Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Hematopoietic Stem Cells VIII In vivo divisional tracking of hematopoietic stem cells Hitoshi Takizawa and Markus G. Manz Division of Hematology, University Hospital Zurich, Zurich, Switzerland Address for correspondence:Hitoshi Takizawa, Division of Hematology, University Hospital Zurich, CH-8091 Zurich, Switzerland. hitoshi.takizawa@usz.ch Hematopoietic stem cell (HSC) division leads to self-renewal, differentiation, or death of HSCs, and adequate balance of this process results in sustained, lifelong, high-throughput hematopoiesis. Despite their contribution to hematopoietic cell production, the majority of cells within the HSC population are quiescent at any given time. Recent studies have tackled the questions of how often HSCs divide, how divisional history relates to repopulating potential, and how many HSCs contribute to hematopoiesis. Here, we summarize these recent findings on HSC turnover from different experimental systems and discuss hypothetical models for HSC cycling and maintenance in steady-state and upon hematopoietic challenge. Keywords: hematopoietic stem cell division; divisional history; labeling technique; quiescence; active cycling Introduction Every second, millions of mature blood cells die and are replenished by newly generated ones over the lifetime of an individual. Homeostatic lev- els of blood cells are ensured by a highly orga- nized hematopoietic hierarchy, where self-renewing hematopoietic stem cells (HSCs) in the bone mar- row (BM) give rise to highly proliferating inter- mediate progenitors with limited self-renewal and restricted lineage potential, which subsequently un- dergo a stepwise differentiation program toward terminal maturation. Given immunesystem com- patibility, HSCs are transplantable from one in- dividual to another, sustaining in both lifelong hematopoiesis, a demonstration of functional excess of HSCs. Additionally, single HSCs possess a high BM-homing and multilineage repopulating capac- ity, as evidenced by in vivo transplantation exper- iments demonstrating that a single HSC can, life- long, reconstitute the hemato-lymphoid system of lethally irradiated recipients. 1–5 The high accessibil- ity, mobility, and robust regenerative potential of HSCs has been used for clinical HSC transplanta- tion to cure inborn genetic disorders and acquired hematopoietic malignancies. Besides self-renewal activity and multilineage blood-forming potential, a characteristic of HSCs is thought to be the relative quiescent cell cycle status of a majority of HSCs. Although recently debated, 6–9 HSC quiescence might lead to protec- tion of metabolic toxicity and genotoxic events, and might be a reservoir for emergency hematopoiesis, for example, upon infection or massive blood loss. 10 However, it remains to be determined how often HSCs divide, to what extent their divisional history affects their function (i.e, their self-renewing and re- populating ability), and how many HSC clones con- tribute to hematopoiesis at any given time. To deter- mine aspects of these issues in vivo, several groups have established different experimental techniques to track HSC division and differentiation. Here, we summarize technical aspects of the dif- ferent in vivo HSC-tracking systems and integrate experimental findings from those approaches to propose and discuss theoretical models for steady- state and demand-adapted HSC turnover. Experimental approaches to determine in vivo steady-state HSC division DNA and DNA complex labeling Many studies have analyzed HSC cell cycle state with DNA-staining dyes, such as 7AAD (7-amino- actinomycin D), PI (propidium iodide), DAPI (4 ′ , 6-diamidino-2-phenylindole), and Hoechst, partly doi: 10.1111/j.1749-6632.2012.06500.x 40 Ann. N.Y. Acad. Sci. 1266 (2012) 40–46 c  2012 New York Academy of Sciences.