External signals regulate continuous transcriptional states in hematopoietic stem cells

Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+,Sca1+) progenitors after in vivo pharmacological perturbation of niche signals interferon, granulocyte-colony stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. External signals induced rapid transitions between HSC states but transcriptional response varied both between external stimulants and within the HSC population for a given perturbation. In contrast to LSK progenitors, HSCs were characterized by a greater link between molecular signatures at baseline and in response to external stressors. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq suggested some HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and LSK progenitor-specific chromatin and transcriptional features that represent determinants of signal receptiveness and regenerative potential during stress hematopoiesis. Overall design: Five male and five female mice were used for each sample. For scATAC-Seq experiment and for HSC replicate 1 scRNA-Seq (HSC_Repl1) mice were left unperturbed. For stimulation scRNA-Seq experiment mice were treated with either 16,16-dimethyl Prostaglandin E2 (dmPGE2), Poly(I:C) (pIC), or G-CSF (GCSF), Indomethacin (indo), or only solvent DMSO as a control (ct). In this experiment HSCs and multipotent progenitors (MPPs) from the same condition (ct, dmPGE2, pIC, GCSF and indo) were extracted from the same mice. The MPP samples were also labeled with hashtag oligonucleotides (HTO) that are part of the Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) methodology. The two control HSC samples (HSC_Repl1 and HSC_ct = repl2) were treated as two independent biological replicates.