Chapter 41 Identafication and Purafication of Murine Hematopoietic Stem Cellsby Flow Cytometry

Abstract

Publisher Summary All the different blood cell types are derived from a common ancestor, the pluripotent hematopoietic stem cell (PHSC). This cell not only has the capacity to differentiate into committed progenitor cells of all the various blood cell lineages but also can renew itself, thus maintaining the hematopoietic organ at a steady-state level throughout life. The production of mature blood cells out of stem cells and committed progenitors requires >10 cell divisions. As a consequence, the stem cells are rare cells, even in hematopoietic organs. Stem cells are detected by the examination of their offspring after in vitro culture or after transplantation into often lethally irradiated syngeneic or congenic recipients. There exists no consensus about the different culture and in vivo transplantation methods with respect to their specificity for pluripotent stem cells. The quantitation of stem cells by those methods may lead to widely different results. Identification of PHSC by flow cytometry (FCM) should be combined with cell culture and transplantation assays for verification. Therefore, FCM methods should employ supravital staining and labeling, which can be combined with sorting, cell culture, and transplantation procedures. This chapter describes various protocols for identifying and sorting spleen colony forming. Most of these employed FCM in combination with other physical and immunological cell separation techniques have served to remove the bulk of mature cells.