人胚胎干细胞生物学性状及向内皮分化过程中ID-1基因作用的研究
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摘要
第一部分由正常和异常原核来源的合子得到的人胚胎干细胞系生物性状的比较
目的：人胚胎干细胞系可建立于正常和异常受精的合子,然而,这两种类型的人胚胎干细胞系之间的性状的异同尚未可知。有文献报道双倍体的人胚胎干细胞系可以由单原核来源的合子建系得到,但形态学为多原核受精的合子是否也可建立正常的人胚胎干细胞系尚无明确报道。为了探索这些问题,我们建立了9株人胚胎干细胞系,分别来源于形态学正常(双原核,2PN)和形态学异常(未见原核,0PN单原核,1PN；三原核,3PN)原核的合子。通过深入比较其性状异同,以期解决以上问题。
方法：建立来源于形态学正常和异常原核的受精合子的hES细胞系及其培养维持。碱性磷酸酶染色及免疫荧光染色检测各株细胞的“干”性,畸胎瘤形成实验检测体内分化能力,RT-PCR分析检测体外分化能力。核型分析检测各株细胞的遗传稳定性,DNA质谱分析检测每株hES细胞的基因组DNA的特异性。流式细胞术分析细胞周期分布情况,BrdU掺入实验分析细胞系增殖潜力。实时定量PCR分析代表分化及未分化基因的表达水平。hES细胞的神经定向诱导分化检测神经分化潜能。
结果：我们由46个临床废弃的新鲜囊胚建立了9株人胚胎干细胞系(细胞株mSDU-hES 1-9),其中5个为2PN来源,2个为0PN来源,1个为1PN来源,1个为3PN来源。由形态学不同原核来源的合子得到的干细胞系显示相似的克隆形态,每株细胞系均能成功冻融,均不存在增殖及自我更新障碍。每一株胚胎干细胞系均能表达碱性磷酸酶活性及“干性”标记：OCT-4,SSEA-3,SSEA-4,TRA-1-60和TRA-1-81。每隔6个月进行一次的核型分析表明,每一株胚胎干细胞系具有正常的双倍体核型,染色体畸变未检测到,每株胚胎干细胞系都有各自独特的遗传特异性标记。严重联合免疫缺陷(SCID)小鼠在接种各株未分化hES细胞10-12周后经解剖证实均有含有三个胚层细胞系的畸胎瘤形成,说明各株干细胞均具有体内分化的全能性。细胞株mSDU-hES 1-8分化得到的拟胚体能够表达OCT4以及代表外胚层、中胚层和内胚层的组织特异性基因,但是细胞株mSDU-hES 9 (2PN)分化得到的拟胚体仅表达代表外胚层和中胚层的基因,而并不表达代表内胚层的基因。hES细胞系的细胞周期分布呈独特的4阶段细胞周期模式,特征为大量细胞处于S期而小部分细胞处于Go／G1和G2／M期。与细胞株mSDU-hES 5(2PN)比较,细胞株mSDU-hES 2(0PN)显示有更多的细胞处于S期,具有较高含量的增殖期细胞；而1PN和3PN合子来源的hES细胞系与细胞株mSDU-hES 5(2PN)具有相似的增殖期细胞含量。mSDU-hES 2 (OPN)比其它细胞株具有较短的细胞周期和较高的增殖率。所检测的所有细胞系均可表达11种分化及未分化标记,但其基因表达含量各不相同。对于大多数未分化的基因OCT4, NANOG, LIN41和SOX2,各株细胞系间无明显差异。然而,2PN来源的细胞株mSDU-hES5中DPPA5的表达显著低于其它细胞系,而3PN来源的mSDU-hES8中UTF1的表达显著高于其它细胞系。各株细胞系对于分化基因的表达也存在较大的差异。这种分化及未分化标记基因相对含量表达差异在正常的hES细胞株之间同样存在。hES细胞经过3周神经定向分化的培养,来源于形态学正常及异常受精合子的干细胞株均可分化出NESTIN阳性神经前体细胞和TUJ1阳性神经元细胞,且2PN合子来源的hES细胞株及0PN、1PN、3PN合子来源的hES细胞株具有相似神经分化潜能。
结论：1.来源于形态学上不同原核的合子的9株hES细胞系具有相似的大致特征。所有9株细胞表达相似的“干性”标记,包括转录因子及糖脂分子标记物：OCT-4,SSEA-3,SSEA-4,TRA-1-60及TRA-1-81；各株细胞的增殖能力及在拟胚体和畸胎瘤中分化为外胚层、内胚层和中胚层细胞系的潜能也具有相似性。在神经定向分化条件下,所有的细胞株均可分化成神经前体和神经元。2.在细胞周期和分化及未分化标记基因的相对表达量等方面存在差异。这些差异在正常来源的不同人胚胎干细胞系之间也同样存在。这种现象可能决定于不同细胞系本身的生物学特性,而非与形态学正常或异常原核来源相关。3.正常的人胚胎干细胞系可以由临床弃用的具有形态学异常原核的受精合子建立。
第二部分ID-1基因在TGF-betal诱导的人胚胎干细胞定向分化为血管内皮细胞中的作用探讨
目的：分化抑制子／DNA结合抑制子-1(ID-1)是内皮细胞(ECs)中活化素受体样激酶-1(ALK1)的特异性下游基因,它介导转化生长因子-β(TGF-β)／ALK1诱导的(Smad依赖的)内皮细胞迁移。然而,ID-1基因在TGF-β1诱导的人胚胎干细胞向内皮分化及血管发生的信号转导通路中的作用,尚未有确切的研究报道。本文以人胚胎干细胞(hESCs)的体外分化作为血管内皮发育的模型,来研究ID-1基因在TGF-β1诱导的人胚胎干细胞定向分化为血管内皮细胞及血管发生的信号转导通路中的作用。
方法：建立TGF-β1诱导的人胚胎干细胞分化为血管内皮细胞及形成血管样结构的模型,用免疫荧光定性分析不同浓度的TGF-β1对血管内皮定向分化及血管发生的作用。利用实时定量PCR技术检测TGF-β1诱导组及对照组中ID-1基因和内皮细胞标志基因PECAM, KDR的表达含量,分析TGF-β1对ID-1基因表达的影响。利用实时定量PCR技术分析TGF-β1诱导的细胞分化和血管发生过程中ID-1基因表达的时间动力学表现。通过小干扰RNA技术和实时定量PCR技术分析ID-1基因在TGF-β1诱导的人胚胎干细胞分化的血管内皮细胞中的功能。利用实时定量PCR技术和western杂交技术分析分化和血管发生过程中TGF-β1受体及信号分子蛋白表达的时间动力学表达。
结果：我们发现在hESCs分化为ECs早期,TGF-β1能够通过抑制ID-1基因的表达诱导人胚胎干细胞向内皮系的定向分化,在分化晚期的血管发生阶段,TGF-β1通过ALK1／Smadl,5／ID-1信号转导通路增强ID-1基因的表达而促进内皮细胞增殖。但是TGF-β1在血管生成过程中对于血管出芽却起抑制作用。另外,TGF-β1诱导的人胚胎干细胞定向分化为血管内皮细胞过程及ID-1基因的表达对于TGF-β1不仅具有时间依赖性,而且具有浓度的依赖性。通过沉默ID-1基因来降调该基因的表达将促进TGF-β1诱导的人胚胎干细胞定向分化为血管内皮细胞而抑制分化的内皮细胞增殖和迁移。
结论：运用TGF-β1诱导的人胚胎干细胞分化模型,我们分析了在向内皮系分化及分化的内皮细胞增殖过程中ID-1基因的功能。我们的数据表明,在分化过程中TGF-β1可以通过ALK5通路刺激内皮方向的分化,而在增殖过程中则通过TGF-β1/ALK1/ID-1,Smad1/Smad5依赖的信号转导途径增强分化的内皮细胞的增殖。本研究运用人胚胎干细胞体外分化的模型,有助于我们了解胚胎发育早期阶段体内血管形成的部分机制。通过进一步研究该基因的调控因子,以促进或抑制其表达水平,对于临床治疗病理性血管生成和促进修复性血管生成有指导借鉴意义。
PARTⅠ. Comparative Evaluation of Human Embryonic Stem Cell Lines Derived from Zygotes with Normal and Abnormal Pronuclei
BACKGROUND AND OBJECTIVE:hES cell lines have been derived from normally or abnormally fertilized zygotes. However, the similar and different properties of these two types of hES cell lines are not well-known. It has been reported that a diploid human embryonic stem cell line was derived from a mononuclear zygote. However, it remains uncertain whether morphologically multi-pronuclei fertilized zygotes can be used for generation of normal hES cell lines. To address these questions, we generated 9 hES cell lines from zygotes containing normal (2PN) and abnormal (OPN,1PN,3PN) pronuclei morphologically. A side-by-side comparison would help us to understand these bewilderments.
METHODS:Derivation and maintenance of hES cell lines from fertilized zygotes with morphologically normal and abnormal pronuclei. Detect alkaline phosphatase (AP) activity and confirm the expression of several undifferentiated (sternness) markers by immunocytochemistry. Test the multilineage differentiation potential in vivo and in vitro by teratomas formation experiment and RT-PCR analysis of differentiated gene expression in EBs. Karyotypic analysis in order to detect the genetic stability, and short tandem repeat (STR) analysis to show distinct identity of each hES cell lines. Compare cell cycle distribution and proliferative potential by flow cytometry and BrdU incorporation assay. Assess undifferentiated and differentiated genes expression by real-time qPCR. Use the neural directed differentiation method to test the neural differentiating potential in vitro culture.
RESULTS:We established nine hES cell lines (mSDU-hES 1-9 cell lines) from 46 fresh blastocysts which were clinically discarded, of which 5 were derived from 2PN-zygotes,2 from OPN-zygotes,1 from 1PN-zygotes, and 1 from 3PN-zygotes. Each cell line of different PN zygotes showed similar round compact colony morphology with a defined border towards the feeder layer. Each of the cell lines was successfully cryopreserved and thawed. A period of replicative crisis was not observed for any of the cell lines. Cells of each hES cell lines possessed high levels of AP activity and were positive to transcription factors and glycolipids markers: OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Karyotyping was performed every 6 month and revealed a normal diploid karyotype in each hES cell line. No chromosome aberration was detected. Each of the nine hES cell lines has its own genetic label. SCID-beige mice were killed ten to twelve weeks after being injected with undifferentiated hES cells of each line to confirm the formation of teratomas containing three embryonic germ lineages, which indicated that each cell line has the totipotential in vivo differentiation. The EBs derived from mSDU-hES 1-8 cell lines expressed OCT4 and the tissue-specific genes representing ectoderm, mesoderm and endoderm. However, the mSDU-hES 9 (2PN)-derived EBs expressed genes representing ectoderm and mesoderm, but not endoderm. The cell cycle distributions of these hES cell lines showed a unique pattern of four cell cycle stages G1, S, G2, and M characterized by a large proportion of cells in S phase and a small proportion of cells in G0/G1 and G2/M phase. Compared to mSDU-hES 5 (2PN) cell line, mSDU-hES 2 (OPN) cell line showed higher proportion of cells in S phase and slightly higher composition of proliferating cells while 1PN and 3PN-zygote-derived hES cell lines showed similar composition of proliferating cells with mSDU-hES 5 (2PN) cell line. mSDU-hES 2 (OPN) cell line may have a shorter cell cycle duration than other cell lines tested due to the higher proliferation rate. hES cell lines derived from normal or abnormal pronuclei zygotes expressed all 11 undifferentiated and differentiated markers, but their gene expression levels were variable. For the most undifferentiated genes OCT4, NANOG, LIN41 and SOX2 there was no significant difference among the hES cell lines. However, compared to other hES cell lines, the expression of DPPA5 was significantly lower in 2PN-derived mSDU-hES 5, while the expression of UTF1 was significantly higher in 3PN-derived mSDU-hES 8. A profound variation in relative abundance of gene expression in differentiated markers of the cell lines was observed. After 3 weeks of neural differentiation of hES cells in vitro culture, both NESTIN+ neural progenitors and TUJ1+ neurons were generated from morphologically normally and abnormally fertilized zygote-derived cell lines. 2PN-zygote-derived and OPN-,1PN- and 3PN-zygote-derived hES cell lines showed similar neural differentiating potential.
CONCLUSION:1. All 9 hES cell lines derived from zygotes with normal and abnormal pronuclei shared the majority of these characteristics. They all showed a similar expression pattern of "stemness" markers, including transcription factors and glycolipids markers:OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81; the ability to proliferate and differentiate into cell lineages in ectoderm, endoderm, and mesoderm in differentiating embryoid bodies and teratomas. Under directed neural differentiation, all the cell lines differentiated into neural progenitors and neurons.2. Differences did exist in cell cycle and in the relative abundance of gene expression in undifferentiated and differentiated markers, but such differences were also found among normal hES cell lines. This phenomenon may be determined by the special biological property of different cell lines, but have no direct connection with the normal or abnormal pronuclei-derivation.3. Normal hES cell lines can be developed from fertilized zygotes with morphologically abnormal (OPN,1PN,3PN) pronuclei which are usually excluded from clinical use.
PartⅡ. Expression and Function of ID-1 Gene during the Differentiation of Human Embryonic Stem Cells to Endothelial Cells Induced by TGF-betal
BACKGROUND AND OBJECTIVE:Inhibitor of differentiation/DNA binding-1 (ID-1) is a specific downstream target gene of activin receptor-like kinase-1 (ALK1) in endothelial cells (ECs) and it can mediate the transforming growth factor-β(TGF-P)/ALK1- induced (and Smad-dependent) migration. However, it remains unclear that how ID-1 plays a part during the differentiation of human embryonic stem cells (hESCs) into ECs induced by TGF-β1. In this study, we used the hESCs differentiation model that recapitulates the developmental steps of vasculogenesis in the early stage of embryo development to explore the role that ID-1 gene plays in the process of TGF-β1 induced hESCs differentiation towards endothelial lineage.
METHODS:hESCs culture and establishment of TGF-β1 induced differentiation model of hESCs differentiating into the endothelial lineage. Then analyze the effect of TGF-β1 of different concentrations on the differentiation and vasculogenesis by immunostaining of EBs (embryoid bodies) and differentiated cells. Compare the expression of ID-1 gene and endothelial cell marker, PECAM (platelet/endothelial cell adhesion molecule-1)、KDR (kinase insert domain receptor) gene in TGF-β1-induced group and control group by qRT-PCR, and then analyze the effect of TGF-β1 on ID-1 gene. Analyze the kinetics of ID-1 expression during differentiation and vasculogenesis induced by TGF-β1 using qRT-PCR technology. Study the function of ID-1 gene during the differentiation of human embryonic stem cells into ECs induced by TGF-β1 by small interfering RNAs and qRT-PCR technology. Analyze the kinetics of TGF-β1 receptors and some signal proteins expression during differentiation and vasculogenesis stages using qRT-PCR and western blot technology.
RESULTS:We have demonstrated that at the early stage of differentiation TGF-β1 may induce the in vitro differentiation of hESCs into ECs by inhibiting expression of ID-1, while at the late stage of differentiation TGF-β1 may stimulate the proliferation and migration of ECs via ALK1/Smad1,5/ID-1 pathway. But TGF-β1 has a negative effect on the angiogenic sprout formation during angiogenesis stage. In addition, in vitro differentiation of hESCs into ECs and expression of ID-1 induced by TGF-β1 are not only time-depended, but also depended on the concentration of TGF-β1. Down-regulation of ID-1 by silence of this gene can lead to accelaration of hESCs differentiation into ECs induced by TGF-β1 and inhibition of proliferation and migration of ECs.
CONCLUSION:Taken together, the use of TGF-β1 induced hESCs differentiation model permitted us to dissect the function of ID-1 gene during differentiation process towards endothelial lineage and proliferation process of differentiated ECs. Our present data suggest that TGF-β1 may stimulate the endothelial-oriented differentiation through activated ALK5 pathway during the differentiation process, whereas enhance the proliferation of differentiated ECs by activated TGF-β1/ALK1/ID-1, Smad1/Smad5-dependent signal transduction during the proliferation process. This study used the in vitro hESCs differentiation model may help us to understand some mechanisms of vasculogenesis in the early stage of embryo development.
目的：人胚胎干细胞系可建立于正常和异常受精的合子,然而,这两种类型的人胚胎干细胞系之间的性状的异同尚未可知。有文献报道双倍体的人胚胎干细胞系可以由单原核来源的合子建系得到,但形态学为多原核受精的合子是否也可建立正常的人胚胎干细胞系尚无明确报道。为了探索这些问题,我们建立了9株人胚胎干细胞系,分别来源于形态学正常(双原核,2PN)和形态学异常(未见原核,0PN单原核,1PN；三原核,3PN)原核的合子。通过深入比较其性状异同,以期解决以上问题。
方法：建立来源于形态学正常和异常原核的受精合子的hES细胞系及其培养维持。碱性磷酸酶染色及免疫荧光染色检测各株细胞的“干”性,畸胎瘤形成实验检测体内分化能力,RT-PCR分析检测体外分化能力。核型分析检测各株细胞的遗传稳定性,DNA质谱分析检测每株hES细胞的基因组DNA的特异性。流式细胞术分析细胞周期分布情况,BrdU掺入实验分析细胞系增殖潜力。实时定量PCR分析代表分化及未分化基因的表达水平。hES细胞的神经定向诱导分化检测神经分化潜能。
结果：我们由46个临床废弃的新鲜囊胚建立了9株人胚胎干细胞系(细胞株mSDU-hES 1-9),其中5个为2PN来源,2个为0PN来源,1个为1PN来源,1个为3PN来源。由形态学不同原核来源的合子得到的干细胞系显示相似的克隆形态,每株细胞系均能成功冻融,均不存在增殖及自我更新障碍。每一株胚胎干细胞系均能表达碱性磷酸酶活性及“干性”标记：OCT-4,SSEA-3,SSEA-4,TRA-1-60和TRA-1-81。每隔6个月进行一次的核型分析表明,每一株胚胎干细胞系具有正常的双倍体核型,染色体畸变未检测到,每株胚胎干细胞系都有各自独特的遗传特异性标记。严重联合免疫缺陷(SCID)小鼠在接种各株未分化hES细胞10-12周后经解剖证实均有含有三个胚层细胞系的畸胎瘤形成,说明各株干细胞均具有体内分化的全能性。细胞株mSDU-hES 1-8分化得到的拟胚体能够表达OCT4以及代表外胚层、中胚层和内胚层的组织特异性基因,但是细胞株mSDU-hES 9 (2PN)分化得到的拟胚体仅表达代表外胚层和中胚层的基因,而并不表达代表内胚层的基因。hES细胞系的细胞周期分布呈独特的4阶段细胞周期模式,特征为大量细胞处于S期而小部分细胞处于Go／G1和G2／M期。与细胞株mSDU-hES 5(2PN)比较,细胞株mSDU-hES 2(0PN)显示有更多的细胞处于S期,具有较高含量的增殖期细胞；而1PN和3PN合子来源的hES细胞系与细胞株mSDU-hES 5(2PN)具有相似的增殖期细胞含量。mSDU-hES 2 (OPN)比其它细胞株具有较短的细胞周期和较高的增殖率。所检测的所有细胞系均可表达11种分化及未分化标记,但其基因表达含量各不相同。对于大多数未分化的基因OCT4, NANOG, LIN41和SOX2,各株细胞系间无明显差异。然而,2PN来源的细胞株mSDU-hES5中DPPA5的表达显著低于其它细胞系,而3PN来源的mSDU-hES8中UTF1的表达显著高于其它细胞系。各株细胞系对于分化基因的表达也存在较大的差异。这种分化及未分化标记基因相对含量表达差异在正常的hES细胞株之间同样存在。hES细胞经过3周神经定向分化的培养,来源于形态学正常及异常受精合子的干细胞株均可分化出NESTIN阳性神经前体细胞和TUJ1阳性神经元细胞,且2PN合子来源的hES细胞株及0PN、1PN、3PN合子来源的hES细胞株具有相似神经分化潜能。
结论：1.来源于形态学上不同原核的合子的9株hES细胞系具有相似的大致特征。所有9株细胞表达相似的“干性”标记,包括转录因子及糖脂分子标记物：OCT-4,SSEA-3,SSEA-4,TRA-1-60及TRA-1-81；各株细胞的增殖能力及在拟胚体和畸胎瘤中分化为外胚层、内胚层和中胚层细胞系的潜能也具有相似性。在神经定向分化条件下,所有的细胞株均可分化成神经前体和神经元。2.在细胞周期和分化及未分化标记基因的相对表达量等方面存在差异。这些差异在正常来源的不同人胚胎干细胞系之间也同样存在。这种现象可能决定于不同细胞系本身的生物学特性,而非与形态学正常或异常原核来源相关。3.正常的人胚胎干细胞系可以由临床弃用的具有形态学异常原核的受精合子建立。
第二部分ID-1基因在TGF-betal诱导的人胚胎干细胞定向分化为血管内皮细胞中的作用探讨
目的：分化抑制子／DNA结合抑制子-1(ID-1)是内皮细胞(ECs)中活化素受体样激酶-1(ALK1)的特异性下游基因,它介导转化生长因子-β(TGF-β)／ALK1诱导的(Smad依赖的)内皮细胞迁移。然而,ID-1基因在TGF-β1诱导的人胚胎干细胞向内皮分化及血管发生的信号转导通路中的作用,尚未有确切的研究报道。本文以人胚胎干细胞(hESCs)的体外分化作为血管内皮发育的模型,来研究ID-1基因在TGF-β1诱导的人胚胎干细胞定向分化为血管内皮细胞及血管发生的信号转导通路中的作用。
方法：建立TGF-β1诱导的人胚胎干细胞分化为血管内皮细胞及形成血管样结构的模型,用免疫荧光定性分析不同浓度的TGF-β1对血管内皮定向分化及血管发生的作用。利用实时定量PCR技术检测TGF-β1诱导组及对照组中ID-1基因和内皮细胞标志基因PECAM, KDR的表达含量,分析TGF-β1对ID-1基因表达的影响。利用实时定量PCR技术分析TGF-β1诱导的细胞分化和血管发生过程中ID-1基因表达的时间动力学表现。通过小干扰RNA技术和实时定量PCR技术分析ID-1基因在TGF-β1诱导的人胚胎干细胞分化的血管内皮细胞中的功能。利用实时定量PCR技术和western杂交技术分析分化和血管发生过程中TGF-β1受体及信号分子蛋白表达的时间动力学表达。
结果：我们发现在hESCs分化为ECs早期,TGF-β1能够通过抑制ID-1基因的表达诱导人胚胎干细胞向内皮系的定向分化,在分化晚期的血管发生阶段,TGF-β1通过ALK1／Smadl,5／ID-1信号转导通路增强ID-1基因的表达而促进内皮细胞增殖。但是TGF-β1在血管生成过程中对于血管出芽却起抑制作用。另外,TGF-β1诱导的人胚胎干细胞定向分化为血管内皮细胞过程及ID-1基因的表达对于TGF-β1不仅具有时间依赖性,而且具有浓度的依赖性。通过沉默ID-1基因来降调该基因的表达将促进TGF-β1诱导的人胚胎干细胞定向分化为血管内皮细胞而抑制分化的内皮细胞增殖和迁移。
结论：运用TGF-β1诱导的人胚胎干细胞分化模型,我们分析了在向内皮系分化及分化的内皮细胞增殖过程中ID-1基因的功能。我们的数据表明,在分化过程中TGF-β1可以通过ALK5通路刺激内皮方向的分化,而在增殖过程中则通过TGF-β1/ALK1/ID-1,Smad1/Smad5依赖的信号转导途径增强分化的内皮细胞的增殖。本研究运用人胚胎干细胞体外分化的模型,有助于我们了解胚胎发育早期阶段体内血管形成的部分机制。通过进一步研究该基因的调控因子,以促进或抑制其表达水平,对于临床治疗病理性血管生成和促进修复性血管生成有指导借鉴意义。
PARTⅠ. Comparative Evaluation of Human Embryonic Stem Cell Lines Derived from Zygotes with Normal and Abnormal Pronuclei
BACKGROUND AND OBJECTIVE:hES cell lines have been derived from normally or abnormally fertilized zygotes. However, the similar and different properties of these two types of hES cell lines are not well-known. It has been reported that a diploid human embryonic stem cell line was derived from a mononuclear zygote. However, it remains uncertain whether morphologically multi-pronuclei fertilized zygotes can be used for generation of normal hES cell lines. To address these questions, we generated 9 hES cell lines from zygotes containing normal (2PN) and abnormal (OPN,1PN,3PN) pronuclei morphologically. A side-by-side comparison would help us to understand these bewilderments.
METHODS:Derivation and maintenance of hES cell lines from fertilized zygotes with morphologically normal and abnormal pronuclei. Detect alkaline phosphatase (AP) activity and confirm the expression of several undifferentiated (sternness) markers by immunocytochemistry. Test the multilineage differentiation potential in vivo and in vitro by teratomas formation experiment and RT-PCR analysis of differentiated gene expression in EBs. Karyotypic analysis in order to detect the genetic stability, and short tandem repeat (STR) analysis to show distinct identity of each hES cell lines. Compare cell cycle distribution and proliferative potential by flow cytometry and BrdU incorporation assay. Assess undifferentiated and differentiated genes expression by real-time qPCR. Use the neural directed differentiation method to test the neural differentiating potential in vitro culture.
RESULTS:We established nine hES cell lines (mSDU-hES 1-9 cell lines) from 46 fresh blastocysts which were clinically discarded, of which 5 were derived from 2PN-zygotes,2 from OPN-zygotes,1 from 1PN-zygotes, and 1 from 3PN-zygotes. Each cell line of different PN zygotes showed similar round compact colony morphology with a defined border towards the feeder layer. Each of the cell lines was successfully cryopreserved and thawed. A period of replicative crisis was not observed for any of the cell lines. Cells of each hES cell lines possessed high levels of AP activity and were positive to transcription factors and glycolipids markers: OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Karyotyping was performed every 6 month and revealed a normal diploid karyotype in each hES cell line. No chromosome aberration was detected. Each of the nine hES cell lines has its own genetic label. SCID-beige mice were killed ten to twelve weeks after being injected with undifferentiated hES cells of each line to confirm the formation of teratomas containing three embryonic germ lineages, which indicated that each cell line has the totipotential in vivo differentiation. The EBs derived from mSDU-hES 1-8 cell lines expressed OCT4 and the tissue-specific genes representing ectoderm, mesoderm and endoderm. However, the mSDU-hES 9 (2PN)-derived EBs expressed genes representing ectoderm and mesoderm, but not endoderm. The cell cycle distributions of these hES cell lines showed a unique pattern of four cell cycle stages G1, S, G2, and M characterized by a large proportion of cells in S phase and a small proportion of cells in G0/G1 and G2/M phase. Compared to mSDU-hES 5 (2PN) cell line, mSDU-hES 2 (OPN) cell line showed higher proportion of cells in S phase and slightly higher composition of proliferating cells while 1PN and 3PN-zygote-derived hES cell lines showed similar composition of proliferating cells with mSDU-hES 5 (2PN) cell line. mSDU-hES 2 (OPN) cell line may have a shorter cell cycle duration than other cell lines tested due to the higher proliferation rate. hES cell lines derived from normal or abnormal pronuclei zygotes expressed all 11 undifferentiated and differentiated markers, but their gene expression levels were variable. For the most undifferentiated genes OCT4, NANOG, LIN41 and SOX2 there was no significant difference among the hES cell lines. However, compared to other hES cell lines, the expression of DPPA5 was significantly lower in 2PN-derived mSDU-hES 5, while the expression of UTF1 was significantly higher in 3PN-derived mSDU-hES 8. A profound variation in relative abundance of gene expression in differentiated markers of the cell lines was observed. After 3 weeks of neural differentiation of hES cells in vitro culture, both NESTIN+ neural progenitors and TUJ1+ neurons were generated from morphologically normally and abnormally fertilized zygote-derived cell lines. 2PN-zygote-derived and OPN-,1PN- and 3PN-zygote-derived hES cell lines showed similar neural differentiating potential.
CONCLUSION:1. All 9 hES cell lines derived from zygotes with normal and abnormal pronuclei shared the majority of these characteristics. They all showed a similar expression pattern of "stemness" markers, including transcription factors and glycolipids markers:OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81; the ability to proliferate and differentiate into cell lineages in ectoderm, endoderm, and mesoderm in differentiating embryoid bodies and teratomas. Under directed neural differentiation, all the cell lines differentiated into neural progenitors and neurons.2. Differences did exist in cell cycle and in the relative abundance of gene expression in undifferentiated and differentiated markers, but such differences were also found among normal hES cell lines. This phenomenon may be determined by the special biological property of different cell lines, but have no direct connection with the normal or abnormal pronuclei-derivation.3. Normal hES cell lines can be developed from fertilized zygotes with morphologically abnormal (OPN,1PN,3PN) pronuclei which are usually excluded from clinical use.
PartⅡ. Expression and Function of ID-1 Gene during the Differentiation of Human Embryonic Stem Cells to Endothelial Cells Induced by TGF-betal
BACKGROUND AND OBJECTIVE:Inhibitor of differentiation/DNA binding-1 (ID-1) is a specific downstream target gene of activin receptor-like kinase-1 (ALK1) in endothelial cells (ECs) and it can mediate the transforming growth factor-β(TGF-P)/ALK1- induced (and Smad-dependent) migration. However, it remains unclear that how ID-1 plays a part during the differentiation of human embryonic stem cells (hESCs) into ECs induced by TGF-β1. In this study, we used the hESCs differentiation model that recapitulates the developmental steps of vasculogenesis in the early stage of embryo development to explore the role that ID-1 gene plays in the process of TGF-β1 induced hESCs differentiation towards endothelial lineage.
METHODS:hESCs culture and establishment of TGF-β1 induced differentiation model of hESCs differentiating into the endothelial lineage. Then analyze the effect of TGF-β1 of different concentrations on the differentiation and vasculogenesis by immunostaining of EBs (embryoid bodies) and differentiated cells. Compare the expression of ID-1 gene and endothelial cell marker, PECAM (platelet/endothelial cell adhesion molecule-1)、KDR (kinase insert domain receptor) gene in TGF-β1-induced group and control group by qRT-PCR, and then analyze the effect of TGF-β1 on ID-1 gene. Analyze the kinetics of ID-1 expression during differentiation and vasculogenesis induced by TGF-β1 using qRT-PCR technology. Study the function of ID-1 gene during the differentiation of human embryonic stem cells into ECs induced by TGF-β1 by small interfering RNAs and qRT-PCR technology. Analyze the kinetics of TGF-β1 receptors and some signal proteins expression during differentiation and vasculogenesis stages using qRT-PCR and western blot technology.
RESULTS:We have demonstrated that at the early stage of differentiation TGF-β1 may induce the in vitro differentiation of hESCs into ECs by inhibiting expression of ID-1, while at the late stage of differentiation TGF-β1 may stimulate the proliferation and migration of ECs via ALK1/Smad1,5/ID-1 pathway. But TGF-β1 has a negative effect on the angiogenic sprout formation during angiogenesis stage. In addition, in vitro differentiation of hESCs into ECs and expression of ID-1 induced by TGF-β1 are not only time-depended, but also depended on the concentration of TGF-β1. Down-regulation of ID-1 by silence of this gene can lead to accelaration of hESCs differentiation into ECs induced by TGF-β1 and inhibition of proliferation and migration of ECs.
CONCLUSION:Taken together, the use of TGF-β1 induced hESCs differentiation model permitted us to dissect the function of ID-1 gene during differentiation process towards endothelial lineage and proliferation process of differentiated ECs. Our present data suggest that TGF-β1 may stimulate the endothelial-oriented differentiation through activated ALK5 pathway during the differentiation process, whereas enhance the proliferation of differentiated ECs by activated TGF-β1/ALK1/ID-1, Smad1/Smad5-dependent signal transduction during the proliferation process. This study used the in vitro hESCs differentiation model may help us to understand some mechanisms of vasculogenesis in the early stage of embryo development.
引文
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