Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming "units" known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit⁺ CD55⁺ CD49f^med/low CD105^med/high CD71^med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71^med CD150^high) and P1-hi (CD71^high CD150^low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit⁺ CD55⁺ CD49f^med/low CD105^med/high CD71^low CD150^high) contains all of the BFU-e progenitors; Population P3 (P3, Kit⁺ CD55⁺ CD49f^med/high CD105^med/low CD150^low CD41^low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit⁺ CD55⁺ CD49f^med/high CD105^med/low CD150^high CD41⁺) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit⁺ CD55⁺ CD49f^med/high CD105^med/low CD150^high CD41^-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.