∗These authors contributed equally to this work. Author contributions: Nicholas Lehman, conception and design, collection and/or assembly of data, data analysis and interpretation; Rochelle Cutrone, collection and/or assembly of data; Amy Raber, collection and/or assembly of data; Robert Perry, collection and/or assembly of data; Wouter Van’t Hof, data analysis and interpretation; Robert Deans, data analysis and interpretation, final approval of manuscript; Anthony E. Ting, data analysis and interpretation; Juliana M. Woda, conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript. Correspondence: Juliana Woda, Athersys Inc., Regenerative Medicine Program, 3201 Carnegie Ave, Cleveland, OH 44115, USA. E-mail jwoda@athersys.com (Received 4 January 2012; accepted 23 April 2012) Development of a surrogate angiogenic potency assay for clinical-grade stem cell production NICHOLAS LEHMAN ∗, ROCHELLE CUTRONE ∗, AMY RABER, ROBERT PERRY , WOUTER VAN’T HOF , ROBERT DEANS, ANTHONY E. TING & JULIANA WODA Athersys Inc., Regenerative Medicine Program, Cleveland, Ohio, USA Abstract Background aims. Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovasculariza- tion through trophic support. Production of clinical-grade stem cell products requires the development of lot-release cri- teria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. Methods and Results. Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors pro- duced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increas- ing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. Conclusions. A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria. Key words: angiogenesis, chemokine (C-X-C motif) ligand 5, interleukin-8, multipotent progenitor cell population, MultiStem, potency assay , vascular endothelial growth factor Introduction The development and use of cellular therapies to treat a variety of disorders, including ischemic injuries, hematopoietic stem cell transplant support, autoim- mune disease and tissue damage, have expanded rap- idly because of their clinical promise. Translation of these therapies to the clinic requires the establishment of consistent and effective cells produced under good manufacturing practice (cGMP) regulations. These regulations require the production of cells that have been demonstrated to be safe, pure, potent, stable and effective for human use. One important aspect of the criteria for establishing the safety and quality of cellular therapies, during production and at the time of lot release, is the use of potency assays to measure the biologic activity. Potency testing, as stipulated by regulatory guidelines, should be a quantitative mea- sure that reflects directly on the fundamental mecha- nistic pathway underlying the therapeutic response to verify the manufacturing process. Unlike defined component therapies with known molecular mecha- nisms, the therapeutic benefit of cellular therapies is often driven by its influence on a combination of mechanistic pathways and is often the result of a complex mixture of factors. Although repeated testing of new cell lots in pre-clinical models to measure improvement in function would provide a degree of certainty with regard to the critical biologic activity, there are two disadvantages to this approach. First, the time for Cytotherapy, 2012; 14: 994–1004 ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2012.688945