Phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time(1). The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2). Both trabecular osteoblasts(2,3) and sinusoidal endothelium(4) constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin(+) osteoblasts(3) and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-1(5) concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity(6). Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands(7) and interferon-α(6) can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin(- )c-Kit(+ )Sca-1(+ )CD150(+ )CD48(- )CD34(-) population has been described(8). Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs(9). A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described(10). In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.