Instant lysis of therapeutic mesenchymal stem cells after intravenous infusion: role of complement system and in vitro prevention strategies
A. R. Muslimov, R. T. Mikhelashvili, E. V. Volchkov, K. V. Lepik, V. S. Sergeev, B. V. Afanasyev
R. M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, The First St. Petersburg State I. Pavlov Medical University, St.Petersburg, Russia.
Multipotent mesenchymal stromal cells (MSCs) harbor great potential for cellular and tissue therapy, and are currently under investigation in numerous clinical trials, the majority thereof relying on systemic infusion. A major drawback in MSC‐therapy appears to be in the incomplete understanding of fate and function of MSCs following systemic administration. In recent years, new evidence indicates a low biocompatibility of MSC during intravenous infusion. Experimental data may suggest that up to 50% of ex vivo cultured MSCs undergo a complement-dependent lysis upon interaction between the cells and intact human serum. Meanwhile, the data concerning this issue is limited and controversial in some instances. The goal of present study was to assess the MSC viability during intravenous infusion, to confirm the complement-mediated lysis of MSCs, searching for possible ways to overcome this damage.
Materials and methods
Bone marrow-derived MSCs were obtained from healthy donors. Parallel cultures of MSCs from each donor were performed in two types of media: 1) MEM-α containing 10% FBS, and 2) MEM-α containing 7,5% platelet lysate (PL). When the cultures became near-confluent (>80%), the cells were detached by treatment with trypsin and EDTA. The harvested cells from different media (either PL-, or FBS-supplied) were divided into 3 sub-groups. Group 1: One-hour MSC incubation with 200µl of native (active) serum (a pool of 4 AB(IV), Rh+ donors). Group 2: One-hour incubation of MSCs with serum pre-treated with anti-C5 monoclonal antibody (Soliris®, Eculizumab, Alexion Pharmaceuticals Inc.) at the dose of 50µg/ml. Group 3: One-hour MSC incubation with EDTA-inactivated serum (controls).
Cytotoxicity analysis was performed by means of flow cytometry (7-AAD was used for the cell viability assays).
In our preliminarily study, we revealed a statistical significant differences between all 3 groups of cells which were cultured in media containing 10% of FBS. About 30% of freshly harvested MSCs were lysed after 1 hour of contact with active AB(IV) serum. This effect was inhibited with EDTA or Eculizumab, a specific C5 inhibitor, thus providing evidence for a complement-dependent cell lysis. In the series incubated incubated with fetal bovine serum, the average cytotoxicity was 27,9% (subgroup 1), 15,4% (subgroup 2), and 10,7% (subgroup 3, p=0,00015). Meanwhile, we could not demonstrate any significant differences between the 3 subgroups of cells cultured with 7,5% human platelet lysate (PL). The average cytotoxicity in the 13.1% for subgroup 1; 15,5% for subgroup 2, and 7,5% for the subgroup 3 (p=0,32).
Our results confirm a phenomenon of pronounced complement-dependent lysis of pre-cultivated MSCs after contact with native serum. By the contrary, MSCs supplemented with human platelet lysate during cultivation showed much lesser (if any) susceptibility to the complement-dependent lysis. Lack of xenoantigens in the PL-containing media may be a possible explanation for this experimental finding.
We also report that pre-treatment of native sera with anti-C5 monoclonal antibody Eculizumab caused a significant reduction of in vitro MSC lysis after their culture in fetal bovine serum, thus suggesting a definite proof for applications of Eculizumab in different areas of cell therapy and transplantation.
Transplantation, cellular lysis, complement-mediated, mesenchymal stem cells