The Research Of The Differentiation Of Embryonic Stem Cells Or Embryonic-like Stem Cells Into Vascular Cells And Related Mechanism

Vascular system is the biggest and the most widely distributed system in human being. Vascular cells include endothelial cells and smooth muscle cells. The disfunction of vascular cells can cause diseases such as atherosclerosis, myocardial infarction, stock and reginal ischemia.Embryonic stem cells (ESCs) are cell lines from inner cell mess of blastula. They can infinitely proliferate and are capable of differentiate into all the cell types of three layers. The in vitro system of differentiation of ESCs into vascular cells is a valuable tool to study vascular development, screen drugs for cardiovascular diseases, or potential cell transplantation in clinic applications. This thesis focuses on several issues that are related to the differentiation of ESCs or ESC-like cells (iPS) to endothelial cells and/or smooth muscle cells as well as the mechanisms of the differentiation, which include the following aspects:1. The promotional effects of conditional medium of endothelial cells to the differentiation of ESCs into endothelial cells; 2. The comparison of ESCs derived endothelial-like cells with adult endothelial progenitors and human umbilical cord vascular endothelial cells; 3. The differentiation of iPS cells into endothelial cells and smooth muscle cells; 4. The screening of the transcriptional factors that are related to smooth muscle differentiation and the mechanisms underneath; 5. ZNF297B regulates differentiation of smooth muscle cells through interaction with myocardin. Chapter 1 The effect of a conditional medium of endothelial cells in inducing the differentiation of mouse embryonic stem cells into endothelial cellsObjective:It has been proved that the conditional medium of mouse bone marrow endothelial cells (mEC-CM) could promote the proliferation and differentiation of embryonic or adult hematopoietic cells; it also could stimulate the growth of bone marrow endothelial cells. The purpose of this experiment is to study the effect of the cytokines in this mEC-CM in inducing the differentiation of mouse embryonic stem cells into endothelial cells.Methods:In this study, we induced the mESCs into endothelial progenitors with mEC-CM, and compared the differentiation efficiency with the induction by cytokine cocktail(VEGF,EGF,bFGF and IGF1). We also handpicked the DiI-Ac-LDL uptaken cells derived from mESCs and analyzed their characteristics. They were a pure cell population with endothelial characteristics.Results:mEC-CM could significantly promote the differentiation of mESCs into Flk1+ cells. The efficiency was similar to the induction by cytokine cocktail. We got pure cobblestone-like cell population by induction by mEC-CM combined with handpick-up Dil-Ac-LDL-positive cells. This cell population could express endothelial marker, bind UEA1, uptake DiI-Ac-LDL and form tube-like structure in vitro.Conclusions:mEC-CM can induce the differentiation of mESCs into endothelial cells. Manual selection of DiI-Ac-LDL-positive cells could be a potential method to isolate endothelial-like cells from differentiated mESCs.Chapter 2 Comparison the embryonic stem cell-derived KDR+ cells with cord blood endothelial progenitors and human umbilical cord vascular endothelial cellsObjective:To study the characteristics of human embryonic stem cell-derived endothelial progenitors (KDR+ cells), and compare them with CBEPCs and HUVECs.Methods:The human ES cell lines that established by our institute were allowed to spontaneously differentiated into 9 day embryoid bodies (9dEBs). Isolated the KDR+ cells from 9dEBs by MACS and cultured them in endothelial medium. Their expression of endothelial markers and their function were tested and compared with CBEPCs and HUVECs.Results:Compared with adult endothelial cells, the 9dEB-derived KDR+ cells expressed higher level of endothelial progenitor markers such as CD133 and CD34, showed endothelial function, but did not express the mature endothelial marker vWF. In the process of in vitro culture, the progenitor markers of KDR+ cells were downregulated, while mature endothelial markers were upregulated.Conclusions:The human ES cell lines established by our institute can differentiated into endothelial cells. The isolated KDR+ cells showed the endothelial progenitor characteristics and could differentiate into mature endothelial cells in vitro. Chapter 3 The induced differentiation of iPS cells into endothelial cells and smooth muscle cellsObjective:The induced pluripotent stem cells (iPS cells) in our experiment were derived from fibroblast cells that had been transfected by four transcriptional factors (Oct4,Nanog,Sox2 and Klf4). iPS is embryonic stem cell like pluripotent cell line. But compared with ESCs, iPS cell experiments avoid the ethical issues and make patient specific pluripotent cell line possible. This experiment studied the differentiation of iPS cells into vascular cells (endothelial cells and smooth muscle cells).Methods:09 iPS cells were induced to differentiate into endothelial cells by monolayer culture, FACS sorting Flk1+ cells, followed by OP9 stromal cell co-culture, and purified by FACS sorting VE-cadherin+ cells(09-EC). Smooth muscle cell differentiation of iPS cells was induced by high dose retinoid acid (RA).The specific marker expression and functional characteristics of the differentiated cells were analyzed. Gene expression during differentiation process were tested by real-time PCR. The differentiation efficiency was compared with ESC differentiation.Results:iPS cells could differentiate into endothelial cells and smooth muscle cells. The endothelial or smooth muscle specific genes were up-regulated in the differentiation process. Four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that induce the generation of iPS cells were significantly down-regulated in this process. The method and efficiency of differentiation were similar with normal mouse ESCs. Conclusions:This study supports that iPS cells can differentiate into endothelial cells and smooth muscle cells in vitro. The vascular differentiation ability of iPS cells is similar with ESCs This procedure may contribute to clinic application of patient-specific iPS cells derived cells in vascular degeneration diseases.Chapter 4 Screening of the co-activators of Myocardin—new discovery in mechanism of regulation of smooth muscle cell differentiationObjective:The proliferation and differentiation of smooth muscle cells is highly related to the mechanisms underneath angiopathological situation such as formation of neointima and coronary stenosis. Myocardin is a key factor in smooth muscle differentiation. In this experiment, we were looking for important transcriptional factors or co-activators in smooth muscle differentiation signaling pathway by screening the proteins that could bind and upregulate myocardin. By studying the function of the protein candidate, we could discover the molecular mechanism of smooth muscle differentiation.Methods:We discovered a list of candidates that could upregulate the expression of myocardin by screening 1170 transcriptional factor candidates by luciferase assay. After further confirmation of the binding between candidates and myocardin by co-IP and analysis with bioinformatics strategy, we got ZNF297B, RAI14 and MAGED1 as the most promising candidates.Results:ZNF297B, RAI14 and MAGED1 were highly expressed in smooth muscle cells and their expression level was upregulated during the differentiation of smooth muscle cells. They could bind to myocardin directly. The overexpression of these candidates promoted the differentiation of ESCs into smooth muscle cells.Conclusions:ZNF297B, RAI14 and MAGED1 could be important transcriptional factors to regulate smooth muscle differentiationChapter 5 ZNF297B regulates differentiation of smooth muscle cells through interaction with myocardinObjective:Discover the expression of ZNF297B in the differentiation of smooth muscle cells and investigate the related mechanism.Methods:The mRNA expression level of ZNF297B in human, rat and mouse smooth muscle cells and the change of ZNF297B expression level in smooth muscle cell differentiation and proliferation was tested by real-time PCR. The ZNF297B-Myc/His plasmid was constructed and transfected into rat smooth muscle cells to test the distribution of ZNF297B in cultured cells. The binding and interaction of ZNF297B and Myocardin was tested by luciferase assay and confirmed by co-immunoprecipitation.Results:ZNF297B was specificly highly expressed in smooth muscle cells. The expression level of ZNF297B was upregulated in the process of RA induced smooth muscle cell differentiation from mouse embryonic stem cells. ZNF297B expression was downregulated in PDGFBB stimulated smooth muscle cell proliferation process. In balloon-injured rat carotid arteries, ZNF297B was upregulated after 6 hours and then downregulated. ZNF297B mainly expressed in nucleus and the cytoplasm around nucleus. ZNF297B can bind and interact with Myocardin in protein level.Conclusions:ZNF297B may play an important role in smooth muscle cell differentiation. This function may related to the interaction between ZNF297B and Myocardin.