Transcriptional landscape of canine hematopoiesis and cross-species comparisons revealed by single-cell RNA sequencing

Hematopoiesis is a complex process that begins with hematopoietic stem cells (HSCs) that progressively differentiate into all the cellular components found in blood. Numerous cell states exist along this continuum and alterations along the differentiation trajectory can lead to disorders, including hematopoietic neoplasms. Dogs are a valuable model for several human diseases, including a variety of leukemias/lymphomas, but comparative studies investigating the transcriptomic programs of canine hematopoiesis are lacking. The goals of this study were to (1) identify bone marrow hematopoietic cell type gene signatures, (2) characterize temporal gene expression patterns, and (3) complete integrated comparisons to human bone marrow. As such, we performed single cell RNA-sequencing (scRNA-seq) on canine bone marrow and fluorescence activated cell sorted CD34 ⁺ hematopoietic precursors. Unsupervised clustering revealed 29 transcriptomically distinct cell populations across 4 major lineages (erythroid, monocytic/dendritic cell, granulocytic, lymphocytic) arising from HSCs. Pseudotemporal analysis enabled the classification of gene expression patterns (as early, intermediate, and late) along lymphocytic, erythroid, and granulocytic lineages. Investigation of transcription factor activity and gene expression during the branch point between granulocytic and monocytic lineages identified features important in cell fate decision, including CIITA and LMO1. Transcriptomic divisions of the granulocytic lineage were subsequently corroborated with immunophenotyping, and the lineage was subjected to comparative analysis with human granulocytes. Comparative transcriptomics revealed 1,522 (78%) conserved and 417 (22%) divergent gene expression patterns along granulopoiesis. Taken together, our analysis characterizes the single-cell transcriptional landscape of canine hematopoiesis which can serve as a reference for the study of hematopoietic malignancies.