Hematopoietic stem cell characterization and isolation.

Hematopoietic stem cells (HSCs) are defined by the capabilities of multi-lineage differentiation and long-term self-renewal. Both these characteristics contribute to maintain the homeostasis of the system and allow the restoration of hematopoiesis after insults, such as infections or therapeutic ablation. Reconstitution after lethal irradiation strictly depends on a third, fundamental property of HSCs: the capability to migrate under the influence of specific chemokines. Directed by a chemotactic compass, after transplant HSCs find their way to the bone marrow, where they eventually home and engraft. HSCs represent a rare population that primarily resides in the bone marrow with an estimated frequency of 0.01% of total nucleated cells. Separating HSCs from differentiated cells that reside in the bone marrow has been the focus of intense investigation for years. In this chapter, we will describe in detail the strategy routinely used by our laboratory to purify murine HSCs, by exploiting their antigenic phenotype (KSL), combined with the physiological capability to efficiently efflux the vital dye Hoechst 33342, generating the so-called Side Population, or SP.

Mouse hematopoietic stem cell identification and analysis.

Hematopoietic stem cells (HSCs) remain by far the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains difficult because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in bone marrow is about 0.01% of total nucleated cells and approximately 5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. This prohibits the study of processes in HSCs, which require large amounts of starting material. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. This report will review different hematopoietic stem and progenitor purification strategies and compare flow cytometry profiles for HSC sorting and analysis on different instruments. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.