All-trans retinoic acid prevents apoptosis of human marrow CD34 + cells deprived of haematopoietic growth factors Olivier Herault, 1,2 Jorge Domenech, 1,2 Marie Therese Georget, 2 Nathalie Clement, 1 Philippe Colombat 1,3 and Christian Binet 1,2 1 EA-3249 ‘Cellules He ´matopoı ¨e ´tiques, He ´mostase et Greffe’ Faculty of Medicine, 2 Department of Haematology and 3 Department of Oncology and Blood Diseases, University Hospital, Tours, France Received 23 January 2002; accepted for publication 30 January 2002 Summary. The regulation of apoptosis plays a key role in haematopoiesis. It has been demonstrated that haemato- poietic progenitor cells progressively undergo apoptotic cell death in the absence of appropriate growth factors. We studied the effects of pharmacological doses of all-trans reti- noic acid (ATRA) on the apoptosis of human adult marrow CD34 + progenitor cells cultured for 7 d in a serum-free medium. We quantified CD34 + cells, clonogenic progenitors and 5 week colony-forming cells (CFC) before and after ATRA exposure. Moreover, we defined the apoptotic status of the CD34 + cell fraction by analysis of phosphatidylserine externalization (using annexin V), the relative membrane permeability to 7-aminoactinomycin D (7AAD) and the mitochondrial membrane potential [using 3,3¢-dihexyloxa- carbocyanine iodide, DiOC 6 (3)]. In the drastic experimental conditions used, a decrease in viable CD34 + cells, granulo- cyte–macrophage colony-forming units (CFU-GM), eryth- roid burst-forming units (BFU-E) and 5 week CFC were observed. Exposure to ATRA partially prevented the decrease in viable CD34 + , without a concomitant effect on the clonogenic and more immature progenitors. ATRA- treated CD34 + cells displayed changes in apoptotic status compared with control cultures, particularly in lower annexin V-binding. These results were confirmed using 7AAD and DiOC 6 (3). Our results demonstrate that ATRA exerts a protective effect on CD34 + cells exposed to such apoptotic stress. Keywords: all-trans retinoic acid, apoptosis, human marrow CD34 + cells, annexin V, 7-aminoactinomycin D. Haematopoietic progenitor cells require defined growth factors for survival, differentiation and proliferation (Sachs, 1996). Withdrawal of these growth factors leads to apop- tosis, and such cell death is involved in the control of cell numbers in both haematopoietic precursors and their fully differentiated cell populations (Williams et al, 1990). All- trans retinoic acid (ATRA), a natural metabolite of retinol, plays an important role in the growth and differentiation of haematopoietic cells (Blomhoff & Smeland, 1994). The differentiating activity of ATRA has been demonstrated in vivo in acute promyelocytic leukaemia (Huang et al, 1988; Castaigne et al, 1990; Chen et al, 1991). Although it has been clearly established that ATRA-terminally differen- tiated cells undergo apoptosis (Martin et al, 1990), little is known regarding the effects of ATRA on the apoptosis of human marrow CD34 + cells. The purpose of our study was, therefore, to characterize the apoptotic status of adult marrow CD34 + cells exposed in vitro to ATRA during deprivation of growth factors. We quantified CD34 + cells, clonogenic progenitors and 5 week colony-forming cell (CFC) production before and after exposure to ATRA, and we defined the morphological and phenotypic changes during serum-free liquid culture. In addition, the apoptotic status of the CD34 + cells was evaluated using three different methods. We characterized the externalization of phosphatidylserines (PS), the relative permeability of the plasma membrane to high doses of 7-aminoactinomycin D (7AAD) and the mitochondrial membrane potential (DYm). Our results demonstrate that exposure to ATRA decreases the number of normal marrow CD34 + cell showing signs of ongoing apoptotsis when deprived of haematopoietic growth factors. MATERIALS AND METHODS Isolation of CD34 + cells. This study was performed with CD34 + cells obtained from bone marrow aspirates of 14 donors without haematological disorders. All donors gave informed consent. Mononuclear cells were separated by Correspondence: O. He ´rault, MD PhD, Service d’He ´matologie, Ho ˆpital Bretonneau, CHU, 2 boulevard Tonnelle ´, 37000 Tours, France. E-mail: olivier.herault@med.univ-tours.fr British Journal of Haematology, 2002, 118, 289–295 Ó 2002 Blackwell Science Ltd 289