Chapter 15 Analysis and Sorting of Hematopoietic Stem Cells from Mouse Bone Marrow

Abstract

It has become clear that a combination of a number of labels and dyes is needed to unambiguously distinguish the pluripotent hematopoietic stem cell (PHSC) from the other cells in the heterogeneous bone marrow. This chapter describes combinations that are of use to demonstrate heterogeneity of Day 12 CFU (colony-forming unit )-S and that, therefore, may be of help to unravel further the true identity of the pluripotent stem cell. Flow cytometry and cell sorting have contributed significantly to the purification of PHSC and, concomitantly, this has introduced uncertainty about the validity of existing methods for stem cell enumeration. For many years the spleen colony assay has been regarded as the clonogenic test for stem cells. Colonies of hematopoietic cells that appear on the surface of spleens of mice 8–14 days after lethal irradiation and bone marrow transplantation were thought to be each derived from a pluripotent stem cell in the graft that based itself in the spleen. Magli et al. demonstrated that the spleen colony assay was more complicated than thought, because Day 8 and Day 12 spleen colonies were not necessarily related.