应用小鼠胚胎干细胞（ESCs）裂解液逆转化胎儿成纤维细胞（MEFs）为诱导性多能干细胞（iPSCs）的研究
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摘要
诱导已分化体细胞发生去分化并形成类似于ESCs多能性细胞特征的过程称作“体细胞重编程”。从ESCs裂解液中提取活性物质诱导已分化体细胞重编程为多能干细胞的过程,既没有诱导性干细胞基因组染色质的直接参与,也没有被诱导体细胞基因组DNA结构序列的变化；研究ESCs提取物中有活性细胞因子的作用,有利于发现和识别与重编程相关的细胞因子及其表达调控机制,并进而研究其相互作用关系；因为ESCs的提取物直接诱导靶细胞使其发生了生物学变化,而没有引起靶细胞基因组碱基序列发生结构学变化,不会使靶细胞发生遗传学变异,因此,应用这项技术对体细胞重编程所诱导形成的多能干细胞具有更好的生物安全性；以上种种优势赋予这项研究内容在生物医学领域具有重要的理论价值和广阔的应用前景。
本研究通过优化小鼠体内受精囊胚、杂合二倍体孤雌囊胚、人工重组异期卵母细胞核二倍体孤雌囊胚(含有速激核成熟新生鼠生长初期卵母细胞与成熟卵母细胞基因组二倍体小鼠孤雌囊胚)的获取途径,获得不同囊胚来源的ESCs。采用形态学观察法、碱性磷酸酶(AKP)染色法、核型分析法、Oct-4和SSEA-1免疫细胞化学检测法、Oct-4基因和Sox-2基因表达检测法、类胚体(EB)分化能力检测等方法对其ESCs的特性进行鉴定。用于被诱导的受体MEFs,采用曲古抑菌素A(TSA)和5-氮杂-2’-脱氧胞苷(5-aza-dc)预处理,然后再经过适宜浓度链球菌溶血素O(SLO)渗透化处理；用于起诱导作用的不同囊胚来源的小鼠ESCs,采用液氮反复冻融和离心的方法,得到不同类型ESCs的无细胞裂解液；将受体细胞孵育在无细胞裂解液的培养液中进行重编程逆转化性诱导培养,获得来源于体细胞的多能干细胞。对诱导所得多能干细胞进行多能干细胞特性的鉴定。借助双重PCR性别鉴定的方法,分选出来源于雌性的有性生殖ESCs,孤雌胚胎干细胞(pESCs)和重构pESCs,三种雌性胚胎干细胞分别通过冻融和离心的方法,获得三种不同类型雌性ESCs(供体细胞)的无细胞裂解液,将此作为诱导剂重编程逆转化雄性MEFs(受体细胞),获得多能干细胞后,依据供体细胞和受体细胞的性别差异,采用双重PCR性别鉴定的方法确定重编程所得多能干细胞集落的来源。最后以鸡成纤维细胞作为重编程的受体细胞,探讨小鼠ESCs裂解液进行种间体细胞重编程、逆转化鸡胚成纤维细胞形成多能干细胞的可行性,并通过核型分析鉴定其所得多能干细胞的来源。
通过上述研究,旨在比较不同来源ESCs裂解液介导体细胞重编程逆转化为多能干细胞的可能性及其效率,筛选适宜的共孵育方式,验证重编程体系的可靠性,比较有性生殖ESCs和无性生殖pESCs裂解液的作用效果,进一步研究父本基因缺失情况下对体细胞重编程逆转化效率的作用和对异种动物体细胞诱导形成多能干细胞的可能性。这项研究结果将为今后进一步探索ESCs和pESCs裂解液中重编程有效成分的筛选、鉴定和物种间多能干细胞的研究奠定基础。研究结果如下：
1.用于分离小鼠ESCs(?)勺三种囊胚有效生成途径研究
1.1小鼠体内受精囊胚内细胞团(ICM)集落获取的条件优化
本研究首先通过比较不同胎龄MEFs的生长状况和连续传代过程中细胞的生长情况,研究影响MEFs分离和培养效率的因素,旨在为早期胚胎共培养体系选择更加有效的MEFs的分离培养体系,用于MEFs饲养层的制备、ESCs的分离培养和被诱导受体细胞的筛查处理。然后,采集3.5d、4d和4.5d胎龄的胚胎,分别移入DMEM培养液、DMEM+白血病抑制因子(LIF)培养液和包含有MEFs贴壁生长细胞的共培养体系中进行体外培养,比较三种培养体系中ICM孵出的时间、孵化囊胚贴壁比率和ICM集落形成比率,旨在筛选获取优质ICM集落的实验方法,为ESCs(?)勺分离培养奠定基础。结果表明：采用12.5-14.5d胎龄的小鼠胎儿分禺MEFs,传代培养后选取第3代MEFs制备饲养层,采集4d的小鼠囊胚置于其MEFs饲养层的共培养体系中,经体外培养3-4d,可以获取优质的ICM,这为体内受精囊胚来源ESCs的分离培养奠定了基础。
1.2小鼠杂合二倍体孤雌囊胚生成途径的优化
本研究采用5种化学激活方法即乙醇、乙醇+6-二甲基氨基嘌呤(6-DMAP)、乙醇+6-DMAP+细胞松弛素B (CB)、SrCl2、SrCl2+CB处理小鼠成熟卵母细胞,通过观察激活卵母细胞第二极体排出情况和核型分析,探讨不同激活方法所得激活卵母细胞染色体倍性的倾向类型,比较不同染色体倍性激活卵母细胞的体外发育能力,筛选有利于形成杂合二倍体孤雌囊胚的激活方法。结果表明：在含有10%乙醇培养液中激活卵母细胞5min的基础上,添加6-DMAP有利于抑制第二极体的排出,促进双原核(2PN)杂合二倍体孤雌胚胎的形成,获取更高的过二细胞阻滞率和桑葚胚/囊胚发育率；采用含有10mM SrCl2和CB处理卵母细胞4h,也能够获取较多的双原核(2PN)杂合二倍体和较高的桑葚胚/囊胚发育率,因此,以上两种孤雌激活方法适宜用于获取小鼠杂合二倍体孤雌囊胚。
1.3人工重组异期卵母细胞核二倍体孤雌囊胚(含有速激核成熟新生小鼠生长初期卵母细胞和M Ⅱ期卵母细胞基因组的二倍体孤雌胚胎)的构建
本研究分别研究了CB对生发泡(GV)期卵母细胞去核效果的影响、去除成年小鼠MⅠ期卵母细胞GV的方法选择、新生小鼠生长初期卵母细胞与去核GV期卵母细胞黏合体系的筛选、电融合条件选择以及重构胚化学激活方法的筛选等,旨在构建含有速激核成熟新生小鼠生长初期卵母细胞和MⅡ期卵母细胞基因组的二倍体孤雌胚胎,用于ESCs的分离。结果表明：预先采用10μg/mL CB溶液处理GV期卵母细胞,0.5%链霉蛋白酶去除GV期卵母细胞的透明带,显微操作仪上去除生发泡,将去除生发泡的GV期卵母细胞与新生小鼠生长初期卵母细胞移入自制凹窝内进行黏合,选择1.5KV/cm-2.0KV/cm的电场强度、40μS的脉冲宽度对黏合细胞进行电击,发生电融合和电激活,获得排出了第一极体的速激核成熟重构卵母细胞。利用显微操作仪将重构卵母细胞的核区移植到去除第一极体的MⅡ期成熟卵母细胞的透明带下,通过电融合使两者发生融合,形成含有两个MⅡ期卵母细胞核的重构卵细胞,再经过对其进行7%乙醇(5min)联合2.5mmol/L6-DMAP (4h)(?)勺激活后用于体外培养,可以构建出含有速激核成熟新生小鼠生长初期卵母细胞和MⅡ期卵母细胞基因组的二倍体孤雌胚胎。
2.小鼠ESCs、pESCs和重构pESCs(?)勺分离、培养与鉴定
2.1小鼠体内受精囊胚ESCs的分离、培养与鉴定
本研究采集13.5d胎龄的胎儿分离MEFs,选取第3代MEFs制备饲养层；分别采用全胚法和免疫外科法从4d胚龄的囊胚中获取ICM,将ICM小团块移到饲养层上进行增殖培养,观察集落形态,并对细胞集落进行多能性鉴定。结果表明：采用全胚法和免疫外科法均能分离得到小鼠ESCs集落,其集落形成率差异不显著(30.26±2.77%vs32.92±4.37%,P>0.05)。两种方法所得ESCs集落具有典型的ESCs形态特征,AKP染色阳性,ESCs的染色体为二倍体,具有正常核型,Oct-4和SSEA-1免疫细胞化学检测为阳性,RT-PCR检测表明ESCs表达多能性基因：Oct-4和Sox-2, RT-PCR检测显示EB中三个胚层的标记基因(Fgf-5、Bra T和Afp)均为阳性。
2.2昆明小鼠孤雌囊胚来源pESCs的分离、培养与鉴定
鉴于孤雌囊胚于体外培养时间过长,胚胎难以突破透明带引起囊胚孵化率和贴壁率降低的情况,本研究选用免疫外科法分离单倍体pESCs(源自SrCl2激活卵母细胞4h所得囊胚)和二倍体pESCs(源自10mM SrCl2+5μg/mL CB激活卵母细胞4h所得囊胚),并对所得细胞进行多能性鉴定,旨在探讨孤雌囊胚染色体倍性对pESCs分离和培养效率的影响,筛选适宜倍性的孤雌囊胚获取pESCs。(?)吉果表明：本研究所得到的pESCs集落具有与体内受精囊胚来源ESCs类似的多能性鉴定效果。从二倍体孤雌囊胚分离所得pESCs的集落形成率显著高于单倍体孤雌囊胚处理组(13.54±4.43%vs7.17±4.45%,P<0.05),宜选择有利于提高二倍体孤雌囊胚形成率的激活方法进行pESCs的分离。
2.3异期二倍染色体重构胚胎来源pESCs的分离、培养与鉴定
本研究通过构建包含有新生小鼠生长初期卵母细胞和MⅡ期卵母细胞的二倍染色体重构核型孤雌胚胎,经体外培养获得囊胚,采用全胚法分离pESCs,并对所得重构pESCs进行多能性鉴定。结果表明：将融合后发育至桑葚胚期和囊胚期的180枚胚胎转移到MEFs饲养层上,48h后从透明带中孵出并贴壁于饲养层上,96h后ICM长出并开始增殖,5d后ICM增殖成柱状,挑出消化离散成细胞小团块后,5d后可见形成了26个pESCs集落,其集落形成率为14.43±4.36%。所得集落具有与体内受精囊胚来源ESCs类似的多能性鉴定效果。
3.应用小鼠ESCs裂解液重编程逆转化MEFs为多能干细胞的研究
3.1不同浓度SLO对MEFs生长状态的影响
本试验采用不同浓度SLO处理MEFs,通过比较SLO处理下MEFs的细胞密度、存活率和贴壁率,并绘制适宜浓度SLO处理后MEFs的生长曲线,研究不同浓度SLO对MEFs生长状态的影响,探讨SLO对体细胞的损伤是否具有剂量依赖性,以便筛选适宜浓度的SLO用于重编程体细胞的渗透化处理。结果表明：25USLO处理组的细胞密度和经CaCl2封膜后继续培养的细胞贴壁率分别与10U SLO处理组之间差异不显著(p>0.05),分别极显著地高于50U和100U SLO处理组(p<0.01)。第3代MEFs经25USLO渗透化处理后细胞仍具有正常分裂增殖的生长模式,说明该浓度的SLO对MEFs的分裂增殖影响较小。因此,宜选用25U SLO对重编程MEFs进行渗透化处理。
3.2昆明小鼠ESCs裂解液的制备
本研究比较了两种机械性裂解法即超声波处理法和液氮反复冻融法对ESCs裂解液制备效果的影响,并对所得裂解液进行体外培养试验和加入未经SLO渗透化处理的MEFs后的Oct-4免疫细胞化学染色,尽可能地排除ESCs完整细胞或细胞碎片残留等造成裂解液诱导体细胞重编程的假阳性,旨在探讨适宜的ESCs裂解方法,以获得用于体细胞重编程的高效ESCs裂解液。结果表明：液氮反复冻融7次可使ESCs细胞绝大部分发生裂解,获得的总蛋白质浓度显著高于超声波裂解细胞处理组(35.6mg/mL vs14.3mg/mL)。裂解液经体外培养后未见MEFs饲养层上生长有ESCs集落,AKP染色也未见红棕色细胞,Oct-4免疫细胞化学染色MEFs为阴性反应。因此,宜选择液氮反复冻融7次的方法制备用于体细胞重编程的ESCs裂解液。
3.3小鼠ESCs裂解液与MEFs共孵育体系的研究
本研究首先探讨了供、受体细胞的性别属性对ESCs裂解液诱导体细胞重编程效率的影响,结果表明供、受体细胞性别对ESCs裂解液诱导MEFs重编程所得多能干细胞集落数量无显著差异。然后,比较了三种不同来源囊胚分离所得雌性ESCs裂解液重编程雄性MEFs或经过TSA和5-aza-dC预处理的雄性MEFs为多能干细胞的效率,以雄性MEFs与其自身裂解液共孵育和未经裂解液处理的雄性MEFs作为对照组,并对所得多能干细胞进行鉴定,旨在探讨适宜的裂解液诱导体细胞重编程体系。结果表明：体内受精囊胚来源并经双重PCR性别鉴定为雌性的ESCs裂解液、孤雌囊胚ESCs裂解液和重构囊胚来源ESCs裂解液均能够重编程雄性MEFs为多能干细胞,这些多能干细胞集落具有与体内受精囊胚来源ESCs类似的多能性鉴定指标表现。在重编程所得多能干细胞集落数量上,体内受精囊胚来源雌性ESCs裂解液处理组(64.33±3.7个、80.67±2.08个)>重构囊胚来源ESCs裂解液处理组(45.00±4.36个、53.67±3.06个)>孤雌囊胚处理组(35.67±2.52个47.67±4.16个)；TSA和5-Aza-dC的预处理有助于促进雄性MEFs的重编程,但是,在没有ESCs裂解液的诱导作用下,仅采用TSA和5-Aza-dC预处理雄性MEFs并不能获得多能干细胞集落；无ESCs裂解液参与的单纯重编程操作过程和雄性的MEFs裂解液并不能使雄性MEFs发生重编程,因此,促使雄性MEFs发生重编程的物质来源于ESCs的裂解液。
4. ESCs裂解液诱导MEFs重编程所得多能干细胞集落的来源鉴定
采用双重PCR性别鉴定方法对体内受精囊胚来源经性别鉴定为雌性ESCs裂解液、孤雌囊胚来源pESCs裂解液和重构孤雌囊胚来源ESCs裂解液诱导雄性MEFs重编程所得到的多能干细胞进行性别鉴定,鉴定所得多能干细胞的来源,判定细胞提取物诱导的重编程体系的可靠性。结果表明：体内受精囊胚来源经性别鉴定为雌性ESCs的裂解液、孤雌囊胚来源pESCs裂解液和重构孤雌囊胚来源的ESCs裂解液均能够诱导雄性MEFs重编程为多能干细胞,所得多能干细胞的基因组DNA中具有250bp的Sry基因序列片段和339bp的ZFX基因序列片段,为雄性细胞。因此,pESCs裂解液重编程所得具有多能性特征的细胞是源自于雄性MEFs,它们所表现出的多能性检测指标的阳性反应并不是由于裂解液制备过程中的ESCs污染所致。
5. ESCs裂解液诱导种间体细胞重编程的可行性研究
本研究以鸡胚成纤维细胞作为重编程的受体细胞,采用小鼠体内受精囊胚来源ESCs裂解液与其进行共孵育,通过形态学观察,研究其对鸡胚成纤维细胞的逆转化效果,对所获得干细胞集落进行多能性检测,旨在探讨小鼠ESCs裂解液作用于异种体细胞后,使其重编程逆转化为多潜能干细胞的可行性。结果表明：与小鼠ESCs裂解液共孵育的鸡胚成纤维细胞均变为圆形细胞,未见梭形的贴壁细胞,形成了42.33±4.5个细胞集落。细胞集落经AKP染色和Oct-4和SSEA-1免疫细胞化学染色,均呈阳性反应。表明小鼠ESCs裂解液重编程鸡胚成纤维细胞后,所获得的细胞集落表现出一定的多能干细胞特性,经核型分析表明这些细胞来源于鸡胚成纤维细胞。单独的重编程操作过程和与鸡胚成纤维细胞裂解液共孵育,均无法将鸡胚成纤维细胞重编程为具有ESCs多能性特征的干细胞,说明参与重编程鸡胚成纤维细胞的物质来源于小鼠ESCs的裂解液。结果表明,ESCs裂解液诱导体细胞重编程逆转化为多能干细胞的作用也能够在小鼠和鸡的种间发生。
总结论：通过共孵育(或者共培养)途径应用ESCs的无细胞裂解液作为诱导剂,能够在较短的时间内(处理后1天内呈现细胞形态学显著变化成为圆形细胞；4天内形成边界清晰的细胞集落；共孵育后第3天细胞集落表现AKP活性,第7天细胞表达Oct-4、Sox-2和Nanog基因)逆转化经过SLO、TSA和5-Aza-dC预处理的MEFs成为多能干细胞；应用这个逆转化体细胞成为多能干细胞的细胞培养体系,使用同样的预处理体细胞的方法,也能够诱导异种鸡胚成纤维细胞成为多能干细胞。
The process of de-differentiating somatic cells to a pluripotent state whereby they adopt characteristics of embryonic stem cells is referred to as'cellular reprogramming' The advantages of cell extracts-mediated reprogramming of somatic cells are:(1) Chromosome reprogramming of imported pluripotent cells does not exist,(2) Manipulating cellular composition of the extract to identify cytokines which are relevant to reprogramming,(3) Since the extract is derived from cells, its role is biological, rather than chemical, so security is higher. More and more researchers pay attention to this method, which is very worthwhile to study and develop in depth, and there are bright prospects of application.
Embryonic stem cells were isolated from the high-quality inner cell mass (ICM) from blastocysts derived from in vivo fertilization, parthenogenetic activation, and reconstruction of embryo using somatic cell nuclear transfer, and were identified with morphological observation, alkaline phosphatase assay, karyotype analysis, immunocytochemistry staining for Oct-4and SSEA-1, RT-PCR analysis for Oct-4and Sox-2, and examination the in vivo differentiation of embryoid body. MEFs were pretreated with trochostatin A (TSA) and5-Aza-deoxycytidine (5-Aza-dc) and permeabilized with streptolysin O, then those cells were incubated with reprogramming cell-free extracts which were prepared by repeatedly freezing and thawing ESCs in liquid nitrogen. Reprogrammed MEFs were continually cultured in ESCs medium and were identified whether those cells were reprogrammed to became a pluripotential stem cells or not. To rule out the possibility of ESCs contamination during the preparation of ESCs extracts, further pluripotential stem cells generation were performed using male MEFs which were identified by dual PCR for sex identification system of a different gender from extract-donor ESCs. The donors of ESCs-derived extract were the parthenogenetic embryonic stem cells (pESCs) from the oocytes which been parthenogenetic activated and female ESCs with the identified by dual PCR for sex identification system from the blastocystosts derived from in vivo fertilization. Therefore, we can decided the origin of the pluripotential stem cells according to their gender using dual PCR for sex identification system. Finally, chicken embryo fibroblasts which being act as the target cells of reprogramming and were incubated with mouse ESCs extracts, to explore the feasibility of reprogramming between the different species, the origin of cells resulted from reprogramming mediated by mouse ESCs extracts was identified by karyotype analysis.
The aim of this study was to screening the appropriate incubation method to improve the efficiencies of reprogramming which being mediated by ESCs extracts, to assess the reliability of the reprogramming system, and to explore the reprogramming mechanism about the effects of paternal gene on the efficiencies of reprogramming and The feasibility of reprogramming which be induced with the effective components in the ESCs extracts between the different species etc. The main results in this paper were as follows:
1. Effective generation pathways of blastocysts which were used to isolate the ESCs
1.1Optimization of generation pathways of ICM from mouse blastocysts which produced from in vivo fertilization
The effects of different gestational age and continuous passage on isolation, culture and growth status of MEFs were explored in this study in order to establish a effective system for the isolation and culture of MEFs, and to obtain the high-quality MEFs for preparing the feeder layer and acting as the target cells of reprogramming.3.5dpc (days post coitum),4dpc and4.5dpc mouse embryos were collected and cultured in different culture system including DMEM, DMEM+LIF and co-culture with MEFs respectively, the hatching time of ICM, attachment efficiencies of hatching blastocysts and formation efficiencies of ICM outgrowth were compared, in order to find out an effective method for obtaining high-quality ICM outgrowth for the isolation of the ESCs. Results indicated that12.5dpc to14.5dpc mouse embryos were appropriate for isolating the primary MEFs, the third generation MEFs were suitable for preparing the feeder layer.4dpc embryos were collected and co-cultured with MEFs for3days to4days, high-quality ICM outgrowth could be obtained for the isolation of ESCs.
1.2Optimization of generation pathways of mouse heterozygous diploid blastocysts
Oocytes were activated with five methods of parthenogenetic activation, including ethanol, ethanol+6-DMAP, ethanol+6-DMAP+CB, SrCl2, SrCl2+CB. Then, activated oocytes were observed under the inverted microscope and karyotype analysis of activated oocytes were done to identify the parthenogenetic activation types of activated oocytes, including uniform haploid, heterozygous diploid with one pronucleus, mosaic haploid, and heterozygous diploid with two pronuclei. The development rate of morula and blastocyst from activated oocytes with different parthenogenetic activation types were compared to screen the appropriate activated method to obtain heterozygous diploid blastocysts. Results indicated that when oocytes were treated with10%alcohol for5min united with6-DMAP (6-dimethylaminopyridine) or with lOmM SrCl2and5μg/mL CB for4h, the extrusion of second polar body would be inhibitted, higher rate of diploid parthenogenetic embryos, overcome the two-cell block and generation of morula and blastocyst were produced. But, the morula and blastocyst development rate of oocytes were treated with lOmM SrCl2and5u g/ml CB for4h was higher than that treated with10%alcohol for5min united with6-DMAP. Therefore, those two parthenogenetic activation methods are the appropriate method to obtain the higher rate of mouse heterozygous diploid blastocysts.
1.3Reconstruction of mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte
The selection of suitable method for enucleating the germinal vesicle (GV) of MⅠ oocyte, GV oocyte being treated with cytochalasin B (CB) at appropriate concentrations before being enucleated, adhesion system between primary oocyte of new-born mouse and GV oocyte without GV, electrofusion parameteres for fusion of reconstructed oocyte and chemical activation of reconstructed embryo were explored in this study, in order to investigate the method for reconstructing mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte. Results indicated that GV oocyte was pretreated with10μg/mL CB, GV oocyte was treated with0.5%pronase for removing the zona pellucida, GV oocyte without germinal vesicle was adhesived to primary oocyte of new-born mouse in the hollowness culture system, the adhesived oocyte was treated with the appropriate parameters (electric field strength was between1.5KV/cm and2.0KV/cm and the pulse width was40μs). The nuclear region of reconstructed oocyte which had been expelled the first polar body was transferred into the perivitelline space of MⅡ oocyte which the first polar body had been enucleated. The nuclear transfer embryo was fused by electrofusion, reconstructed embryo was activated with7%ethanol for5min united with2.5mmol/L6-DMAP for4h, then we can obtain the mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte.
2. Isolation, culture and identification of ESCs from blastocysts derived from in vivo fertilization, parthenogenetic activation, and reconstruction of embryo
2.1Isolation, culture and identification of ESCs from blastocysts derived from in vivo fertilization
13.5dpc mouse embryos were collected for the isolation of MEFs. Mitomycin C-treated MEFs at the third passages for preparing the feeder layer. ICMs were isolated from4dpc mouse embryos with intact embryo method and immunosurgury method, then they were transferred on the feeder layer and cultured in ESCs medium, finally cell colonies on the feeder layer were identified. Results indicated that there were no significantly differences in colony formation rate between intact embryo method and immunosurgury method (30.26±2.77%vs32.92±4.37%, P>0.05). The pluripotent nature of ESCs isolated with intact embryo method and immunosurgury method was identified by typical cellular and colonial morphology of ESCs, positive AKP staining, normal karyotype, positive Oct-4and SSEA-1immunocytochemistry staining, expression of Oct-4and Sox-2. These ESCs could also spontaneously differentiate into embryoid bodies which could express Fgf-5、Bra T and Afp.
2.2Isolation, culture and identification of ESCs from blastocysts derived from parthenogenetic activation
Considering it was not easy for parthenogenetic blastocyst to hatch out from zona pellucida because of long time operation in vitro, immunosurgury method was used to isolated pESCs from uniform haploid blastocysts which developed from oocytes activated with SrCl2for4h, and pESCs from heterozygous diploid blastocysts which developed from oocytes activated with lOmM SrCl2and5μg/mL CB for4h, then cell colonies on the feeder layer were identified. Isolation efficiencies of pESCs from uniform haploid blastocysts and heterozygous diploid blastocysts were compared in order to select parthenogenetic blastocysts with suitable chromosome ploidy for the isolation of pESCs. Results indicated that the pluripotent nature of pESCs obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization. The colonies formation rate of pESCs from heterozygous diploid blastocysts was significantly higher than that from uniform haploid blastocysts (13.54±4.43%vs7.17±4.45%, P<0.05)。 Therefore, we should select the parthenogenetic activation method which could result in higher formation rate of heterozygous diploid blastocysts for the isolation of pESCs.
2.3Isolation, culture and identification of pESCs from blastocysts derived from reconstructed embryos
Intact embryo method was used to isolated pESCs from mouse diploid parthenogenetic blastocysts contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MII oocyte. Results indicated that180morula and blastocyst derived from reconstructed embryos, after being cultured for48h morula and blastocyst developed into hatched blastocyst, after being cultured for96h ICM outgrowth were generated on the MEFs feeder layer,26pESCs colonies were observed after the ICM were dissociated into clusters of cells and transferred on the MEFs feeder layer, the pESCs colonies formation rate was14.43±4.36%. The pluripotent nature of pESCs obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization.
3. Cell-free ESCs extracts or-mediated derivation of multipotent stem cells from MEFs
3.1Effects of SLO at different concentration on growth state of MEFs
The aim of this study was to explore the comparatively suitable SLO concentration for reversible permeabilization of MEFs before those cells were used to reprogram into pluripotent stem cells using cell-free extracts, MEFs were incubated in PBS without Ca2+and Mg2+supplemented with SLO which was added to a final concentration of10U,25U,50U and100U SLO, cells density, survival rate and adherence rate of MEFs were measured. Results were as follows:No significant difference was found in cells density of MEFs between25U SLO treated group and10U SLO treated group (p>0.05), but the cells density of MEFs permeabilized with10U SLO or25U SLO was significantly higher than those permeabilized with50U SLO or100U SLO respectively (p<0.01). There was no significantly difference in the survival rate of MEFs permeabilized with25U SLO compared with the0U or10U SLO group respectively (p>0.05), but was significantly higher than those permeabilized with50U SLO (p<0.05). The adherence rate of MEFs permeabilized with25U SLO and resealed in2mM CaCl2was significantly higher than that with50U or100U SLO (p＜0.01). Proliferation of MEFs permeabilized with25U SLO was normal. Therefore,25U SLO was better suited to permeabilize MEFs considering both improving the efficiency of permeabilization and reducing the damage to MEFs.
3.2Preparation of ESCs extract from Kunming mouse blastocysts derived from in vivo fertilization
Effects of ultrasonic waves treatment and repeatedly freezing and thawing in liquid nitrogen on preparation of ESCs extract were explored in this study to obtain the high-efficiency lysis method to prepare the ESCs extract for programming of somatic cells. In order to detect and reduce the possibility of ESCs contamination during the preparation of ESCs extracts, ESCs extracts were transferred on the feeder layer and cultured in vitro, and MEFs without being permeablizated with streptolysin O were added to the ESCs extracts and were used to perform immunocytochemistry staining for Oct-4. Results were as follows:the vast majority of ESCs could be lysed after being repeatedly frozen and thawed in liquid nitrogen for seven times, the protein concentration were higher than that in ultrasonic waves treatment group (35.6mg/ml vs14.3mg/ml). There were no ESCs colonies observed on the feeder layer, MEFs without being permeablizated with streptolysin O revealed negative reaction to Oct-4immunocytochemstry staining. Therefore, ESCs were lysed and intracellular substances were destroyed as less as possible after ESCs were repeatedly freezing and thawing in liquid nitrogen for seven times, it provided a incubated culture system for reprogramming of somatic cells.
3.3Studies on incubated system for mouse ESCs extract and MEFs
Efficiencies of male MEFs which being pretreated with TSA and5-aza-dC or not being reprogrammed into multipotent stem cells by three kinds of female ESCs extracts respectively were compared in this study, male MEFs treated with male MEFs extracts and male MEFs without being treated with extracts were acted as controls, the pluripotent nature of multipotent stem cells obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization, the aim of the study was to optimize the reprogramming system which mediated by ESCs extracts. Results were as follows:male MEFs could be reprogrammed into multipotent stem cells by the female ESCs which be identified by dual PCR for sex identification system extracts from blastocysts derived from in vitro fertilization, parthenogenetic activation, and reconstruction of embryo respectively. The pluripotent nature of multipotent stem cells obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization. The number of colonies of multipotent stem cells from male MEFs which being treated with female ESCs extracts from blastocysts derived from in vivo fertilizationwere largest (64.33±3.7、80.67±2.08), following that of blastocysts derived from reconstruction of embryo (45.00±4.36、53.67±3.06), and that of blastocysts derived from parthenogenetic activation (35.67±2.52、47.67±4.16). Reprogramming of male MEFs could be improved by the pretreatment with TSA and5-Aza-dC, but it could not obtain the colonies of multipotent stem cells from male MEFs only pretreated with TSA and5-Aza-dC. Colony of multipotent stem cells was not be observed in control groups. Therefore, components which could reprogram the male MEFs into multipotent stem cells were from the ESCs extracts.
4. Identification of the origin of multipotent stem cells from MEFs which been reprogrammed by ESCs extracts
To rule out the possibility of ESCs contamination during the preparation of ESCs extracts, and assess the reliability of the reprogramming system, further pluripotential stem cells generation were performed using male MEFs which were identified by dual PCR for sex identification system(which can amplify250bp Sry gene and399bp ZFX gene) of a different gender from extract-donor ESCs. The donors of ESCs-derived extract were a parthenogenetic embryonic stem cells (pESCs) from the oocytes (which can only amplify399bp ZFX gene) which been parthenogenetic activated and the female ESCs identified with dual PCR for sex identification system from the blastocyst produced from in vivo fertilization. Therefore, we can decided the origin of the pluripotential stem cells according to their gender using dual PCR for sex identification system. Results indicated that40colonies of multipotent stem cells from8batch reprogramming (5colonies of multipotent stem cells were randomly selected in every batch) which mediated by pESCs extracts, pESCs from the reconstructed embryos extract and female extract from in vivo fertilization were used to perform dual PCR for sex identification. Multipotent stem cells which had been amplified250bp Sry gene and399bp ZFX gene, that was there were male colonies. Therefore, the origin of multipotent stem cells obtained in our study were male MEFs, ESCs extracts were not the contaminated by donor ESCs during the preparation of pESCs extracts.
5. Feasibility of interspecies somatic cells reprogramming-mediated by ESCs extracts
Chicken embryonic fibroblasts which being act as the target cells of reprogramming and were incubated with mouse ESCs extracts from Kunming mouse blastocysts derived from in vivo fertilization, morphology changes were observed, and the pluripotent nature of cells obtained from chicken embryonic fibroblasts after being reprogrammed by mouse ESCs extracts were identified, in order to explore the feasibility of reprogramming between the different species. Results indicated that after be incubated with mouse ESCs extracts, reprogrammed chicken embryonic fibroblasts turned to be round, fusiform adherent cells were not observed, and42.33±4.5colonies were generated. The pluripotent nature of the colonies was identified by typical cellular and colonial morphology of ESCs, positive AKP staining, and positive Oct-4and SSEA-1immunocytochemistry staining, and results from karyotype analysis indicated that those cells were derived from chicken embryonic fibroblasts. Chicken embryonic fibroblasts incubated with chicken embryonic fibroblasts extracts or without incubated with any extracts were not appeared the pluripotent nature, Therefore, components which could reprogram the chicken embryonic fibroblasts into multipotent stem cells were from the mouse ESCs extracts. Interspecies somatic cells reprogramming between mouse ESCs and chicken embryonic fibroblasts could be mediated by ESCs extracts.
Conclusions:MEFs pretreated with SLO, TSA and5-Aza-dC could be reprogram ed into multipotent stem cells with cell-free ESCs extract in a shorter period. One day after treated with ESCs extract, MEFs formed the round cells, colonies with defined edges developed and resembled ESCs colonied within4days, these colonies showed the AKP activity on day3and they express Oct-4, Sox2and Nanog on day7. Chicken embryonic fibroblasts could be reprogrammed into multipotent stem cells using the reprogramming system mediated by mouse ESCs extract.
本研究通过优化小鼠体内受精囊胚、杂合二倍体孤雌囊胚、人工重组异期卵母细胞核二倍体孤雌囊胚(含有速激核成熟新生鼠生长初期卵母细胞与成熟卵母细胞基因组二倍体小鼠孤雌囊胚)的获取途径,获得不同囊胚来源的ESCs。采用形态学观察法、碱性磷酸酶(AKP)染色法、核型分析法、Oct-4和SSEA-1免疫细胞化学检测法、Oct-4基因和Sox-2基因表达检测法、类胚体(EB)分化能力检测等方法对其ESCs的特性进行鉴定。用于被诱导的受体MEFs,采用曲古抑菌素A(TSA)和5-氮杂-2’-脱氧胞苷(5-aza-dc)预处理,然后再经过适宜浓度链球菌溶血素O(SLO)渗透化处理；用于起诱导作用的不同囊胚来源的小鼠ESCs,采用液氮反复冻融和离心的方法,得到不同类型ESCs的无细胞裂解液；将受体细胞孵育在无细胞裂解液的培养液中进行重编程逆转化性诱导培养,获得来源于体细胞的多能干细胞。对诱导所得多能干细胞进行多能干细胞特性的鉴定。借助双重PCR性别鉴定的方法,分选出来源于雌性的有性生殖ESCs,孤雌胚胎干细胞(pESCs)和重构pESCs,三种雌性胚胎干细胞分别通过冻融和离心的方法,获得三种不同类型雌性ESCs(供体细胞)的无细胞裂解液,将此作为诱导剂重编程逆转化雄性MEFs(受体细胞),获得多能干细胞后,依据供体细胞和受体细胞的性别差异,采用双重PCR性别鉴定的方法确定重编程所得多能干细胞集落的来源。最后以鸡成纤维细胞作为重编程的受体细胞,探讨小鼠ESCs裂解液进行种间体细胞重编程、逆转化鸡胚成纤维细胞形成多能干细胞的可行性,并通过核型分析鉴定其所得多能干细胞的来源。
通过上述研究,旨在比较不同来源ESCs裂解液介导体细胞重编程逆转化为多能干细胞的可能性及其效率,筛选适宜的共孵育方式,验证重编程体系的可靠性,比较有性生殖ESCs和无性生殖pESCs裂解液的作用效果,进一步研究父本基因缺失情况下对体细胞重编程逆转化效率的作用和对异种动物体细胞诱导形成多能干细胞的可能性。这项研究结果将为今后进一步探索ESCs和pESCs裂解液中重编程有效成分的筛选、鉴定和物种间多能干细胞的研究奠定基础。研究结果如下：
1.用于分离小鼠ESCs(?)勺三种囊胚有效生成途径研究
1.1小鼠体内受精囊胚内细胞团(ICM)集落获取的条件优化
本研究首先通过比较不同胎龄MEFs的生长状况和连续传代过程中细胞的生长情况,研究影响MEFs分离和培养效率的因素,旨在为早期胚胎共培养体系选择更加有效的MEFs的分离培养体系,用于MEFs饲养层的制备、ESCs的分离培养和被诱导受体细胞的筛查处理。然后,采集3.5d、4d和4.5d胎龄的胚胎,分别移入DMEM培养液、DMEM+白血病抑制因子(LIF)培养液和包含有MEFs贴壁生长细胞的共培养体系中进行体外培养,比较三种培养体系中ICM孵出的时间、孵化囊胚贴壁比率和ICM集落形成比率,旨在筛选获取优质ICM集落的实验方法,为ESCs(?)勺分离培养奠定基础。结果表明：采用12.5-14.5d胎龄的小鼠胎儿分禺MEFs,传代培养后选取第3代MEFs制备饲养层,采集4d的小鼠囊胚置于其MEFs饲养层的共培养体系中,经体外培养3-4d,可以获取优质的ICM,这为体内受精囊胚来源ESCs的分离培养奠定了基础。
1.2小鼠杂合二倍体孤雌囊胚生成途径的优化
本研究采用5种化学激活方法即乙醇、乙醇+6-二甲基氨基嘌呤(6-DMAP)、乙醇+6-DMAP+细胞松弛素B (CB)、SrCl2、SrCl2+CB处理小鼠成熟卵母细胞,通过观察激活卵母细胞第二极体排出情况和核型分析,探讨不同激活方法所得激活卵母细胞染色体倍性的倾向类型,比较不同染色体倍性激活卵母细胞的体外发育能力,筛选有利于形成杂合二倍体孤雌囊胚的激活方法。结果表明：在含有10%乙醇培养液中激活卵母细胞5min的基础上,添加6-DMAP有利于抑制第二极体的排出,促进双原核(2PN)杂合二倍体孤雌胚胎的形成,获取更高的过二细胞阻滞率和桑葚胚/囊胚发育率；采用含有10mM SrCl2和CB处理卵母细胞4h,也能够获取较多的双原核(2PN)杂合二倍体和较高的桑葚胚/囊胚发育率,因此,以上两种孤雌激活方法适宜用于获取小鼠杂合二倍体孤雌囊胚。
1.3人工重组异期卵母细胞核二倍体孤雌囊胚(含有速激核成熟新生小鼠生长初期卵母细胞和M Ⅱ期卵母细胞基因组的二倍体孤雌胚胎)的构建
本研究分别研究了CB对生发泡(GV)期卵母细胞去核效果的影响、去除成年小鼠MⅠ期卵母细胞GV的方法选择、新生小鼠生长初期卵母细胞与去核GV期卵母细胞黏合体系的筛选、电融合条件选择以及重构胚化学激活方法的筛选等,旨在构建含有速激核成熟新生小鼠生长初期卵母细胞和MⅡ期卵母细胞基因组的二倍体孤雌胚胎,用于ESCs的分离。结果表明：预先采用10μg/mL CB溶液处理GV期卵母细胞,0.5%链霉蛋白酶去除GV期卵母细胞的透明带,显微操作仪上去除生发泡,将去除生发泡的GV期卵母细胞与新生小鼠生长初期卵母细胞移入自制凹窝内进行黏合,选择1.5KV/cm-2.0KV/cm的电场强度、40μS的脉冲宽度对黏合细胞进行电击,发生电融合和电激活,获得排出了第一极体的速激核成熟重构卵母细胞。利用显微操作仪将重构卵母细胞的核区移植到去除第一极体的MⅡ期成熟卵母细胞的透明带下,通过电融合使两者发生融合,形成含有两个MⅡ期卵母细胞核的重构卵细胞,再经过对其进行7%乙醇(5min)联合2.5mmol/L6-DMAP (4h)(?)勺激活后用于体外培养,可以构建出含有速激核成熟新生小鼠生长初期卵母细胞和MⅡ期卵母细胞基因组的二倍体孤雌胚胎。
2.小鼠ESCs、pESCs和重构pESCs(?)勺分离、培养与鉴定
2.1小鼠体内受精囊胚ESCs的分离、培养与鉴定
本研究采集13.5d胎龄的胎儿分离MEFs,选取第3代MEFs制备饲养层；分别采用全胚法和免疫外科法从4d胚龄的囊胚中获取ICM,将ICM小团块移到饲养层上进行增殖培养,观察集落形态,并对细胞集落进行多能性鉴定。结果表明：采用全胚法和免疫外科法均能分离得到小鼠ESCs集落,其集落形成率差异不显著(30.26±2.77%vs32.92±4.37%,P>0.05)。两种方法所得ESCs集落具有典型的ESCs形态特征,AKP染色阳性,ESCs的染色体为二倍体,具有正常核型,Oct-4和SSEA-1免疫细胞化学检测为阳性,RT-PCR检测表明ESCs表达多能性基因：Oct-4和Sox-2, RT-PCR检测显示EB中三个胚层的标记基因(Fgf-5、Bra T和Afp)均为阳性。
2.2昆明小鼠孤雌囊胚来源pESCs的分离、培养与鉴定
鉴于孤雌囊胚于体外培养时间过长,胚胎难以突破透明带引起囊胚孵化率和贴壁率降低的情况,本研究选用免疫外科法分离单倍体pESCs(源自SrCl2激活卵母细胞4h所得囊胚)和二倍体pESCs(源自10mM SrCl2+5μg/mL CB激活卵母细胞4h所得囊胚),并对所得细胞进行多能性鉴定,旨在探讨孤雌囊胚染色体倍性对pESCs分离和培养效率的影响,筛选适宜倍性的孤雌囊胚获取pESCs。(?)吉果表明：本研究所得到的pESCs集落具有与体内受精囊胚来源ESCs类似的多能性鉴定效果。从二倍体孤雌囊胚分离所得pESCs的集落形成率显著高于单倍体孤雌囊胚处理组(13.54±4.43%vs7.17±4.45%,P<0.05),宜选择有利于提高二倍体孤雌囊胚形成率的激活方法进行pESCs的分离。
2.3异期二倍染色体重构胚胎来源pESCs的分离、培养与鉴定
本研究通过构建包含有新生小鼠生长初期卵母细胞和MⅡ期卵母细胞的二倍染色体重构核型孤雌胚胎,经体外培养获得囊胚,采用全胚法分离pESCs,并对所得重构pESCs进行多能性鉴定。结果表明：将融合后发育至桑葚胚期和囊胚期的180枚胚胎转移到MEFs饲养层上,48h后从透明带中孵出并贴壁于饲养层上,96h后ICM长出并开始增殖,5d后ICM增殖成柱状,挑出消化离散成细胞小团块后,5d后可见形成了26个pESCs集落,其集落形成率为14.43±4.36%。所得集落具有与体内受精囊胚来源ESCs类似的多能性鉴定效果。
3.应用小鼠ESCs裂解液重编程逆转化MEFs为多能干细胞的研究
3.1不同浓度SLO对MEFs生长状态的影响
本试验采用不同浓度SLO处理MEFs,通过比较SLO处理下MEFs的细胞密度、存活率和贴壁率,并绘制适宜浓度SLO处理后MEFs的生长曲线,研究不同浓度SLO对MEFs生长状态的影响,探讨SLO对体细胞的损伤是否具有剂量依赖性,以便筛选适宜浓度的SLO用于重编程体细胞的渗透化处理。结果表明：25USLO处理组的细胞密度和经CaCl2封膜后继续培养的细胞贴壁率分别与10U SLO处理组之间差异不显著(p>0.05),分别极显著地高于50U和100U SLO处理组(p<0.01)。第3代MEFs经25USLO渗透化处理后细胞仍具有正常分裂增殖的生长模式,说明该浓度的SLO对MEFs的分裂增殖影响较小。因此,宜选用25U SLO对重编程MEFs进行渗透化处理。
3.2昆明小鼠ESCs裂解液的制备
本研究比较了两种机械性裂解法即超声波处理法和液氮反复冻融法对ESCs裂解液制备效果的影响,并对所得裂解液进行体外培养试验和加入未经SLO渗透化处理的MEFs后的Oct-4免疫细胞化学染色,尽可能地排除ESCs完整细胞或细胞碎片残留等造成裂解液诱导体细胞重编程的假阳性,旨在探讨适宜的ESCs裂解方法,以获得用于体细胞重编程的高效ESCs裂解液。结果表明：液氮反复冻融7次可使ESCs细胞绝大部分发生裂解,获得的总蛋白质浓度显著高于超声波裂解细胞处理组(35.6mg/mL vs14.3mg/mL)。裂解液经体外培养后未见MEFs饲养层上生长有ESCs集落,AKP染色也未见红棕色细胞,Oct-4免疫细胞化学染色MEFs为阴性反应。因此,宜选择液氮反复冻融7次的方法制备用于体细胞重编程的ESCs裂解液。
3.3小鼠ESCs裂解液与MEFs共孵育体系的研究
本研究首先探讨了供、受体细胞的性别属性对ESCs裂解液诱导体细胞重编程效率的影响,结果表明供、受体细胞性别对ESCs裂解液诱导MEFs重编程所得多能干细胞集落数量无显著差异。然后,比较了三种不同来源囊胚分离所得雌性ESCs裂解液重编程雄性MEFs或经过TSA和5-aza-dC预处理的雄性MEFs为多能干细胞的效率,以雄性MEFs与其自身裂解液共孵育和未经裂解液处理的雄性MEFs作为对照组,并对所得多能干细胞进行鉴定,旨在探讨适宜的裂解液诱导体细胞重编程体系。结果表明：体内受精囊胚来源并经双重PCR性别鉴定为雌性的ESCs裂解液、孤雌囊胚ESCs裂解液和重构囊胚来源ESCs裂解液均能够重编程雄性MEFs为多能干细胞,这些多能干细胞集落具有与体内受精囊胚来源ESCs类似的多能性鉴定指标表现。在重编程所得多能干细胞集落数量上,体内受精囊胚来源雌性ESCs裂解液处理组(64.33±3.7个、80.67±2.08个)>重构囊胚来源ESCs裂解液处理组(45.00±4.36个、53.67±3.06个)>孤雌囊胚处理组(35.67±2.52个47.67±4.16个)；TSA和5-Aza-dC的预处理有助于促进雄性MEFs的重编程,但是,在没有ESCs裂解液的诱导作用下,仅采用TSA和5-Aza-dC预处理雄性MEFs并不能获得多能干细胞集落；无ESCs裂解液参与的单纯重编程操作过程和雄性的MEFs裂解液并不能使雄性MEFs发生重编程,因此,促使雄性MEFs发生重编程的物质来源于ESCs的裂解液。
4. ESCs裂解液诱导MEFs重编程所得多能干细胞集落的来源鉴定
采用双重PCR性别鉴定方法对体内受精囊胚来源经性别鉴定为雌性ESCs裂解液、孤雌囊胚来源pESCs裂解液和重构孤雌囊胚来源ESCs裂解液诱导雄性MEFs重编程所得到的多能干细胞进行性别鉴定,鉴定所得多能干细胞的来源,判定细胞提取物诱导的重编程体系的可靠性。结果表明：体内受精囊胚来源经性别鉴定为雌性ESCs的裂解液、孤雌囊胚来源pESCs裂解液和重构孤雌囊胚来源的ESCs裂解液均能够诱导雄性MEFs重编程为多能干细胞,所得多能干细胞的基因组DNA中具有250bp的Sry基因序列片段和339bp的ZFX基因序列片段,为雄性细胞。因此,pESCs裂解液重编程所得具有多能性特征的细胞是源自于雄性MEFs,它们所表现出的多能性检测指标的阳性反应并不是由于裂解液制备过程中的ESCs污染所致。
5. ESCs裂解液诱导种间体细胞重编程的可行性研究
本研究以鸡胚成纤维细胞作为重编程的受体细胞,采用小鼠体内受精囊胚来源ESCs裂解液与其进行共孵育,通过形态学观察,研究其对鸡胚成纤维细胞的逆转化效果,对所获得干细胞集落进行多能性检测,旨在探讨小鼠ESCs裂解液作用于异种体细胞后,使其重编程逆转化为多潜能干细胞的可行性。结果表明：与小鼠ESCs裂解液共孵育的鸡胚成纤维细胞均变为圆形细胞,未见梭形的贴壁细胞,形成了42.33±4.5个细胞集落。细胞集落经AKP染色和Oct-4和SSEA-1免疫细胞化学染色,均呈阳性反应。表明小鼠ESCs裂解液重编程鸡胚成纤维细胞后,所获得的细胞集落表现出一定的多能干细胞特性,经核型分析表明这些细胞来源于鸡胚成纤维细胞。单独的重编程操作过程和与鸡胚成纤维细胞裂解液共孵育,均无法将鸡胚成纤维细胞重编程为具有ESCs多能性特征的干细胞,说明参与重编程鸡胚成纤维细胞的物质来源于小鼠ESCs的裂解液。结果表明,ESCs裂解液诱导体细胞重编程逆转化为多能干细胞的作用也能够在小鼠和鸡的种间发生。
总结论：通过共孵育(或者共培养)途径应用ESCs的无细胞裂解液作为诱导剂,能够在较短的时间内(处理后1天内呈现细胞形态学显著变化成为圆形细胞；4天内形成边界清晰的细胞集落；共孵育后第3天细胞集落表现AKP活性,第7天细胞表达Oct-4、Sox-2和Nanog基因)逆转化经过SLO、TSA和5-Aza-dC预处理的MEFs成为多能干细胞；应用这个逆转化体细胞成为多能干细胞的细胞培养体系,使用同样的预处理体细胞的方法,也能够诱导异种鸡胚成纤维细胞成为多能干细胞。
The process of de-differentiating somatic cells to a pluripotent state whereby they adopt characteristics of embryonic stem cells is referred to as'cellular reprogramming' The advantages of cell extracts-mediated reprogramming of somatic cells are:(1) Chromosome reprogramming of imported pluripotent cells does not exist,(2) Manipulating cellular composition of the extract to identify cytokines which are relevant to reprogramming,(3) Since the extract is derived from cells, its role is biological, rather than chemical, so security is higher. More and more researchers pay attention to this method, which is very worthwhile to study and develop in depth, and there are bright prospects of application.
Embryonic stem cells were isolated from the high-quality inner cell mass (ICM) from blastocysts derived from in vivo fertilization, parthenogenetic activation, and reconstruction of embryo using somatic cell nuclear transfer, and were identified with morphological observation, alkaline phosphatase assay, karyotype analysis, immunocytochemistry staining for Oct-4and SSEA-1, RT-PCR analysis for Oct-4and Sox-2, and examination the in vivo differentiation of embryoid body. MEFs were pretreated with trochostatin A (TSA) and5-Aza-deoxycytidine (5-Aza-dc) and permeabilized with streptolysin O, then those cells were incubated with reprogramming cell-free extracts which were prepared by repeatedly freezing and thawing ESCs in liquid nitrogen. Reprogrammed MEFs were continually cultured in ESCs medium and were identified whether those cells were reprogrammed to became a pluripotential stem cells or not. To rule out the possibility of ESCs contamination during the preparation of ESCs extracts, further pluripotential stem cells generation were performed using male MEFs which were identified by dual PCR for sex identification system of a different gender from extract-donor ESCs. The donors of ESCs-derived extract were the parthenogenetic embryonic stem cells (pESCs) from the oocytes which been parthenogenetic activated and female ESCs with the identified by dual PCR for sex identification system from the blastocystosts derived from in vivo fertilization. Therefore, we can decided the origin of the pluripotential stem cells according to their gender using dual PCR for sex identification system. Finally, chicken embryo fibroblasts which being act as the target cells of reprogramming and were incubated with mouse ESCs extracts, to explore the feasibility of reprogramming between the different species, the origin of cells resulted from reprogramming mediated by mouse ESCs extracts was identified by karyotype analysis.
The aim of this study was to screening the appropriate incubation method to improve the efficiencies of reprogramming which being mediated by ESCs extracts, to assess the reliability of the reprogramming system, and to explore the reprogramming mechanism about the effects of paternal gene on the efficiencies of reprogramming and The feasibility of reprogramming which be induced with the effective components in the ESCs extracts between the different species etc. The main results in this paper were as follows:
1. Effective generation pathways of blastocysts which were used to isolate the ESCs
1.1Optimization of generation pathways of ICM from mouse blastocysts which produced from in vivo fertilization
The effects of different gestational age and continuous passage on isolation, culture and growth status of MEFs were explored in this study in order to establish a effective system for the isolation and culture of MEFs, and to obtain the high-quality MEFs for preparing the feeder layer and acting as the target cells of reprogramming.3.5dpc (days post coitum),4dpc and4.5dpc mouse embryos were collected and cultured in different culture system including DMEM, DMEM+LIF and co-culture with MEFs respectively, the hatching time of ICM, attachment efficiencies of hatching blastocysts and formation efficiencies of ICM outgrowth were compared, in order to find out an effective method for obtaining high-quality ICM outgrowth for the isolation of the ESCs. Results indicated that12.5dpc to14.5dpc mouse embryos were appropriate for isolating the primary MEFs, the third generation MEFs were suitable for preparing the feeder layer.4dpc embryos were collected and co-cultured with MEFs for3days to4days, high-quality ICM outgrowth could be obtained for the isolation of ESCs.
1.2Optimization of generation pathways of mouse heterozygous diploid blastocysts
Oocytes were activated with five methods of parthenogenetic activation, including ethanol, ethanol+6-DMAP, ethanol+6-DMAP+CB, SrCl2, SrCl2+CB. Then, activated oocytes were observed under the inverted microscope and karyotype analysis of activated oocytes were done to identify the parthenogenetic activation types of activated oocytes, including uniform haploid, heterozygous diploid with one pronucleus, mosaic haploid, and heterozygous diploid with two pronuclei. The development rate of morula and blastocyst from activated oocytes with different parthenogenetic activation types were compared to screen the appropriate activated method to obtain heterozygous diploid blastocysts. Results indicated that when oocytes were treated with10%alcohol for5min united with6-DMAP (6-dimethylaminopyridine) or with lOmM SrCl2and5μg/mL CB for4h, the extrusion of second polar body would be inhibitted, higher rate of diploid parthenogenetic embryos, overcome the two-cell block and generation of morula and blastocyst were produced. But, the morula and blastocyst development rate of oocytes were treated with lOmM SrCl2and5u g/ml CB for4h was higher than that treated with10%alcohol for5min united with6-DMAP. Therefore, those two parthenogenetic activation methods are the appropriate method to obtain the higher rate of mouse heterozygous diploid blastocysts.
1.3Reconstruction of mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte
The selection of suitable method for enucleating the germinal vesicle (GV) of MⅠ oocyte, GV oocyte being treated with cytochalasin B (CB) at appropriate concentrations before being enucleated, adhesion system between primary oocyte of new-born mouse and GV oocyte without GV, electrofusion parameteres for fusion of reconstructed oocyte and chemical activation of reconstructed embryo were explored in this study, in order to investigate the method for reconstructing mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte. Results indicated that GV oocyte was pretreated with10μg/mL CB, GV oocyte was treated with0.5%pronase for removing the zona pellucida, GV oocyte without germinal vesicle was adhesived to primary oocyte of new-born mouse in the hollowness culture system, the adhesived oocyte was treated with the appropriate parameters (electric field strength was between1.5KV/cm and2.0KV/cm and the pulse width was40μs). The nuclear region of reconstructed oocyte which had been expelled the first polar body was transferred into the perivitelline space of MⅡ oocyte which the first polar body had been enucleated. The nuclear transfer embryo was fused by electrofusion, reconstructed embryo was activated with7%ethanol for5min united with2.5mmol/L6-DMAP for4h, then we can obtain the mouse diploid parthenogenetic embryo contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MⅡ oocyte.
2. Isolation, culture and identification of ESCs from blastocysts derived from in vivo fertilization, parthenogenetic activation, and reconstruction of embryo
2.1Isolation, culture and identification of ESCs from blastocysts derived from in vivo fertilization
13.5dpc mouse embryos were collected for the isolation of MEFs. Mitomycin C-treated MEFs at the third passages for preparing the feeder layer. ICMs were isolated from4dpc mouse embryos with intact embryo method and immunosurgury method, then they were transferred on the feeder layer and cultured in ESCs medium, finally cell colonies on the feeder layer were identified. Results indicated that there were no significantly differences in colony formation rate between intact embryo method and immunosurgury method (30.26±2.77%vs32.92±4.37%, P>0.05). The pluripotent nature of ESCs isolated with intact embryo method and immunosurgury method was identified by typical cellular and colonial morphology of ESCs, positive AKP staining, normal karyotype, positive Oct-4and SSEA-1immunocytochemistry staining, expression of Oct-4and Sox-2. These ESCs could also spontaneously differentiate into embryoid bodies which could express Fgf-5、Bra T and Afp.
2.2Isolation, culture and identification of ESCs from blastocysts derived from parthenogenetic activation
Considering it was not easy for parthenogenetic blastocyst to hatch out from zona pellucida because of long time operation in vitro, immunosurgury method was used to isolated pESCs from uniform haploid blastocysts which developed from oocytes activated with SrCl2for4h, and pESCs from heterozygous diploid blastocysts which developed from oocytes activated with lOmM SrCl2and5μg/mL CB for4h, then cell colonies on the feeder layer were identified. Isolation efficiencies of pESCs from uniform haploid blastocysts and heterozygous diploid blastocysts were compared in order to select parthenogenetic blastocysts with suitable chromosome ploidy for the isolation of pESCs. Results indicated that the pluripotent nature of pESCs obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization. The colonies formation rate of pESCs from heterozygous diploid blastocysts was significantly higher than that from uniform haploid blastocysts (13.54±4.43%vs7.17±4.45%, P<0.05)。 Therefore, we should select the parthenogenetic activation method which could result in higher formation rate of heterozygous diploid blastocysts for the isolation of pESCs.
2.3Isolation, culture and identification of pESCs from blastocysts derived from reconstructed embryos
Intact embryo method was used to isolated pESCs from mouse diploid parthenogenetic blastocysts contained genetic materials from primary oocyte with quickly activated and matured nucleus of new-born mouse and MII oocyte. Results indicated that180morula and blastocyst derived from reconstructed embryos, after being cultured for48h morula and blastocyst developed into hatched blastocyst, after being cultured for96h ICM outgrowth were generated on the MEFs feeder layer,26pESCs colonies were observed after the ICM were dissociated into clusters of cells and transferred on the MEFs feeder layer, the pESCs colonies formation rate was14.43±4.36%. The pluripotent nature of pESCs obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization.
3. Cell-free ESCs extracts or-mediated derivation of multipotent stem cells from MEFs
3.1Effects of SLO at different concentration on growth state of MEFs
The aim of this study was to explore the comparatively suitable SLO concentration for reversible permeabilization of MEFs before those cells were used to reprogram into pluripotent stem cells using cell-free extracts, MEFs were incubated in PBS without Ca2+and Mg2+supplemented with SLO which was added to a final concentration of10U,25U,50U and100U SLO, cells density, survival rate and adherence rate of MEFs were measured. Results were as follows:No significant difference was found in cells density of MEFs between25U SLO treated group and10U SLO treated group (p>0.05), but the cells density of MEFs permeabilized with10U SLO or25U SLO was significantly higher than those permeabilized with50U SLO or100U SLO respectively (p<0.01). There was no significantly difference in the survival rate of MEFs permeabilized with25U SLO compared with the0U or10U SLO group respectively (p>0.05), but was significantly higher than those permeabilized with50U SLO (p<0.05). The adherence rate of MEFs permeabilized with25U SLO and resealed in2mM CaCl2was significantly higher than that with50U or100U SLO (p＜0.01). Proliferation of MEFs permeabilized with25U SLO was normal. Therefore,25U SLO was better suited to permeabilize MEFs considering both improving the efficiency of permeabilization and reducing the damage to MEFs.
3.2Preparation of ESCs extract from Kunming mouse blastocysts derived from in vivo fertilization
Effects of ultrasonic waves treatment and repeatedly freezing and thawing in liquid nitrogen on preparation of ESCs extract were explored in this study to obtain the high-efficiency lysis method to prepare the ESCs extract for programming of somatic cells. In order to detect and reduce the possibility of ESCs contamination during the preparation of ESCs extracts, ESCs extracts were transferred on the feeder layer and cultured in vitro, and MEFs without being permeablizated with streptolysin O were added to the ESCs extracts and were used to perform immunocytochemistry staining for Oct-4. Results were as follows:the vast majority of ESCs could be lysed after being repeatedly frozen and thawed in liquid nitrogen for seven times, the protein concentration were higher than that in ultrasonic waves treatment group (35.6mg/ml vs14.3mg/ml). There were no ESCs colonies observed on the feeder layer, MEFs without being permeablizated with streptolysin O revealed negative reaction to Oct-4immunocytochemstry staining. Therefore, ESCs were lysed and intracellular substances were destroyed as less as possible after ESCs were repeatedly freezing and thawing in liquid nitrogen for seven times, it provided a incubated culture system for reprogramming of somatic cells.
3.3Studies on incubated system for mouse ESCs extract and MEFs
Efficiencies of male MEFs which being pretreated with TSA and5-aza-dC or not being reprogrammed into multipotent stem cells by three kinds of female ESCs extracts respectively were compared in this study, male MEFs treated with male MEFs extracts and male MEFs without being treated with extracts were acted as controls, the pluripotent nature of multipotent stem cells obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization, the aim of the study was to optimize the reprogramming system which mediated by ESCs extracts. Results were as follows:male MEFs could be reprogrammed into multipotent stem cells by the female ESCs which be identified by dual PCR for sex identification system extracts from blastocysts derived from in vitro fertilization, parthenogenetic activation, and reconstruction of embryo respectively. The pluripotent nature of multipotent stem cells obtained in this study was identified by the criteria as listed in the identification of ESCs from blastocysts derived from in vivo fertilization. The number of colonies of multipotent stem cells from male MEFs which being treated with female ESCs extracts from blastocysts derived from in vivo fertilizationwere largest (64.33±3.7、80.67±2.08), following that of blastocysts derived from reconstruction of embryo (45.00±4.36、53.67±3.06), and that of blastocysts derived from parthenogenetic activation (35.67±2.52、47.67±4.16). Reprogramming of male MEFs could be improved by the pretreatment with TSA and5-Aza-dC, but it could not obtain the colonies of multipotent stem cells from male MEFs only pretreated with TSA and5-Aza-dC. Colony of multipotent stem cells was not be observed in control groups. Therefore, components which could reprogram the male MEFs into multipotent stem cells were from the ESCs extracts.
4. Identification of the origin of multipotent stem cells from MEFs which been reprogrammed by ESCs extracts
To rule out the possibility of ESCs contamination during the preparation of ESCs extracts, and assess the reliability of the reprogramming system, further pluripotential stem cells generation were performed using male MEFs which were identified by dual PCR for sex identification system(which can amplify250bp Sry gene and399bp ZFX gene) of a different gender from extract-donor ESCs. The donors of ESCs-derived extract were a parthenogenetic embryonic stem cells (pESCs) from the oocytes (which can only amplify399bp ZFX gene) which been parthenogenetic activated and the female ESCs identified with dual PCR for sex identification system from the blastocyst produced from in vivo fertilization. Therefore, we can decided the origin of the pluripotential stem cells according to their gender using dual PCR for sex identification system. Results indicated that40colonies of multipotent stem cells from8batch reprogramming (5colonies of multipotent stem cells were randomly selected in every batch) which mediated by pESCs extracts, pESCs from the reconstructed embryos extract and female extract from in vivo fertilization were used to perform dual PCR for sex identification. Multipotent stem cells which had been amplified250bp Sry gene and399bp ZFX gene, that was there were male colonies. Therefore, the origin of multipotent stem cells obtained in our study were male MEFs, ESCs extracts were not the contaminated by donor ESCs during the preparation of pESCs extracts.
5. Feasibility of interspecies somatic cells reprogramming-mediated by ESCs extracts
Chicken embryonic fibroblasts which being act as the target cells of reprogramming and were incubated with mouse ESCs extracts from Kunming mouse blastocysts derived from in vivo fertilization, morphology changes were observed, and the pluripotent nature of cells obtained from chicken embryonic fibroblasts after being reprogrammed by mouse ESCs extracts were identified, in order to explore the feasibility of reprogramming between the different species. Results indicated that after be incubated with mouse ESCs extracts, reprogrammed chicken embryonic fibroblasts turned to be round, fusiform adherent cells were not observed, and42.33±4.5colonies were generated. The pluripotent nature of the colonies was identified by typical cellular and colonial morphology of ESCs, positive AKP staining, and positive Oct-4and SSEA-1immunocytochemistry staining, and results from karyotype analysis indicated that those cells were derived from chicken embryonic fibroblasts. Chicken embryonic fibroblasts incubated with chicken embryonic fibroblasts extracts or without incubated with any extracts were not appeared the pluripotent nature, Therefore, components which could reprogram the chicken embryonic fibroblasts into multipotent stem cells were from the mouse ESCs extracts. Interspecies somatic cells reprogramming between mouse ESCs and chicken embryonic fibroblasts could be mediated by ESCs extracts.
Conclusions:MEFs pretreated with SLO, TSA and5-Aza-dC could be reprogram ed into multipotent stem cells with cell-free ESCs extract in a shorter period. One day after treated with ESCs extract, MEFs formed the round cells, colonies with defined edges developed and resembled ESCs colonied within4days, these colonies showed the AKP activity on day3and they express Oct-4, Sox2and Nanog on day7. Chicken embryonic fibroblasts could be reprogrammed into multipotent stem cells using the reprogramming system mediated by mouse ESCs extract.
引文
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