始出现CK7表达的细胞。ABC,scale bar=200μm,DEF,scale bar=100μm。
图2将分化的滋养外胚层样细胞与EPS按照一定比例组合,在3D培养体系下,通过IVC1/LCDM培养,形成类囊胚结构。(A)人EPS细胞在体外第0天至第5天逐渐形成类囊胚结构的过程;(B)人EPS细胞在体外诱导形成类囊胚结构的形态,B,scale bar=100μm。
图3本发明生成的类囊胚结构与人类受精的囊胚的不同胚层的分子标记物免疫荧光染色结果。(A)白光记录EPS构建的类囊胚结构形态,可见类似于人类囊胚的结构,红色箭头为ICM样细胞团,a1 scale bar=200μm,a2 scale bar=100μm;(B)人类正常囊胚的结构形态,scale bar=100μm;(C)免疫荧光检测可见囊腔表达GATA3和CK7,ICM样组织成分表达OCT4。以上图片scale bar=50μm。
图4利用3D体外培养技术可将本发明制备的类囊胚结构培养到第10天的分子标记物免疫荧光染色结果。(A)类囊胚结构延时培养至8天(d1)和10天(d2)的形态;(B)人囊胚延时培养至10天的形态;(C)免疫荧光检测显示其能正常发育且表达OCT4、GATA3以及CK7。以上图片scale bar均为100μm。
具体实施方式
下面结合具体实施例,进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。
N2B27-LCDM培养基:45%DMEM/F12(Thermo Fisher Scientific,Catalog#11330-032),45%Neurobasal(Thermo Fisher Scientific,Catalog#21103-049),5%knockoutserum replacement(Thermo Fisher Scientific,Catalog#A3181502),0.5%N2supplement(Stemcell,Catalog#07152),1%B27supplement(Stemcell,Catalog#05711),1%penicillin-streptomycin(Thermo Fisher Scientific,Catalog#15140-122),1%GlutaMAX supplement(Thermo Fisher Scientific,Catalog#35050-061),1%MEM non-essential amino acids solution(Thermo Fisher Scientific,Catalog#11140-050),0.1mMβ-mercaptoethanol(Thermo Fisher Scientific,Catalog#21985-023),10ng/mlrecombinant human LIF(L,Peprotech,Catalog#300-05),1μM CHIR 99021(C,Tocris,Catalog#4423),2μM(S)-(+)-Dimethindene maleate(D,Tocris,Catalog#1425),2μMMinocycline hydrochloride(M,Santa Cruz Biotechnology,Catalog#sc-203339),1μMIWR-endo-1(Selleckchem,Catalog#S7086)and 2μM Y-27632(Tocris,Catalog#1254)。
BMP4培养基:45%DMEM/F12(Thermo Fisher Scientific,Catalog#11330-032),45%Neurobasal(Thermo Fisher Scientific,Catalog#21103-049),5%knockout serumreplacement(Thermo Fisher Scientific,Catalog#A3181502),0.5%N2 supplement(Stemcell,Catalog#07152),1%B27supplement(Stemcell,Catalog#05711),1%penicillin-streptomycin(Thermo Fisher Scientific,Catalog#15140-122),1%GlutaMAX supplement(Thermo Fisher Scientific,Catalog#35050-061),1%MEM non-essential amino acids solution(Thermo Fisher Scientific,Catalog#11140-050),0.1mMβ-mercaptoethanol(Thermo Fisher Scientific,Catalog#21985-023),25ng/mLrecombinant human BMP4(Thermo Fisher Scientific,Catalog#PHC9531)and 2mM Y-27632(Tocris,Catalog#1254)。
IVC1培养基:75%Advanced DMEM/F12(Thermo Fisher Scientific,Catalog#12634010),20%Fetal Bovine Serum(Corning,Catalog#35-081-CV),1%penicillin-streptomycin(Thermo Fisher Scientific,Catalog#15140-122),2mM GlutaMAXsupplement(Thermo Fisher Scientific,Catalog#35050-061),1%Insulin-Transferin-Selenium-X100(Thermo Fisher Scientific,Catalog#51500-056),8nMβ-estradiol(Sigma-Aldrich,Catalog#E8875-250MG),200ng/ml progesterone(Sigma-Aldrich,Catalog#P0130-25G),25μM N-acetyl-l-cysteine(Sigma-Aldrich,Catalog#A7250-5G)。
IVC2培养基:68%Advanced DMEM/F12(Thermo Fisher Scientific,Catalog#12634010),30%knockout serum replacement(Thermo Fisher Scientific,Catalog#A3181502),1%penicillin-streptomycin(Thermo Fisher Scientific,Catalog#15140-122),2mM GlutaMAX supplement(Thermo Fisher Scientific,Catalog#35050-061),1%Insulin-Transferin-Selenium-X100(Thermo Fisher Scientific,Catalog#51500-056),1%sodium pyruvate solution(Sigma-Aldrich,Catalog#S8636),0.37%sodium DL-lactate solution(Sigma-Aldrich,Catalog#L7900),8nMβ-estradiol(Sigma-Aldrich,Catalog#E8875-250MG),200ng/ml progesterone(Sigma-Aldrich,Catalog#P0130-25G),25μM N-acetyl-l-cysteine(Sigma-Aldrich,Catalog#A7250-5G)。
实施例1
BMP4诱导EPS细胞向滋养外胚层样细胞分化
一种利用人潜能扩展的多能干细胞诱导的滋养外胚层样细胞,本实施例的主要步骤包括:
①EPS细胞的培养传代时以1.0×105/四孔板的量接种于0.5%明胶铺板的培养皿,使用BMP4(25ng/ml)诱导培养基;
②诱导3-5天,每天更换诱导培养基,诱导过程中细胞逐渐变平、变大,PBS清洗后,加入4%甲醛,室温固定30分钟,PBS清洗后加入0.3%Triton-x100(Sigma-Aldrich,Catalog#X100-100mL),室温条件下破膜1小时;PBS清洗后加入2%BSA(Sigma-Aldrich,Catalog#V900933-100G),室温封闭30分钟;
③配置OCT4(Human Oct-4A mouse mAb,小鼠来源,购买于R&D,Catalog#MAB17591,使用比例1:200)和GATA3(Anti-GATA3antibody[EPR16651],兔来源,购买于Abcam,Catalog#ab199428,使用比例1:200),置入已利用BMP4诱导的EPS分化形成的滋养外胚层样细胞后放置于4℃过夜孵育。
④次日PBS清洗3次,配置二抗anti-mouse 555(Anti-mouse IgG Fab2AlexaFluor(R)555Molecular Probes,购买于Thermo Fisher Scientific,Catalog#4409S,使用比例1:500)、anti-rabbit 488(Goat anti-Rabbit IgG(H+L)Highly Cross-AdsorbedSecondary Antibody,Alexa Fluor Plus 488,购买于Thermo Fisher Scientific,Catalog#A32731,使用比例1:500)后,置入滋养外胚层样细胞后于室温孵育2小时;
⑤PBS清洗3次,配置CK7直染抗体(Anti-Cytokeratin7antibody[EPR1619Y]-Cytoskeleton Marker(Alexa647),购买于Abcam,Catalog#ab192077,使用比例1:200)和DAPI(ProLong Gold Antifade Mountant with DAPI,购买于Invitrogen,Catalog#P36941)混合溶液,置入已利用BMP4诱导的EPS分化形成的滋养外胚层样细胞后于室温孵育1小时;
⑥PBS清洗后行激光共聚焦显微镜拍摄记录(图1)。
实施例2类囊胚结构的构建和检测
一种基于体外诱导人潜能扩展的多能干细胞发育形成的人工类囊胚结构的技术体系,其步骤包括:
(1)类囊胚结构的构建
①本次培养皿使用AggreWellTM400(Stemcell,Catalog#34415)六孔培养板,其每孔内含7000个小凹槽,便于细胞聚集成团生长,以每小槽10-15个细胞计算每个孔的细胞量7×104-1.0×105,使用前需要抗黏附剂(Stemcell,Catalog#07010)室温处理15分钟,用PBS清洗干净后使用;
②用TrypLE消化酶(Thermo Fisher Scientific,Catalog#12605010)将生长状态好的EPS和BMP4诱导的滋养外胚层样细胞消化成单细胞,以10万:50万/孔的细胞量混合,1300g离心5分钟,使细胞聚集于小槽底部,置于37℃5%O2培养箱中培养,第一天使用N2B27-LCDM培养基培养。
③将N2B27-LCDM培养基和IVCI培养基按1.5:1比例混合成重组胚胎培养基,第二天更换重组胚胎培养基,此时小心操作,避免细胞团受振动的影响而聚集更换重组胚胎培养基培养。
④第三天可见细胞已经聚集成小团于小槽底部,部分细胞团开始形成腔,更换重组胚胎培养基继续培养;
⑤第四、五天细胞团增大容易聚集成群,细胞团的腔继续扩大,类囊胚初步形成,更换重组胚胎培养基继续培养;
⑥第六天,使用口吸管在显微镜进行挑选收集类囊胚结构,拍摄记录细胞形态(图2)。
实施例3类囊胚结构OCT4、GATA3以及CK7表达的免疫荧光检测
①体视镜下用口吸管挑选形态结构完整的类囊胚结构,转移至含4%甲醛四孔板内,室温固定30分钟,PBS清洗类囊胚后置入0.3%Triton-x100(Sigma-Aldrich,Catalog#X100-100mL),室温条件下破膜1小时;PBS清洗类囊胚结构后置入2%BSA(Sigma-Aldrich,Catalog#V900933-100G),室温封闭30分钟;
②配置OCT4(Human Oct-4A mouse mAb,小鼠来源,购买于R&D,Catalog#MAB17591,使用比例1:200)和GATA3(Anti-GATA3antibody[EPR16651],兔来源,购买于Abcam,Catalog#ab199428,使用比例1:200)一抗,置入类囊胚结构后放置于4℃过夜孵育。
③进一步地,所述分化二抗为:。
④次日PBS清洗类囊胚结构,配置二抗anti-mouse 555(Anti-mouse IgG Fab2Alexa Fluor(R)555Molecular Probes,购买于Thermo Fisher Scientific,Catalog#4409S,使用比例1:500)、anti-rabbit 488(Goat anti-Rabbit IgG(H+L)Highly Cross-Adsorbed Secondary Antibody,Alexa Fluor Plus 488,购买于Thermo FisherScientific,Catalog#A32731,使用比例1:500)后,置入类囊胚后于室温孵育2小时;
⑤PBS清洗类囊胚,在CK7直染抗体(Anti-Cytokeratin7antibody[EPR1619Y]-Cytoskeleton Marker(Alexa647),购买于Abcam,Catalog#ab192077,使用比例1:200)和DAPI(ProLong Gold Antifade Mountant with DAPI,购买于Invitrogen,Catalog#P36941)混合溶液中置入类囊胚结构后于室温孵育1小时;
⑥PBS清洗类囊胚结构后行激光共聚焦显微镜拍摄记录(图3)。
实施例4类囊胚结构的延时培养及检测
①向八孔板中加入50μL/孔未稀释的Geltrex/Matrigel(Corning,Catalog#354277)并均匀铺满孔底,置入37℃培养箱孵育30分钟,再向每孔中加入150μL IVC1,体视镜下用口吸管挑选形态结构完整的类囊胚结构,转移至八孔板内培养。
②延迟培养第1天(人造胚胎第七天),类囊胚结构尚处于未贴壁状态,使用IVC1培;
③第二天(人造胚胎第八天),观察类囊胚结构贴壁生长的状况,没有贴壁生长继续使用IVC1培养基,如果已经贴壁生长则更换IVC2培养基;
④第三天(人造胚胎第九天),类囊胚结构几乎全部贴壁生长,更换IVC2培养基;
⑤第四至七天(人造胚胎第十至十三天),观察类囊胚结构生长情况,每天更换IVC2培养基。贴壁后根据需要获取不同时间的延迟培养的胚胎,通过免疫荧光实验对其与正常人胚胎发育状态进行比较(图4)。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,包括如下步骤:
A.利用BMP4诱导EPS细胞分化为滋养外胚层样细胞;
B.将滋养外胚层样细胞和EPS细胞按比例使用N2B27-LCDM培养基和IVCI培养基的混合培养基培养为类胚囊结构。
2.根据权利要求1所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,步骤A中,所述滋养外胚层样细胞的分化过程如下:
1)将EPS细胞使用BMP4诱导培养,经清洗、破膜、封闭处理后,置于分化一抗中过夜孵育;
2)次日清洗后,置于分化二抗中室温孵育,孵育完成后,清洗;
3)清洗完成后的干细胞置于CK7直染抗体和DAPI混合溶液中室温孵育,清洗。
3.根据权利要求2所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,步骤1)中,将EPS细胞使用25ng/ml的BMP4诱导培养基培养3-5天,每天更换培养基。
4.根据权利要求2所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,步骤1)中,所述清洗、破膜、封闭处理的过程如下:将培养完成的细胞用PBS清洗后,加入4%甲醛,室温固定30分钟,PBS清洗后加入0.3%Triton-x100,室温条件下破膜1小时;PBS清洗后加入2%BSA,室温封闭30分钟。
5.根据权利要求2所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,所述分化一抗为:OCT4和GATA3一抗;
所述分化二抗为:anti-mouse 555、anti-rabbit 488。
6.根据权利要求1所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,步骤B中,所述类囊胚结构的培养过程如下:将滋养外胚层样细胞和EPS细胞按比例用消化酶消化成单细胞,经两次培养后,挑选收集类囊胚结构。
7.根据权利要求6所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,所述滋养外胚层样细胞和EPS细胞按比例用TrypLE消化酶消化成单细胞,其中滋养外胚层样细胞和EPS细胞以50万:10万/孔的细胞量混合,1300g离心5分钟,使细胞聚集于小槽底部,置于37℃5%O2培养箱中培养。
8.根据权利要求7所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,所述两次培养过程如下:第一天使用N2B27-LCDM培养基进行第一次培养;第二天更换重组胚胎培养基进行培养,连续培养五天。
9.根据权利要求8所述基于体外诱导EPS发育形成类囊胚结构的方法,其特征在于,所述重组胚胎培养基为按1.5:1比例混合N2B27-LCDM培养基和IVCI培养基制成重组胚胎培养基。
10.一种将EPS细胞诱导分化为滋养外胚层样细胞的方法,其特征在于,分化过程如下:
a)将EPS细胞使用BMP4诱导培养,经清洗、破膜、封闭处理后,置于分化一抗中过夜孵育;
b)次日清洗后,置于分化二抗中室温孵育,孵育完成后,清洗;
c)清洗完成后的干细胞置于CK7直染抗体和DAPI混合溶液中室温孵育,清洗。
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