Interleukin-6 (IL-6) and Low O 2 Concentration (1%) Synergize to Improve the Maintenance of Hematopoietic Stem Cells (Pre-CFC) MILICA KOVAC ˇ EVIC ´ -FILIPOVIC ´ , 1 MARIJANA PETAKOV, 2 FRANCIS HERMITTE, 3 CHRISTELLE DEBEISSAT, 3 ALEKSANDRA KRSTIC ´ , 2 GORDANA JOVC ˇ IC ´ , 2 DIJANA BUGARSKI, 2 XAVIER LAFARGE, 4 PAVLE MILENKOVIC ´ , 2 VINCENT PRALORAN, 3 AND ZORAN IVANOVIC ´ 4 * 1 Faculty of Veterinary Medicine, University of Belgrade, Serbia 2 Institute for Medical Research, University of Belgrade, Serbia 3 CNRS UMR 5164, 2nd University of Bordeaux, Bordeaux, France 4 French Blood Establishment, Regional Aquitaine-Limousin Center, Bordeaux, France Low O 2 concentration (1%) favors the self-renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL-6 influences both stem cells and committed progenitors at 20% O 2 , we studied its effects in cultures at 1% O 2 . The pre-CFC activity in LinS population of mouse bone marrow was analyzed following 10 days of serum-free culture in medium (LC1) supplemented with IL-3 with and without IL-6, at 20 and 1% O 2 and phenotypic differentiation and proliferative history monitored. The IL-6 receptor expression and initiation of VEGF-A synthesis were also investigated. At 20% O 2 , the effects of IL-6 on pre-CFC were negligible but effects on CFC were apparent; conversely, at 1% O 2 , the IL-6 enhances activity of pre-CFC but not of CFC. Unlike at 20% O 2 , at 1% O 2 a subpopulation of cells remained LinS in spite of extensive proliferation. However, the absolute number of LinS cells, did not correlate with pre-CFC activity. A relative increase in VEGF transcripts at 1% O 2 in presence of IL-3 alone was enhanced by the addition of IL-6. IL-6 enhanced pre-CFC activity at 1% O 2 and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O 2 does not exist in tissues in vivo, further studies in vitro at lower O 2 concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells. J. Cell. Physiol. 212: 68–75, 2007. ß 2007 Wiley-Liss, Inc. Sustained production of mature blood cells depends on the survival, self-renewal and commitment of a small population of primitive pluripotent stem cells that reside in extensively hypoxic endosteal bone marrow (BM) niches (Chow et al., 2001). Indeed, O 2 concentrations in the BM microenvironment are far below those in the air (20%), varying from <0.1% close to the endosteum to 4–5% in perivascular areas. Our in vitro results (Cipolleschi et al., 2000; Ivanovic ´ et al., 2000a,b, 2002, 2004; Brunet de la Grange et al., 2003; Hermitte et al., 2006) and those of others (Cipolleschi et al., 1993; Koller et al., 1992a,b; Danet et al., 2003 and see review: Csete, 2005) show that these low O 2 concentrations represent an important regulatory mechanism that allows the balance between self-renewal and differentiation of stem cells and influences proliferation, differentiation and maturation of committed progenitors. Liquid cultures usually performed at 20% O 2 concentration lead to the exhaustion of the stem cell potential. Conversely, the stem cell populations (pre-CFC: pre-Colony Forming Cells, MRA: Marrow Repopulating Ability, CRA: Culture Repopulating Ability, SRC: Scid Repopulating Cells) are better maintained at low O 2 concentration (1, 1.5, and 3% O 2 ) (Cipolleschi et al., 1993, 2000; Ivanovic ´ et al., 2000a,b, 2002, 2004; Brunet de la Grange et al., 2003; Danet et al., 2003) whatever the source (human or mice, BM, mobilized blood cells or cord blood cells), selection procedures (human CD34þ, murine LinS, nonadherent BM cells), combination of cytokines or medium (serum depleted or serum free). The level of oxygenation also influences committed progenitor cells whose expansion at 1% O 2 is reduced, but at 3% O 2 reaches the same level as at 20% O 2 , and is even higher at 5% O 2 (Bradley et al., 1978; Lu and Broxmeyer, 1985; Katahira and Mizoguchi, 1987; Pennathur-Das and Levitt, 1987; Koller et al., 1992a,b) with an increased production of differentiated granulocytes (Hevehan et al., 2000) and megacaryocytes in short term liquid culture systems (Mostafa et al., 2000). The pleiotropic cytokine Interleukin-6 (IL-6) activates its target cells through a two step process: it first dimerizes with Contract grant sponsor: French and Serbian governments, grant for bilateral cooperation ‘‘Pavle Savic’’; Contract grant number: 079524UF. Contract grant sponsor: Ministry of Science and Ecology of Republic of Serbia; Contract grant number: 145048. Contract grant sponsor: Ligue contre le Cancer (Comite ´ Aquitaine-Charente); Contract grant number: 1205-241. Contract grant sponsor: Etablissement franc ¸ais du sang Aquitaine- Limousin. Contract grant sponsor: Annual funds for research and development for the years 2003 and 2004. *Correspondence to: Zoran Ivanovic ´, Etablissement Franc ¸ais du Sang Aqitaine-Limousin, 5 Place Ame ´lie Raba Le ´on, BP 24, 33035 Bordeaux Cedex, Bordeaux, France. E-mail: zoran.ivanovic@efs.sante.fr Received 22 September 2006; Accepted 1 December 2006 DOI: 10.1002/jcp.21003 ORIGINAL ARTICLE 68 Journal of Journal of Cellular Physiology Cellular Physiology ß 2007 WILEY-LISS, INC.