Hematopoietic stem cell characterization and isolation

Abstract

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.

Fig. 1

Example of an SP population from an unenriched whole bone marrow sample. In order to visualize the characteristic SP pattern of bone marrow cells, the emission of Hoechst 33342 must be displayed bimodally as Hoechst Red vs. Hoechst Blue, both in a linear scale. The cells concentrated at the lower left corner represent red blood cells and cellular debris, while the rest of the sample is mainly grouped on the upper right side of the acquisition window. The SP gate is drawn around the tail that diagonally emerges from the main population and usually represents 0.02–0.05% of whole bone marrow cells.

Fig. 2

Sorting strategy for SP ^KLS (SP c-Kit ⁺ Lineage⁻ Sca-1⁺) cells. (a) SP gate: the first step consists in displaying the Hoechst 33342 efflux pattern is linear mode (as Hoechst Red vs. Hoechst Blue) and gating the SP population. (b) Morphological characteristics: display the SP cells gated in the first panel as FSC (forward scatter) vs. SSC (side scatter) and draw a second gate as shown in figure. (c) Lineage staining (PE-Cy5): gate out cells that express markers of hematopoietic terminal differentiation and select Lineage-negative cells. (d) c-Kit vs. Sca-1: the last panel shows the expression of the stem cell markers c-Kit (PE) and Sca-1 (FITC) in SP/Lineage-negative cells. This is the sorting gate, comprising the SP ^KLS population. (e–h) The panels on the right show, by comparison, how unenriched bone marrow cells (gated only on the live population from (e)) distribute on the same parameters.