Developmental Hematopoiesis Related Cytokines: The Regulatory Effects On Hemangioblast In Mouse Agm Region

Hemangioblast is a stem cell which has a potential of bidirectional differentiation.and can differentiate into hematopoietic cells and endothelial cells in the development process in vivo and vitro.The hemangioblast theory has been tested by ES differentiation model,that is embryonic stem cells differentiate into EB after 2.5-3.5 days,and form a special kind of blast colony in the particular differentiation system,which has the capacity of generating endothelial cells and hematopoietic cells simultaneously,so these cells that formed blast-like coloy are known as BL-CFC(the blast colony-forming cell).Nowadays,hemangioblast studies have made great progress in many aspects , such as the origin,molecular biological characteristics and differentiation in vitro. Previous studies find that in the AGM region, the original part of definitive hematopoietic, also exists cells like hemangioblast precursor HPP-HA. After that , the existence of BL-CFC is found in mouse AGM region,and the special model of hemangioblast BL-CFC is set up. This model has been used to study development and regulation of the hemangioblast in mouse AGM region. For example, the classical hematopoietic growth factor IL-3 can also promote hemangioblast to grow and differentiate.However, as we all know,serum-free culture system has complex nutrients,and it is not fit for study the real role of some cytokines on the hemangioblast. So,in this study, we establish a BL-CFC serum-free incubation culture model in mouse AGM region based on the established BL-CFC model,and optimize the conventional BL-CFC culture system. And on this basis, study the regulatory effects of developmental hematopoiesis related cytokines on hemangioblast in mouse AGM region.First of all,this research establishes BL-CFC serum-free incubation culture model in the mouse AGM region. Obtain E10.5 mouse AGM region, and digest it into single cells, after 12h incubation in serum-free incubation culture system,then culture colonies in the semi-solid medium. After 3-3.5 days'culture in the presence of bFGF,SCF,VEGF,IL-6,IGF-I and LIF, we can see a special form colonies begin to appear. The morphological and differentiation characteristics of blast colony is similar to that from E9.5-12.5 mouse AGM region. Second,identify the hematopoietic function and endothelial differentiation function of BL-CFC. 4-5 days later, pick a single colony to culture hematopoietic colony. As expected,all the cells from an individual blast colony are capable of forming various kinds of hematopoietic colonies, including CFU-E,CFU-GM and CFU-Mix.The adherent population is continue to cultivate in EGM2 endothelial cells complete medium. On the 7-10th day,it appears a large number of adherent cells, cobblestone-like endothelial cells and several mature hematopoietic cells. Then vascular function in vitro is detected . The result shows that the adherent population could incorporate LDL, the adherent cells could form tube-like and network structures in Matrigel in vitro.Third,we test the hematopoietic and endothelial cells functions of BL-CFC on the OP9 stromal cells. Pick a single colony and cuture it on OP9 stromal cells for 7-10 days, all the blast colonies show the potential of differentiating into hematopoietic cells(CD45+), and 30% HA can form the tube-like structure(CD31+).In order to study reactions of different molecular protein in serum and serum-free culture system, we study three kinds of cytokines associated with the development of hematopoietic,that is IL-3 ,SCF and NGF ,with the method of AGM region cells incubated in vitro. These three cytokines and cells from AGM region together incubated for 12 hours in the SR and FBS respectively, and inoculate BL-CFC. The results show that IL-3 could amplify BL-CFC under the two systems,but the NGF had no effect on BL-CFC under the two systems, only in the SR system can SCF amplify the BL-CFC significantly.In summary, this study set up a serum-free incubation culture model of BL-CFC in the mouse AGM region, optimize the conventional BL-CFC culture system. Based on this model, we further study the regulatory effects of developmental hematopoiesis related cytokines on hemangioblast in mouse AGM region,that is IL-3, NGF and SCF,in two culture model.Compared to the serum culture model, serum-free culture model can reflect the role of cytokine on hemangioblast more sensitively and accurately, thus it shows the advantages of serum-free culture system.