Phenotypic Analysis of the Mouse Hematopoietic Hierarchy Using Spectral Cytometry: From Stem Cell Subsets to Early Progenitor Compartments.

Phenotypic analysis by flow cytometry is one of the most utilized primary tools to study the hematopoietic system. Here, we present a complex panel designed for spectral flow cytometry that allows for the in-depth analysis of the mouse hematopoietic stem and progenitor compartments. The developed panel encompasses the hematopoietic stem cell (HSC) compartment, an array of multipotent progenitors with early marks of lineage specification and a series of progenitors associated with lymphoid, granulo-macrophagic, megakaryocytic and erythroid lineage commitment. It has a built-in redundancy for key markers known to decipher the fine architecture of the HSC compartment by segregating subsets with different functional potential. As a resource, we used this panel to provide a snapshot view of the evolution of these phenotypically defined hematopoietic compartments during the life of the animals. We show that by using a spectral cytometer, this panel is compatible with the analysis of GFP-expressing gene-reporter mice across the hematopoietic system. We leverage this tool to determine how previously described markers such as CD150, CD34, CD105, CD41, ECPR, and CD49b define specific HSC subsets and confirm that high expression of the transcription factor Gfi1 is a hallmark of the most primitive HSC compartment. Altogether, our results provide a convenient protocol to obtain in one analysis a more extensive view of the hematopoietic architecture in mouse models. Our results could also serve as a base for further development of high-end panels leveraging spectral flow cytometry beyond the 15-fluorochrome panel presented in this report.