Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Summary

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Abstract

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-time¹. 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 endothelium⁴ constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin⁺ osteoblasts³ and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-1⁵ concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity⁶. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands⁷ and interferon-α⁶ 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 described⁸. 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-HSCs⁹. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described¹⁰. 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.

Protocol

1. Preparation of Cell Suspension from Bone Marrow

1. Euthanize the mouse and place the animal in a stainless pan and spray 70% ethanol onto its abdomen and back. Collect femur and tibia from hind legs, and spinal column.
2. Accurately remove all soft tissue residues from the spinal column with a sharp tip scissors and from legs bones with a gauze. Store bones in a 50 mL Falcon tube with 30 mL of RPMI medium containing 10% heat inactivated fetal bovine serum (FBS), supplemented with 50 μM β-mercaptoethanol, 100 μM MEM Non-Essential Amino Acids, 1 mM MEM Sodium Pyruvate, 2 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin.
3. Cover t....

Discussion

The method described here enables rapid and accurate analysis of hematopoiesis in individual mice (Figure 1). This analysis in various experimental settings, including murine models of inflammation, autoimmunity, immunodeficiency, degenerative diseases, metabolic disorders and cancer, allows addressing the impact of pathological conditions on hematopoiesis. Figure 3 shows the analysis of cell cycling activity on electronically gated LKS CD34^- cells from healthy mice.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Gibco	42401
MEM NEAA 100X	Gibco	11140
Sodium Pyruvate	Gibco	11360
PenStrep	Gibco	15070
PBS	Gibco	20012
FBS	Gibco	16000
Cell Strainer 40 μm	BD Falcon	352340
7-AAD Staining solution	BD Pharmingen	559925
Lyse/Fix buffer	BD Pharmingen	558049
Perm buffer III	BD Pharmingen	558050
Ki-67	BD Pharmingen	556026
DAPI	Invitrogen	D21490
CD4 (GK1.5)	eBioscience	150041
CD8 (53-6.7)	eBioscience	150081
CD3 (145-2C11)	eBioscience	150031
CD45R (RA3-6B2)	eBioscience	150452
CD19 (6D5)	eBioscience	150193
Gr1 (RB6-8C5)	eBioscience	155931
Tre119 (TER-119)	eBioscience	155921
NK-1.1 (PK136)	eBioscience	455941
c-Kit (2B8)	eBioscience	171171
Sca-1 (D7)	eBioscience	135981
CD34 (RAM34)	eBioscience	110341
FcγR (2.4G2)	eBioscience	553145
Anti-APC MicroBeads	Miltenyi Biotec	130-090-855
LS Columns	Miltenyi Biotec	130-042-401