Low Doses of Bone Morphogenetic Protein 4 Increase the Survival of Human Adipose-Derived Stem Cells Maintaining Their Stemness and Multipotency Marı ´a A. Vicente Lo ´ pez, 1, * Miriam N. Va ´ zquez Garcı ´a, 1, * Ana Entrena, 1 Susana Olmedillas Lopez, 2 Mariano Garcı ´a-Arranz, 2 Damia ´ n Garcı ´a-Olmo, 3 and Agustin Zapata 4 Mesenchymal stem cells (MSCs) have emerged as important tools for cell therapy; therefore, identification of factors capable of governing their ex vivo expansion become essential. In this study we demonstrate that human adipose-derived stem cells (ASCs) express all components of the bone morphogenetic protein (BMP)=BMP receptor signaling pathway and respond to BMP4 inducing upregulated expression of its specific target genes Id1–Id4. Moreover, ASCs grown in a medium reduced in serum produce endogenous BMP4 that could affect autocrinely ASC growth. On the contrary, dorsomorphin, an inhibitor of BMP signaling pathway, decreases cell numbers yielded from ASC cultures in correlation with increased apoptosis and decreased cycling cells. Therefore, BMP4 emerges as a possible factor for ex vivo expanding human ASCs. Our results demonstrate that, as other morphogens, BMP4 effects on human MSCs are dose dependent. High doses significantly increased apoptosis and drastically reduced cell proliferation, whereas low doses of BMP4 (0.01–0.1 ng=mL) significantly increase culture cell content, reduce the number of apoptotic cells, and increase that of cycling cells. Further, treatment of human ASCs with low doses of BMP4 does not modify expression of Nanog and Oct4, two transcription factors involved in self-renewal and pluripotency of stem cells or avoid their osteogenic or oste- oblastic differentiation capacities when cultured in adequate inducing media, as shown by the induction of specific gene expression (CEBP, PPARg, and RUNX2). Our results therefore support BMP4 as a promising factor for expanding human adipose tissue-derived MSCs maintaining their properties of stemness and multipotency. Introduction M esenchymal stem cells (MSCs) were initially de- scribed as a bone marrow-derived mononuclear cell fraction that, after culture ex vivo, adhered to plastic and acquired a fibroblast-like morphology [1]. These cells were distinct from hematopoietic stem cells and exhibited ca- pacity to differentiate into multiple mesodermal cell line- ages, largely to bone, cartilage, and adipocytes [2]. Apart from bone marrow, MSCs with similar biological proper- ties, although slight phenotypic differences, have been isolated from numerous tissues, suggesting that they re- side within the connective tissue of most organs [3]. Of special interest is the adipose tissue since it constitutes an abundant and easily accessible source of MSCs [4]. These stem cells have been variously termed, but a recent con- sensus has settled on the term ‘‘adipose-derived stem cells’’ (ASCs) [5]. However, a critical problem of MSCs is their limited ex vivo growth rate that makes mandatory their expansion in vitro to obtain a sufficient number of cells for cell therapy. Currently, MSCs destined for clinical therapy are expanded before transplantation in the culture medium supplemented with 10%–20% fetal bovine serum (FBS), increasing the po- tential risk of transferring animal proteins to human cells. To drastically reduce the animal-derived serum concentration or to use reagents of human origin become, therefore, critical issues for improving the therapeutical applications of MSCs. It is important to identify growth factors capable of inducing MSC expansion maintaining their properties of survival and multilineage differentiation capacities to an extensive thera- peutic application of MSCs. In the last years, several factors, especially different members of transforming growth factor b superfamily, have been used with the proposal of increase in vitro the number of MSCs. Remarkably, MSCs survive at low density in the absence of serum [6], a phenomenon that has 1 Department of Cell Biology, School of Medicine, Complutense University, Madrid, Spain. 2 Cell Therapy Unit and 3 Surgery Department, La Paz University Hospital, Madrid, Spain. 4 Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain. *These two authors contributed equally to this work. STEM CELLS AND DEVELOPMENT Volume 20, Number 6, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089=scd.2010.0355 1011