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Differentiation of Human Embryonic Stem Cells into Endothelial Cells and the Mechanism of hESC-derived Endothelial Cells Migration

Author: ChenTong
Tutor: XieYi;Zack Wang
School: Fudan University
Course: Internal Medicine
Keywords: human embryonic stem cell differentiation endothelial cell mesoderm embryo body vasculogenesis migration VEGF SDF-1 CXCR4
CLC: R329.2
Type: PhD thesis
Year: 2006
Downloads: 373
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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.

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CLC: > Medicine, health > Basic Medical > Human morphology > Human histology > Human cytology
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