Human Neonatal Thymus–Derived Mesenchymal Stromal Cells: Characterization, Differentiation, and Immunomodulatory Properties Matthias Siepe, M.D., 1,* Andreas R. Thomsen, M.D., 2,* Natalie Duerkopp, Ph.D., 2 Ulf Krause, M.D., 3 Katharina Fo ¨ rster, M.D., 1 Philip Hezel, M.D., 1 Friedhelm Beyersdorf, M.D., Ph.D., 1 Christian Schlensak, M.D., 1 Norbert P. Su ¨ dkamp, M.D., Ph.D., 4 Roland Bosse, Ph.D., 2 and Philipp Niemeyer, M.D. 4 Mesenchymal stromal cells represent an attractive cell population for cell transplantation and tissue engineering purposes. The aim of this study was to search for neonatal thymus–derived mesenchymal stromal cells (nTMSC) and further characterize the differentiation and immunomodulatory properties thereof. The thymus glands of 13 infants undergoing congenital cardiac surgery were removed. After in vitro isolation and expansion, we iden- tified adherent stromal cells with substantial proliferation potential. As characterized by FACS, the pattern of surface antigen expression of nTMSC resembled bone marrow stromal cells. Full mesenchymal differentiation potential is maintained during proliferation as confirmed by cultures for osteogenic, chondrogenic, and adi- pogenic lineages. After 5-azacytidine enrichment, morphological characteristics of cardiomyocytes were achieved. For immunologic investigations, the influence of nTMSC on the proliferative behavior of peripheral blood mononuclear cells was studied as a measure of the immune response. The nTMSC did not stimulate an allogeneic reaction in this coculture. Further, the expression of immunologically relevant markers was measured. Alike MSC from other origins, nTMSC did not express MHC-II. In contrast to mature MSC, some nTMSC even lack the expression of MHC-I. Our results confirm that the neonatal thymus contains mesenchymal stromal cells (nTMSC) with full mesenchymal differentiation potential and immunomodulatory properties. Introduction D uring embryonic development, the human thymus originates from the third branchial pouch and groove and thus consists of epithelial tissue. During the ninth week of gestation, thymocytic precursor cells migrate from the bone marrow, spleen, and liver. Maturation of T-cells in the thymus proceeds later with an interplay of thymocytes and supporting epithelial cells. The thymus reaches full immu- nological competence at birth, whereby the organ weighs approximately 15 g. During neonatal cardiac surgery (e.g., transposition of the great arteries, and hypoplastic left heart syndrome), the thymus gland of affected neonates is frequently excised to allow unrestricted access to the heart and great arteries. The whole-gland tissue or just one half of it (right hemi- thymectomy) might be discarded. Studies focusing on the immunological outcome of these babies have identified no severe adverse effect associated with this procedure. 1,2 However, the numbers of T helper cells are lower than those of neonates with intact thymus glands. This does not result in more infections or immune defects, because extrathymic T-cell maturation is compensatorily enhanced. 1,2 Mesenchymal stromal cells (MSC, formerly known as mesenchymal stem cells) are an attractive cell source for cell transplantation and tissue engineering. MSC are capable of giving rise to specific mesenchymal tissues in vitro and tend to acquire tissue-specific characteristics when cocultured with specialized cell types or exposed to tissue extracts in vitro. 3–5 These cells are usually obtained from bone marrow (BMSC). However, other sources have recently been de- scribed to contain corresponding cell populations. MSC with characteristics similar to bone marrow–derived cells can be derived from human adipose tissue, ligaments, and the lung. However, up to now there is no investigation on the occur- rence of MSC in the human thymus. Animal studies sug- gest that most adult tissues contain different amounts of MSC. Variations in immunophenotype and differentiation 1 Department of Cardiovascular Surgery, University Medical Center Freiburg, Freiburg, Germany. 2 CellGenix Technologie Transfer GmbH, Freiburg, Germany. 3 National Center for Tumor Diseases, Heidelberg, Germany. 4 Department of Orthopedic Surgery and Traumatology, University Medical Center Freiburg, Freiburg, Germany. *These authors contributed equally to this work. TISSUE ENGINEERING: Part A Volume 15, Number 7, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2008.0356 1787