TRANSPLANTATION AND CELLULAR ENGINEERING Two steps to functional mesenchymal stromal cells for clinical application Christina Bartmann, Eva Rohde, Katharina Schallmoser, Peter Pürstner, Gerhard Lanzer, Werner Linkesch, and Dirk Strunk BACKGROUND: Ex vivo expansion of multipotent mes- enchymal stromal cells (MSCs) is a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. Even large volumes of starting material and extended culture periods, however, do not necessarily produce 2 ¥ 10 6 MSCs per kg per adult patient. A new two-step procedure has been devised to propagate more than 1 ¥ 10 8 MSCs from small marrow volumes within fewer than 4 weeks. STUDY DESIGN AND METHODS: The influence of log fold decreased MSC seeding (2500, 250, 25, 2.5/cm 2 ) on clinical-scale expansion, MSC phenotype, and immunomodulatory function combined with multiplex cytokine display was analyzed. Maintenance of MSC characteristics was tested in fibroblast colony-forming unit and differentiation assays. RESULTS: Reduced seeding density boosted MSC propagation. Low-density expanded MSCs were CD29+, CD73+, CD90+, CD105+, CD14–, CD34–, CD45–, HLA-DR–; retained their differentiation potential; and inhibited lymphocyte proliferation. This was accompanied by deregulated cytokine production. Seeding 0.7 ¥ 10 6 to 1 ¥ 10 6 MSCs derived from a 10- to 13-day primary culture at a low density of 28 to 40 per cm 2 permitted propagation of 1.5 ¥ 10 8 to 3.7 ¥ 10 8 functional MSCs within a 13- to 15-day secondary expansion step. CONCLUSION: Primary seeding of only 10-mL marrow aspirates on approximately 0.2-m 2 culture area (Step 1) followed by expansion on 2.5 m 2 (Step 2) is sufficient to consistently generate at least 1.5 ¥ 10 8 MSCs in fetal bovine serum–supplemented medium within less than 4 weeks. The efficiency of this two-step procedure for clinical-scale MSC propagation may facilitate rational clinical testing of MSC-based therapies. M ammalian bone marrow (BM) contains a heterogeneous population of nonhemato- poietic cells including fibroblasts, adipo- cytes, osteogenic precursors, and reticular endothelial cells. 1 Adhesion of mononuclear cells from BM aspirates to tissue culture plastic followed by the removal of nonadherent cells during the first 3 days of culture selects for an enriched population of proliferating CD45–, CD29+, CD73+, CD90+ spindle-shaped nonhe- matopoietic cells. 2 Because expanded BM stromal cells bear the capacity for self-renewal and can be induced to differentiate into osteo-, chondro-, and adipogenic progeny they are usually referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). 3 Despite extensive in vitro characterization, little is known about their in vivo biology and clinical applicability. 4 Friedenstein and coworkers 5-7 first described fibro- blast precursors derived from guinea pig BM and various other tissues in a fibroblast colony-forming unit (CFU-F) ABBREVIATIONS: a-MEM = alpha-modified minimum essen- tial medium; BM = bone marrow; CFU-F = fibroblast colony- forming unit; MSC(s) = mesenchymal stromal cell(s); RS cell(s) = recycling stem cell(s). From the Department of Internal Medicine, Division of Hema- tology and Stem Cell Transplantation, the Department of Blood Group Serology and Transfusion Medicine, the Department of Gynecology and Obstetrics, and StemCell Cluster, Medical Uni- versity, Graz, Austria. Address reprint requests to: Dirk Strunk, MD, Department of Internal Medicine, Division of Hematology and Stem Cell Transplantation, Medical University, Auenbrugger Pl. 38, A-8036 Graz, Austria; e-mail: dirk.strunk@klinikum-graz.at. This work has been supported by The Adult Stem Cell Research Foundation. Received for publication November 24, 2006; revision received January 26, 2006, and accepted January 31, 2006. doi: 10.1111/j.1537-2995.2007.01219.x TRANSFUSION 2007;47:1426-1435. 1426 TRANSFUSION Volume 47, August 2007