Isolation And Biological Characterization Of Mice Hematopoietic Stem Cells At Different Development Stages Of Hematopoiesis In Multi-Anatomical Sites

Hematopoietic Stem Cells(HSCs)through controlling balanced turnover of all types of cells in blood and immune system by maintaining their self-renewal,differentiation,quiescence and dormancy properties and thereby to maintain whole body's homeostasis.In early embryonic development,to better understand of the regulation of marker molecules and microenvironment in the dynamic evolution of HSCs and the formation mechanism of heterogeneity HSCs,is helpful for the establishment of expansion culture system for HSCs in vitro and provide theoretical basis for the establishment of high efficient directed differentiation system for HSCs from pluripotent stem cells.It has potentially clinical application values in the field of regenerative medicine and stem/immune cell therapy.However,most of the current studies on HSCs only deal with the molecular regulatory mechanisms of self-renewal and differentiation multi-potential of HSCs in vitro.Only a few studies have been concerned with exploring the origin of definitive HSCs and the establishment of high efficient differentiation system for HSCs induced from pluripotent stem cells.In this study,based on the similarity of hematopoietic mechanism and anatomical location between mouse and human embryos,we selected and isolated cells at different developmental stages from different hematopoietic anatomical sites of mouse embryos,including yolk sac E9.5-E14.5,aorta-gonad-mesonephric region E10.5-E14.5,placenta E9.5-E14.5 and fetal liver E11.5-E14.5.The expression patterns and dynamic changes of Cd34,Cd41,Cd45,Runx1 and c-kit genes were analyzed by RT-PCR and flow cytometry in detail.It was found that the expression levels of Cd41,Cd45,c-kit and Runx1 had dynamic changes,but Cd34 had no expression at all stages of development.We also found that Cd41 has potential to be one of the new candidate marker genes in the development of HSCs.In vitro induction experiments,embryonic pluripotent stem cells can differentiate into mesodermal cells and hematopoietic stem cell-like cells in a step-wisely induced culture system.