Expression profiling by high throughput sequencing
Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are at the apex of the hematopoietic hierarchy. The relationship among HSC and MPPs, their heterogeneity and regulation are challenging subjects of continuing study. We performed single-cell RNA-seq on HSC and their immediate progeny MPP1 cells and compared results with bulk RNA-seq data from 18 hematopoietic populations, literature compilations of cell cycle-related genes, lineage specific genes, and putative quiescence genes. HSC and MPP1 were separated into several distinct subsets including actively cycling, quiescent and a putative hibernating subset. The quiescent cells preferentially expressed lymphoid precursor genes, while in actively cycling cells, the relative level of genes specific for individual lineages were differently correlated with stages of cell cycle. Granulocytic/monocytic and megakaryocytic precursor mRNA levels rose in early G1 phase, while erythroid specific expression showed selective augmentation at the G2/M phase. Stimulation of erythropoiesis shifted most HSCs into the active cell fraction without a disproportionate increase in the expression of early erythroid genes. Most transcription factors of the hematopoietic lineages were randomly expressed in a number of the early precursor cells but certain transcription factor mRNAs were detected only in very few cells. The latter genes may act as valves controlling the flow of differentiation. In summary we detect multi-level heterogeneity of the earliest hematopoietic precursor cells, demonstrate novel lineage specific cell cycle effects on the expression of precursor genes, and find that key regulatory genes for stages of differentiation can be identified from their silencing in stem cells.
RNA samples from HSC, MPPs (both from normal and bled mice) and the other early precursors were obtained. Gene expression of those cells were analyzed with single cell- and bulk RNA-seq.