Development of a surrogate angiogenic potency assay for clinical-grade stem cell production.
OBJECTIVE
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 neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency.


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 produced 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 increasing 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.
Athersys Inc., Regenerative Medicine Program, Cleveland, Ohio 44115, USA.
Nicholas
N
Lehman
Rochelle
R
Cutrone
Amy
A
Raber
Robert
R
Perry
Wouter
W
Van't Hof
Robert
R
Deans
Anthony E
AE
Ting
Juliana
J
Woda
D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D064987:Cell- and Tissue-Based Therapy; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology*; D015870:Gene Expression; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000637:transplantation; D009203:Myocardial Infarction / Q000628:therapy; D018919:Neovascularization, Physiologic; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D016207:Cytokines; D016209:Interleukin-8; D043562:Receptors, G-Protein-Coupled; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C091671:XCR1 protein, human