Functional Heterogeneity Of Emerging Hematopoietic Stem Cells In The Early Mouse Embryos

Accompanied with our life,hematopoietic stem/progenitor cells(HSPCs)can be continuously regenerated millions blood cells every second.Hematopoietic progenitor cells(HPCs)can produce blood cells,but having little rigorous self-renewal ability.At the most higher hierarchy hematopoietic system,only hematopoietic stem cells(HSCs)have the ability of self-renewal and multi-lineage differentiation.In the mid-1950 s,increasing researchers focus on the HSC surface marker to solve the origin,development,differentiation and regulation mechanism of HSC in vitro and in vivo.HSC heterogeneity problems have been put forward when new findings are unveiled by enrichment of surface markers with high efficiency.In fact,what we are obtained HSCs by enrichment of surface markers may represent typical HSCs or one subtype of HSCs.Thus,it is obvious that the conclusion from the origin,development,differentiation and regulatory mechanism of HSC research was obscured,so the HSC heterogeneity problem urgent need to study.Previously,foreign researchers found that fetal liver,spleen and bone marrow(from E14.5 to adult)were of various subtypes,focusing on the characteristics and kinetics of those subtype of HSC through function experiments.Based on the endothelial to hematopoietic cell transition(EHT)hypothesis,immature HSC also named pre-hematopoietic stem cells(pre-HSCs)before mature HSC emergence,subdividing into pre-HSC Ⅰ(Type 1)and pre-HSC Ⅱ(Type 2).Collectively,will immature pre-HSC and mature HSC contain more than one type of HSC in the important hematopoietic sites AGM(aorta-gonad-mesonephros)region in the early development of mouse embryos? Whether the functional heterogeneous has already been determined when HSC emergence? And what is the characteristics of various subtypes of pre-HSC and HSC? Furthermore,the emergence of all sorts of HSC and pre-HSC subtypes mechanism and regulation mechanism are to be solved at present.We first through the experiment of the E11 AGM uncultured transplantation by limit dilution assay(LDA).Confirmation of the AGM LDA system can obtain clonal level reconstituted recipients,we performed a retrospective analysis of peripheral blood collection from these recipients.In 40 long-term clonal HSCs of reconstituted recipients,85%(34/40)of the cells showed the myeloid relative deficient characteristic(gammaHSC),another 15%(6/40)of the cells showed the lymphomyeloid balanced output(betaHSC),and no lymphoid relative deficient(alpha-HSC)and myeloid absolute deficient(delta-HSC)subtype were found.In addition,in 34 gamma-HSC,according to its development dynamics,we found a few T cells deficient of lymphocytes(B-dominant gamma-HSC),the remaining was defined as balanced gamma-HSC.Compared with previous reports of fetal liver and adult bone marrow subtypes of HSC,we found that AGM HSC compartment is dramatically different in the early embryonic,and the characteristics of the subtypes are also different.Secondly,we used the OP9-DL1 coculture/transplantation assay to obtain the successful reconstitution recipients in single cell level,and found retroactively the initial subtypes and characteristics of pre-HSC.In long-term single-cell level for more than 20 reconstitution recipients,55%(12/22)of the pre-HSC performed myeloid relative deficient characteristic(gamma-pre-HSC),45%(10/22)for the lymphomyeloid balanced pre-HSC(beta-pre-HSC).Meanwhile,we have not found alpha-pre-HSC and delta-preHSC.Moreover,a similar B-dominant gamma-pre-HSC was discovered.Surprisingly,we found that there is no corresponding relationship between the type of pre-HSC and its subtypes.Taken together,we added HSC and pre-HSC subtype composition and characteristics in and clonal level and single-cell level on the development of the most important site of mouse embryos.Using single cell RNA-seq,we found the lineage-genes expression profile of embryonic period(E11 Type 1,E11 Type 2,E14 fetal liver)compared with the adult stage(8 to 12 weeks bone marrow)was increased obviously,suggesting that fetal stage could become a key step for different subdivides of HSC and its differentiation regulation.Then,we use Sm22a-cre,ROSATMT(RFP),ROSAEYFP mice to investigate the Sm22 acre labeled mesenchymal cells for HSC heterogeneity microenvironment for the importantce time window and site in mouse embryos.Sm22 a is a surface marker of smooth muscles,but we realized that Sm22a-cre expressed stably in AGM region and fetal liver at E10.5-E11.5,and the important EHT process,especially in Tpye1 pre-HSC which is the same as the expression of CD45 + cells in the adult bone marrow(30%).And Sm22a-cre also expressed in HSPC antibodies combination in the important stage of AGM region and fetal liver.But,we did not obtain successful reconstituted recipients from Sm22 a subdivide the endothelial and the mesenchymal cells by OP9-DL1 coculture/transplant experiment.Then,we obtained by sorting the Sm22a-cre labeled mesenchymal cells represented ventral microenvironment of AGM and the Sm22a-cre unlabeled cells represented dorsal AGM,founding that the expression of Bmp4 was low in ventral cells by semi-quantitative PCR analysis.And,we did not discover different proportion of HSCs by Sm22a-cre subdivided Sm22a+ and Sm22a-HSCs in bone marrow by typical transplant experiment.We concluded that Sm22a-cre was difficult to sovle bone marrow HSCs heterogeneity of hematopoietic microenvironment problems.In conclusion,this study first discovered two major kinds of HSC subtypes during the early development of mouse embryos in the important hematopoietic organ: AGM region,which is mainly about myeloid relative deficient HSCs and have no lymphoid relative deficient and myeloid absolute HSCs.Based on the EHT hypothesis,through the HSC mature process,meaning that they are pre-determined in the pre-HSC stage.And,more importantly,through the follow-up of exploring the origin of HSC subtypes mechanism on AGM region,either from the perspective of the spatial heterogeneity of HSC(different mesenchymal cells represent the hematopoietic microenvironment)and the time point of the hematopoietic heterogeneity generate(different time and tissue),we are not getting a satisfactory answer.However,fetal stage could become a pivotal period trough RNA-seq,and the mechanism of lymphoid relative deficient in fetal liver are subject to subsequent research.