| Background:Hematopoietic stem cells(HSCs)sit at the top of the hematopoietic hierarchy.They have the capacity of self-renewal and differentiation to all blood lineages.The self-renewal,proliferation and differentiation of HSCs were regulated by their surrounding cells.HSCs experienced dramatic expansion after they migrate to fetal liver from placenta during early embryogenesis.Then HSCs migrate to bone marrow where they maintain life-long hematopoiesis but mainly keep quiescence.Therefore,fetal liver is an ideal model to study the proliferation of HSCs.In addition,Tet2 loss lead to enhanced self-renewal and altered differentiation of fetal liver HSCs,but whether and how Tet2 regulates HSCs via microenvironments in fetal liver remains to be clarified.Objective:As a typical adult stem cells,HSCs are involved in variants of blood diseases particularly leukemia and genetic diseases.The cellular and molecular mechanisms of HSC have been extensively studied,yet the microenvironment regulation in the early developmental stage of HSC remains to be addressed.The study on microenvironmental regulation of HSCs will provide a theoretical foundation,and will also have a great clinical value in understanding the development mechanism of these blood diseases,and in promoting the effective application of HSC in bone marrow transplantation.We investigated the HSC niche of E14.5 fetal liver to unveil the mechanism of HSC proliferation.Method:Single cell RNA-sequencing(scRNA-seq)achieved huge outcomes in parsing cell heterogeneity and molecular mechanism,but the spatial information of the single cells in the native tissue is lost.Single molecular fluorescence in situ hybridization(smFISH)measures individual RNAs in situ;however,smFISH can only detect a very limited number of genes in parallel of single cells.Multiplexed error-robust fluorescence in situ hybridization(MERFISH)as an excellent tool,maps hundreds to thousands of RNA species simultaneously at single cell resolution,combining smFISH labeling with encoding schemes(modified hamming code).We combined scRNA-seq and MERFISH to study spatial transcriptome for the regulation of HSC niche in fetal liver.Results:According to scRNA-seq,we obtained the cell markers of variants of cell types in fetal liver,and studied the features of HSC niche with MERFISH.We exploited a robust method to label cell membrane and successfully segmented fetal liver tissue sections into single cells.We found that HSCs were closely associated with endothelial cells via cell interaction analysis.Approximately 53%and 75%of HSCs in WT and Tet2-/-fetal liver,respectively,were directly in contact with endothelial cells based on MERFISH imaging.Specifically,6%of the cells in the WT HSC niche are arterial endothelial cells(AEC)compared to average 3%for overall cells in the liver(p=0.0088).This enrichment of endothelial cells is even more significant for the Tet2-/-HSC niches,where 15%of the cells are AEC and 14%are sinusoidal endothelial cells(SEC)compared to overall 4%(p<0.001)and 5%(p<0.001),respectively.In addition,the Notch and Wnt signaling pathway genes were both significantly enriched in Tet2-/-HSC niche.Furthermore,a series of stem cell growth factors such as Kitl,Cxcl12,Fgf2,Igf1,Il6 and Angptl2 were upregulated in Tet2-/-niche compared with WT.Finally,erythroid progenitors were increases overall in the Tet2-/-fetal liver compared to WT(19%vs.10%,p<0.001).Conclusion:1)The rapid expansion of HSCs may be regulated by variants of cytokines secreted by niche endothelial cells.2)Notch and Wnt signaling pathways are elevated in the endothelial niche,which presumably play an important role separately for self-renewal and development of HSCs,and for maintenance of HSCs on quiescence.3)Tet2-/-significantly enhance the output of myeloid lineage in E14.5 fetal liver. |
