Studies On Differentiation Of Human Embryonic Stem Cells Into Endothelial Progenitor Cells And Mechanism Of The Enhancement Of Tumor Growth By Endothelial Cells

Growing interest in using endothelial cells (ECs) for clinical therapeutic purposes has led to explore human embryonic stem cells as a potential source for endothelial progenitor cells (EPCs), which can be induced into ECs. ECs or EPCs play important roles in many clinical therapies, acting as the seed cells in injury of blood vessel, ischemia diseases, etc. But only 0.02-0.1% cells in peripheral blood mononuclear cells are EPCs, which limit its clinical therapy. Human embryonic stem cells (hESCs) are advantageous when compared with other endothelial cell origins, due to their high proliferation capability, pluripotency, and low immunogenity. Differentiation of hESCs into EPCs or ECs also provides scientists an ideal model for stem cell development research. However, there are still many challenges and obstacles to overcome before the vision of using embryonic endothelial progenitor cells.Even though ECs play important roles in regenerative medicine, it is a double-edge sword. Previous studies have suggested that ECs were involved in many kinds of human diseases, such as abnormal growth of blood vessel, cancers, and so on. More and more researches have been focused on exploring the regulation mechanisms of ECs differentiation, development and functional, as well as the possibilities to use ECs as clinical therapeutic targets. The roles of vascular endothelial growth factor (VEGF) in tumorigenesis and tumor progression were identified and the clinical values of VEGF antibody were widely recognized. Previous studies had been suggested that hypoxia, which is one of critical factors in tumor development, played very important roles in VEGF regulation. Hypoxia inducible transcription factor (HIF) is a dimeric complex that plays an integral role in Hypoxia. But it is still not very clear about the roles of HIF in ECs proliferation and biological function.Two major parts were included in this project. 1. Differentiation of human embryonic stem cells into endothelial progenitor cellsAfter digested by collagenase, the hESCs were cocultured with mouse embryonic fibroblasts (MEF), followed by a 4-step induce procedure to induce hESCs into endothelial progenitor cells:cultured in differentiation medium consisting of BMP4 (10ng/ml) for 4 days→cultured in differentiation medium deprived of BMP4 for 6 days→isolation of CD34+progenitor cells using MACS MicroBeads columns→cultured in EGM-2 medium for 7～10 days. Then the adherent cells were harvested by trypsin-treatment and used for further analyses.The results showed that endothelial cell-related genes (CD31% CD34,VE-cad,KDR,vWF,eNOS) could be detected by RT-PCR in primary and passage cells, as well as smooth muscle cell(SMC)-related gene (a-SMA). Expression of CD31,VE-cad and VEGF determined by immunofluorescence staining are positive, however, a-SMA is negative. After the replacement of SMC culture, the cells become broadened and a-SMA gene expression up-regulated more than 22 times folded, which can also be detected by immunofluorescence. There are no differences in gene expression among different passage cells. These results indicated that hESCs can be effectively induced into EPCs which have the potential to differentiate into ECs and SMCs.2. Function and Mechanism of endothelial cells in tumorIn this part, human umbilical vein endothelial cells (HUVECs) were first isolated, cultured and identified. These HUVECs were then used to study the interaction between ECs and cancer cells At the same time, we interfered the gene expression of HIF-1a in ECs (pSicoR-HIF-1α/HUVECs) using SiRNA technology to explore roles of HIF-1αin ECs'biological functions.Our results showed that endothelial cells could promote cancer cell proliferation, migration and invasion. They participated in and promoted cancer angiogenesis. HIF-la in ECs was up regulated when treated with cancer conditioned medium. To further explore the roles of HIF-la in ECs' biological functions, we interfered the gene expression of HIF-la in ECs (pSicoR-HIF-1α/HUVECs) using SiRNA vector. Our results suggested that knock down of HIF-la in ECs decreased their effects on cancer cell migration and invasion in vitro, but didn't changed cancer cell proliferation. Compared with groups which were injected cancer cells lonely, there was no difference in tumor volume and vessel density in groups injected cancer cells with pSicoR-HIF-1α/HUVECs, but lower than in groups injected cancer cells with normal HUVECs. These results indicated that HIF-la play a critical role in tumor angiogenesis.Conclusion:1. Established a system of hESCs differentiation into EPCs effectively, the hESCs-derived EPCs can be largely amplification.2. ECs can promote tumor angiogenesis and cancer cell proliferation, invasive ability, HIF-la play a critical role in this process.