【作者】 陈彤；

【导师】 谢毅； Zack Wang；

【作者基本信息】 复旦大学 ， 内科学， 2006， 博士

【摘要】 人胚胎干细胞向血管内皮细胞的诱导分化及后者迁移机制的研究目的：探讨人胚胎干细胞(hESC)向造血-内皮系诱导分化的能力，将hESC定向分化为高纯度的hESC来源内皮细胞，为hESC应用于构建组织工程血管提供依据。以hESC作为研究载体，建立模仿人胚胎发育过程中血管生成的芽生EB模型，探究SDF-1/CXCR4轴对人类胚胎干细胞来源内皮细胞的迁移和血管生成的影响。材料与方法：1．选用人胚胎干细胞系(NIH人胚胎干细胞登记署登记号为：WA01和09，简称为H1、H9)为研究对象。将未分化hESC培养在经丝裂霉素或照射灭活的小鼠成纤维细胞饲养层上，每日换液一次，第5-7天后进行细胞传代，检测细胞表面期特异性抗原SSEA(Stage specific embryonic antigen)-1、SSEA-4、TRA(tumor rejection antigen)-1-60、TRA-1-81的表达以明确细胞是否属于未分化状态。2．以与小鼠骨髓基质细胞(OP9)共培养或以悬浮类胚体(embryo body，EB)形式进行造血-内皮系诱导分化，每2-3日换液一次。检测分化过程中细胞表面SSEA-1、SSEA-4、和造血-内皮系转录调控基因(GATA-1、SCL、LMO-2、CD31、Tie-2)、以及该二系细胞表面标志的变化，并以半固体培养基进行造血集落的培养。3．以磁珠分选分化后H1来源CD34~+细胞，将这部分细胞置于内皮培养体系再诱导，获取一群类似于成人脐静脉内皮细胞的贴壁细胞，并对这部分细胞进行内皮细胞的性质鉴定，包括CD31、CD34、Flk-1、VE-Cadherin和vWF等表面标记的表达、吞噬LDL的能力以及在Matrigel基质表面形成网状结构的能力等。4．将分化后EB置于胶原基质和VEGF、FGF等血管生成因子的混合体系中，进行芽生EB的诱导，摸索该体系的最佳培养条件，包括EB的时龄、培养的时间、VEGF/FGF的浓度组合等，并进行芽生结构内皮系性质的鉴定，如：CD31荧光标记和血管生成抑制因子对芽状结构的抑制作用等。5．以RT-PCR法和流式细胞仪检测H1分化不同阶段、H1来源内皮细胞中CXCR4和SDF-1的表达水平。将20μl hESC来源内皮细胞置于微孔膜一侧，微孔膜另一侧加入不同浓度梯度的SDF-1，以ChemoTx系统进行H1来源内皮细胞向SDF-1的跨膜迁移能力检测。以外源性SDF-1上调SDF-1/CXCR4轴、或以AMD3100、CXCR4抗体阻断SDF-1/CXCR4的相互结合，观察这些调节作用对H1来源内皮细胞迁移能力、以及hESC来源原始血管生成的影响。结果：1．未分化hESC集落表现为圆形或椭圆形、平坦、细胞排列紧密的细胞团，表达SSEA-4、TRA-1-60、TRA-1-81等未分化hESC的特异性标志，但不表达SSEA-1。随着细胞分化程度的提高，SSEA-4的表达逐渐减弱，而SSEA-1的表达进行性升高。2．HESC与小鼠骨髓基质细胞共培养或以EB形式诱导分化后，原本在未分化状态时不表达的造血-内皮系基因在RT-PCR检测时逐渐显示阳性条带，而未分化基因Oct-4的表达逐渐减弱，中胚层标志基因Brachyury在分化后第6天左右消失。VEGF-R2(Flk-1)在未分化hESC中也有一定程度的表达，随着分化程度的提高，Flk-1的表达程度也逐渐上升。3．细胞在分化后开始表达CD34这一造血/内皮系共同标记，且在第12天左右达到峰值，同期也获得了最多的造血集落数。此后，CD34的阳性率和造血集落的数量逐渐减弱，但粒-巨噬系集落占所有造血集落的比例却逐渐上升。4．我们选择分化后第10天-12天这一CD34表达高峰期为分选时机，以磁珠分选的方法分选hESC来源CD34~+细胞，CD34~+细胞的纯度达到90％以上，这部分细胞同时还表达高水平的CD31、但不表达CD45。将hESC来源CD34~+细胞在内皮细胞培养体系中进一步分化成熟，所获得的贴壁细胞形态类似于成熟人脐静脉内皮细胞，具有成熟内皮细胞的特征，表达高水平的CD31、CD34、Flk-1、VE-Cadherin和vWF，能吞噬Dil-AcLDL，在Matrigel基质表面形成网络状结构。5．将分化第11天的EB置于含有VEGF、FGF的胶原基质中，从EB中可以伸展出有内皮细胞积聚的、类似于血管腔样结构的芽状分支，为芽生EB。芽生EB间还可形成网络状结构，是研究人类胚胎发育过程中血管生成的一种模型。6．HESC的各个分化阶段、hESC来源内皮细胞表面都表达有CXCR4，而且VEGF可以上调细胞表面CXCR4的表达水平。HESC来源内皮细胞可以顺SDF-1浓度梯度出现向SDF-1的跨膜迁移，SDF-1可以增强hESC来源内皮细胞的伸展和相互接触，促进由内皮细胞构成的网络状结构的形成。阻断SDF-1和CXCR4的相互作用可以破坏内皮细胞网络结构、抑制芽状结构的生长、切断芽生EB间的联系。结论：1．在小鼠胚胎成纤维细胞滋养层上，人胚胎干细胞系能够无限增殖并保持其未分化状态。2．在小鼠骨髓基质细胞表面或以悬浮EB形式，未分化hESC能够进入中胚层途径，向造血-内皮系进一步分化。分化后细胞逐渐表达造血/内皮系表面标记，SSEA-4、Oct-4等未分化标志的表达则明显减弱。3．分化后第12天左右，hESC来源的造血细胞大量涌现，随着分化程度的加深，细胞有逐步脱离原始胚胎造血阶段的可能。4．选择分化后第10天-12天这一CD34近高峰期为分选时机，可以获得高纯度的hESC来源CD34阳性细胞，而且这部分细胞可以进一步诱导分化为成熟的血管内皮细胞。因此，我们已经建立了一种将hESC定向分化为成熟血管内皮细胞的方法。5．HESC来源内皮细胞表达有SDF-1/CXCR4这一趋化因子配体/受体对，SDF-1/CXCR4可以调节hESC来源内皮细胞的迁移能力。VEGF可能通过上调细胞表面CXCR4的表达而促进芽生EB的生成，与SDF-1/CXCR4共同介导人胚胎发生过程中原始血管网的形成。更多还原

【Abstract】 Objective: HESCs were differentiated into hemato-endothelial progenitors to determine its ability to derive mature endothelial cells as a potential material to generate tissue-engineered vessels. The molecular mechanisms that regulate human blood vessel formation during early development are largely unknown. Here, we used human embryonic stem cells (hESCs) as an in vitro model to explore early human vasculogenesis. SDF-1/CXCR4 axis was shown to be a morphogen which regulates hESOderived endothelial cells migration in human embryo vasculogenesis.Materials and Methods: 1. Undifferentiated hESCs, H1 and H9, were cultured on mouse embryonic fibroblast cells (MEF) which were inactivated by mitomycin-C or radiation, with a daily change of medium. Cells were passaged on day 5-7. To determine the undifferentiated stage, cells were stained with SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81. 2. To induce hemato-endothelial differentiation of hESCs, cells were cocultured with mouse bone marrow stroma cells (0P9) or treated by dispase to generate suspending hEBs, with a medium change of every 2-3 days. Expression of SSEA-1, SSEA-4, hematopoietic or endothelial lineage markers (GATA-1 SCL LMO-2 CD31 Tie-2), and transcript genes which regulate hemato-endothelial lineage differentiation, were detected by flow cytometry or RT-PCR. Hematopoietic colonies were generated in semi-solid culture system. 3. To purify endothelial cells from hESCs, differentiated CD34~+ cells were sorted by magnet beads, and were further cultured in endothelial medium. The attached cells resembling human umbilical vein endothelial cells (HUVECs) morphologically were identified with the endothelial property. The hESC-derived CD34~+ attached cells were also plated on Matrigel matrix to form tubular structure. 4. Differentiated hEBs can produce sprouting structures in the presence of collagen, VEGFand FGF. We determined the culturing condition appropriate to induce sprouting EBs, including the differentiated EBs age, culturing time, and the concentration of VEGF and FGF. To reveal the endothelial property of the sprouting structure, we stained the sprouting EB with anti-CD31, and the angiogeneic inhibitors were added into the culture system. 5. Expression of SDF-1/CXCR4 on differentiated hEB cells and hESC-derived endothelial cells was clarified by flow cytometry or by RT-PCR analysis. We used a 96-well Boyen ChemoTx System to investigate cell mobility in respond to SDF-1. Twenty μ1 of hESC-derived endothelial cells was plated on the top of the membrane. Different working concentration of SDF-1 was added in the lower chamber. The migrated cells were counted by microscope. Other regulators of SDF-1/CXCR4, including exogenic SDF-1, anti-CXCR4 antibody, and AMD3100, the antagonist of CXCR4, were tested in the assays of Matrigel tubular formation and Collagen sprouting EBs.Results: 1. Undifferentiated hESCs formed round, flat, loose aggregates, which expressed SSEA-4, TRA-1-60, and TRA-1-81. Along with the differentiation of hESC, SSEA-4 was downregulated whereas SSEA-1 was upregulated. 2. Differentiated hESCs by coculture or EB induction were detected to express hemato-endothelial transcription genes, including GATA-1, SCL, LMO-2, CD31, Tie-2 from day-12 and from day-16. Oct-4, the pluripotent gene, was decreased and the mesodermal marker, Brachyury, was undetectable after 6 days of differentiation. Flk-1 was highly expressed on Undifferentiated hESCs, and the expression was moderately increased after 12 days of differentiation. 3. The common marker of hemato-endothelial lineages, CD34, was expressed after the onset of differentiation, and peaked around day 12 when the number of hematopietic colonies was also at the highest. Both of CD34 expression and hematopoietic colonies were decreased afterwards. However, the percentage of CFU-G and CFU-GM in hematopoietic colonies was increased after day 25 of differentiation. 4. We chose CD34 as a candidated marker to isolate hESC-derived EPCs between day 10 and day 12. The purity of isolated CD34~+ cells was greater than 90%. Majority of CD34~+ cells also expressed CD31 but not CD45. To mature hESC-derived endothelial cells,the isolated CD34~+ cells were cultured in the presence of VEGF to grow into the cells morphologically resembling HUVECs, which express endothelial markers, including CD31, CD34, Flk-1, VE-Cadherin and vWF, uptake LDL, and form tubular structure on Matrigel. 5. We set up sprouting EB assay, a model which mimics human embryo vasculogenesis in the presence of VEGF and FGF. The microtubule outgrowths were indeed endothelial origin in nature which stained by CD31 and revealed a cylindrical organization of cord-like vessel. 6. CXCR4 was expressed on different stages of EBs and hESC-derived endothelial cells. VEGF can induce CXCR4 expression on hESC-derived endothelial cell surface. The derived endothelial cells performed a dose-dependent pattern of transmigration to SDF-1, which can be shown to enhance the cell-to-cell stretching and contacting to form microtubule on Matrigel. Blockage of interaction of SDF-1 and CXCR4 can abolish the tubular structure, inhibit the sprouting outgrowth, and diminish the endothleial network between sprouting EBs.Conclusion: 1. HESCs sustain the ability of self-renew and Undifferentiated stage on MEF feeder layers. 2. HESC can differentiate mesodermally into hemato-endothelial lineages by coculture with 0P9 or induction of EBs. Differentiated hESCs express hemato-endothelial lineage markers whereas decrease expression of pluripotent markers, SSEA-4 and Oct-4. 3. HESC-derived hematopoietic cells emerged largely in day12 EB. Increasing percentage of CFU-G/GM colonies on day 25 revealed that profound differentiation of hESCs at that time may leave the primitive way which resembling first wave of hematopoiesis during human embryogenesis. 4. We choose day10-day12 as the candidated timepoint to select hESC-derived CD34~+ cells. By further cultured with VEGF, hESC-derived CD34~+ cells can differentiate into mature endothelial cells. 5. SDF-1/CXCR4 was expressed in hESC-derived endothelial cells. Function of VEGF on upregulating surface CXCR4 expression suggested that the enhancement of sprouting EBs by VEGF is due to the increase of CXCR4 expression. VEGF and SDF-1/CXCR4 axis may collaborate in the vasculogenesis during early human embryo development.更多还原