ORIGINAL RESEARCH REPORT A Mouse Bone Marrow Stromal Cell Line with Skeletal Stem Cell Characteristics to Study Osteogenesis In Vitro and In Vivo Sebastian Raeth, 1 Benedetto Sacchetti, 2 Georg Siegel, 3 Ulrike A. Mau-Holzmann, 4 Jan Hansmann, 5 Gabriele Vacun, 5 Thomas G. Hauk, 1 Klaus Pfizenmaier, 1 and Angelika Hausser 1 Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells, which are able to differentiate in vitro into osteocytes, adipocytes, and chondrocytes. Mouse BMSCs (mBMSCs) are a versatile model system to investigate factors involved in BMSC differentiation in vitro and in vivo as a variety of transgenic mouse models are available. In this study, mBMSCs were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multi- potency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcuta- neously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to im- prove current strategies in bone regenerative therapy. Introduction S tem cells have the capacity to self-renew and dif- ferentiate into cells of various lineages. The bone marrow (BM) stroma contains progenitor cells of all tissues found in the skeleton, such as bone, cartilage, fibrous tissue, and fat [1]. In addition, these BM-derived progenitor cells give rise to cells that constitute the hematopoietic microenvironment (HME) [2] and are thus called BM stromal stem cells or skeletal stem cells [3]. Different laboratories have identified, under partly different isolation or culture conditions (eg, oxygen concentration), multipotent stromal cells [also called ‘‘mesenchymal stromal cell’’ (MSC)] from a panel of tissues, such as BM, adipose tissue, and umbilical cord. The differ- ences in tissue origin, isolation method, and culture condi- tions likely contribute to diverse phenotypes and functions ascribed to MSCs [4]. Thus, for better characterization of MSCs, the International Society of Cellular Therapy defined MSCs by the following three criteria. First, MSCs must be adherent to plastic under standard tissue culture conditions. Second, MSCs must express certain cell surface markers, such as CD73, CD90, and CD105, and lack expression of other markers, including CD45, CD34, CD14, or CD11b, CD79alpha or CD19, and HLA-DR surface molecules. Finally, MSCs must have the capacity to differentiate into osteoblasts, adipocytes, and chondroblasts under in vitro conditions [5]. Still, these criteria are based on the phenotype of culture-expanded MSCs and reliable markers specific for both native and cultured MSCs remain to be identified (re- viewed in Ref. [6]). The understanding of skeletal progenitor cells was extended by a study that demonstrates that in vivo transplantation of progenitor cells is the standard assay for confirming their multipotency. The authors postulated that only bone marrow stromal cells (BMSCs) are capable to differentiate into skeletal tissue and support the hematopoi- etic environment in vivo and are thus bona fide skeletal stem cells [7]. The definition and origin of MSCs and specifically the scope of potential clinical indications for MSC-based ther- apy is currently under heavy debate [8–12]. In particular, 1 Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany. 2 Department of Molecular Medicine, Sapienza University, Rome, Italy. 3 Institute of Clinical and Experimental Transfusion Medicine (IKET), University Hospital Tu ¨bingen, Tu ¨bingen, Germany. 4 Department of Medical Genetics, University of Tu ¨bingen, Tu ¨bingen, Germany. 5 Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany. STEM CELLS AND DEVELOPMENT Volume 00, Number 00, 2014 Ó Mary Ann Liebert, Inc. DOI: 10.1089/scd.2013.0367 1