Study On The Regulation Of Endothelial Cells On The Expansion Of Hematopoietic Stem Cells In The Bone Marrow Microenvironment

Hematopoietic stem cells(HSCs)are a group of cells in the hematopoietic system that have the ability to self-renew and differentiate into all mature blood lineages.They are especially important for maintaining the hematopoietic homeostasis and immune function of the body.HSCs transplantation has been recognized as an effective treatment for some hematopoieticmalignancies,hematopoieticdisorders,and immunodeficiency.However,there are still clinical limitations,such as difficulty in matching HSCs,donor deficiency,and immune rejection.The transplantation of cord blood HSCs has overcome these difficulties.However,the number of HSCs in a single umbilical cord is limited and insufficient to meet the patient's hematopoietic reconstitution needs.In vitro expansion of HSCs is the most direct and effective way to address this difficulty.However,due to the lack of a good culture system,the in vitro expansion of HSCs is far from meeting the clinical requirements.Studieshaveshownthatendothelialcellsinthe aorta-gonad–mesonephros(AGM)region and near the yolk sac artery can support the in vitro expansion of HSCs.Endothelial cells isolated from other adult tissues do not have this ability.After constant experimentation,we determined the serum-free co-culture system.After co-culturing the yolk sac endothelial cells with LSK cells(Lin~-Sca1~+ckit~+)for nine days,the co-cultured LSK cells and HSCs(Lin~-Sca1~+ckit~+CD150~+CD48~-)were compared with the control group in which LSK cells were cultured alone.The amount of amplification was significantly higher than that of the control group.The number of LSK cells can be amplified from 3 x 10~3nine days later to about 1.3 x 10~5cells.Amplification was about 43 times.The control group was amplified from 3x10~3nine days later to about 6.7x10~3.Amplification is about 2 times.After nine days of co-cultivation,the number of HSCs can be amplified to approximately 3x10~3.The number of HSCs in the control group was only about 100.The difference was significant from the co-culture experiment group.We collected all cells from the co-culture for nine days for competitive bone marrow transplantation experiments.Chimeric rate and lineage differentiation were analyzed at 1,2,3,and 4 months after transplantation.It was found that HSCs expanded in vitro had stronger hematopoietic remodeling ability and favored lymphoid cell differentiation.The above experiments indicate that yolk sac endothelial cells can promote the effective expansion of HSCs.Through contact co-culture and non-contact co-culture(Transwell)experiments,we initially demonstrated that yolk sac endothelial cells can regulate the in vitro expansion of HSCs through membrane-type factors and secreted factors.Membrane factors may play a more important role.Specific factors and regulatory mechanisms will be further studied.The Notch signaling pathway has been repeatedly demonstrated to be closely related to the self-renewal and proliferation of HSCs.We constructed a yolk sac endothelial cell line with different degrees of activation Notch signaling.In in vitro co-culture experiments,we found that the Notch signaling pathway in endothelial cells could not amplify LSK cells,whether activated or inhibited.It is speculated that the role of microenvironment stromal cells or the role of CXCL12,IL11 and other cytokines may be absent in our culture system.This study provides a scientific basis for further revealing the regulation of HSCs by cellular signals in the bone marrow microenvironment.It provides a new idea for solving the problem of in vitro expansion of HSCs.