Application Of Single Cell Technology In Hematopoietic System Research

In recent years,the related research in the field of single cell biology has attracted extensive attention of scientists.The rapid development of technologies related to single cell separation and single cell sequencing technology provides a powerful biological means for accurate and reliable analysis of hematopoietic cell heterogeneity.Hematopoietic stem cells,as a kind of tissue-specific stem cells with the most in-depth research,can produce all mature blood cell types and have self-renewal ability.For this highly heterogeneous group,single cell level research is indispensable.The research team has recently developed the single cell Index sorting technology and used this flow technology to study the mouse embryo blood endothelial cells.A large number of single cell RNA-seq data of Hematopoietic Stem Cell(HSC)accumulated in the early stage provide important clues for further exploring and enriching the endothelial population of the blood at birth.Previous studies have found that surface molecule CD47 is expressed on some vascular endothelial cells in AGM region and all pre-HSC(precursor of hematopoietic stem cells).Through endothelial cells enriched with CD47-,CD47+ and pre-HSC respectively,through in vitro induction incubation and mouse transplantation experiments,it is found that cells with hematopoietic stem cell activity are all enriched in CD47+ cell population,suggesting that CD47 can be further used as a marker for hematopoietic stem cell-related population earlier in development stage.In this study,the retrospective function of flow cytometry Index Sorting technology is applied,i.e.CD47 molecules are simultaneously labeled on the basis of the above sorted population markers,and then the expression of CD47 molecules concerned with important cell populations can be traced according to the results of in vitro incubation and culture.Analysis showed that about 60%of the endothelial cell population of CD41"CD43"CD45"CD31 KIT+ expressed CD47.In addition,CD41-CD43-CD45-CD31+KIT+ cells with endothelial and blood-producing potential are all enriched in the cell population of CD47+.The results suggest that the expression of CD47 can significantly improve the accuracy of the blood-producing endothelial cell population by 1.5 times.According to the results of in vitro incubation and induction culture,it is further found that more than 60%of the endothelium population of CD47+still does not have the ability to produce endothelium or blood,suggesting that the established flow cytometry Index Sorting technique can be used to further explore new surface marker molecules of blood-producing endothelial cells in the future,so as to further improve the accuracy of enriching blood-producing endothelial cells.In addition,a single cell transplantation technique was established,i.e.single cell sorting technique was used to separate single bone marrow hematopoietic stem cell,and the accuracy and reliability of single cell sorting technique were verified through mouse hematopoietic stem cell transplantation experiments.Based on this,we studied the heterogeneity of mouse bone marrow hematopoietic stem cell population at the single cell level.Flow single cell sorting was applied to single bone marrow hematopoietic stem cell with Lin"CD45+CD201hiCD48-GFP+ immunophenotype.Single cell sorting mode was adopted to mix the sorted single cells with 2x104 bone marrow protective cells respectively and transplant them directly into mice irradiated with lethal dose.After 3 independent repeated experiments,week blood outside recipient mice was detected in 4th week,6th week,8th week,12th week,14th week and 16th week respectively to observe the chimerism rate and lineage distribution of donor cells in recipient mice.Of the 60 transplant recipients,5 long-term reconstruction recipients were obtained for subsequent analysis.According to the differentiation pedigree of hematopoietic stem cells,two of the five successfully reconstructed recipient mice are Beta-HSC type,two are Gamma-HSC type and one is delta-HSC type,which functionally verified the heterogeneity of hematopoietic stem cells.To sum up,firstly,the flow cytometry Index Sorting technique is applied to divide aortic endothelial cells with single molecular phenotype of CD41-CD43-CD45-CD31+KIT+,and the aortic endothelial cells are incubated with stromal cells OP9-DL1 for induction and culture for 7 days,and then the effects of KIT and CD47 on enriching blood-producing endothelial cells are comprehensively analyzed retrospectively in combination with the flow sorting characteristic parameters of individual endothelial cells and the efficiency of generating CD45+ hematopoietic cells and CD31+ endothelial cells after induction.Using the function of flow cytometry Index Sorting technology,it was found for the first time that CD47 molecule can significantly enrich mouse embryonic blood endothelial cells,providing an important technical means for further accurate enrichment and graduate study of blood endothelial cells.Secondly,single bone marrow hematopoietic stem cell was sorted and a single cell transplantation system of mouse bone marrow was established.Through the single cell transplantation data of mouse bone marrow,the contribution of donor cells to each line of recipient mice was observed.Furthermore,the heterogeneity of hematopoietic stem cells was verified by in vivo reconstruction analysis after single cell transplantation.The above experiments show that single cell sorting technology is a necessary means to accurately analyze the heterogeneity of hematopoietic stem cell development.