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

Abstract

Abstract Hematopoiesis is a complex process that begins with hematopoietic stem cells (HSCs) that progressively differentiate into the cellular components found in blood. Dogs develop spontaneous, biologically similar hematopoietic diseases as humans, yet transcriptomic maps of normal canine hematopoiesis are lacking. Understanding the transcriptional landscape of this process in dogs may advance translational hematology studies. Here, we aimed to (1) identify bone marrow hematopoietic cell type gene signatures, (2) characterize temporal gene expression patterns, and (3) integrated comparisons to human bone marrow. We performed single-cell RNA sequencing (scRNA-seq) on normal canine bone marrow and FACS-isolated CD34⁺ precursors. Unsupervised clustering revealed 29 transcriptionally distinct populations across four major lineages: erythroid, monocytic/dendritic, granulocytic, and lymphocytic. Pseudotemporal analysis classified gene expression dynamics as early, intermediate, or late within lymphoid, erythroid, and granulocytic lineages. Key regulators of cell fate at the granulocytic-monocytic branch point included CIITA and LMO1. Granulocyte subclusters were validated by immunophenotyping and compared to human granulocytes, revealing 1,522 conserved (78%) and 417 divergent (22%) genes during granulopoiesis. Overall, this study provides a high-resolution transcriptional atlas of canine hematopoiesis and highlights conserved regulatory programs between dogs and humans, offering a reference for future studies of hematologic disease and cross-species comparisons.