In Vitro Hematopoietic Differentiation Using Mouse Embryonic Stem Cell

Blood diseases are one of the biggest problems that have been troubling people all the time.Although hematopoietic stem cell transplantation can cure blood diseases at present,it has been troubled by the shortage of donors.The purpose of our study is to establish an in vitro model that induced mouse embryonic stem cells differentiated to hematopoietic stem and progenitor cells.There are two main in vitro differentiation system,embryonic body differentiation system and coculture differentiation system.In vitro hematopoietic differentiated system can help to understand early development of mouse and to study the function of genes.It also provides a feasible direction for the new source of hematopoietic stem cells.The research of my paper is mainly divided into the following three parts:1.The culture and identification of mouse embryonic stem cells.At first we portray the growth curve of mouse embryonic stem cells we gained,and then we calculate the doubling time and find the exponential phase of the ESCs.Then we examined totipotent of our ESCs,we used alkaline phosphatase staining,measure the expression of pluripotent genes,randomly differentiation and teratoma formation experiment to prove our ESCs is totipotency,that can developed to three germ layer（endoderm,mesoderm and ectoderm）.2.Formation of embryonic bodies and in vitro hematopoietic differentiation.Nest we formed embryonic bodies with the same size using low adhesion round-bottom ninety-six well plate.At day 1-2,we added BMP4 grow factor to induce embryonic bodies differentiated to mesoderm,at day 3,we added grow factors Activin A and bFGF to induce mesoderm differentiated to hemangioblast,at day 4-6 we added grow factor VEGF and IL-6 to induce hemangioblast differentiated to hemogenic endothelium and HSPC can produced by endothelial-hematopoietic transition.During the differentiation we used FACS to analyses the marker expression of each stage and used q-PCR to assay the expression of related gene of each stage.The result of FACS analysis showed that hemangioblast appeared at day 2 and rapidly increased at day 3,endothelial cells appeared at day 3 and continuously increase,hematopoietic cells appeared at day 4 and continuously increase.The results of q-PCR were corresponding with FACS.3.In vitro hematopoietic differentiation by coculture systemWe co-cultured mouse embryonic stem cells with OP9 cells.In the progress of experiment,first OP9 cell line is overgrowing and then seeding mouse embryonic stem cells on OP9 cells.Mouse embryonic stem cells can differentiated to hematopoietic stem and progenitor cells with the action of OP9 cell.During the differentiation we used FACS to analyses the marker expression of each stage.The result of FACS analysis showed that Flk-1 which is the marker of hemangioblast can be detected at day 3 and continuously increase,CD41 which is the marker of early hematopoietic cells can be detected at day 8.The results showed that embryonic stem cells can differentiated to hematopoietic stem and progenitor cells by coculture system.The following conclusions can be drawn from the above three experiments.First our embryonic stem cells are totipotent and can be differentiated into three germ layers.Nest embryonic stem cells can be differentiated into hematopoietic cells by using EB system and coculture system.Finally,endothelial-hematipoietic-transition process can be observed by coculture system.