493 Heparinzation of cell surfaces with a heparin-binding peptide-conjugated PEG-lipid for regulation of thromboinflammation in transplantation of human MSC and hepatocyte
Tuesday November 17, 2015 from 11:00 to 12:30
Room 111-112

Yuji Teramura, Japan

Associate Professor

Department of Bioengineering

University of Tokyo

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Heparinzation of cell surfaces with a heparin-binding peptide-conjugated PEG-lipid for regulation of thromboinflammation in transplantation of human MSC and hepatocyte

Yuji Teramura1, Sana Asif2, Elisabet Gustafson3, Kristina Nilsson-Ekdahl2,4, Bo Nilsson2.

1Department of Bioengineering, The University of Tokyo, Tokyo, Japan; 2Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Uppsala, Sweden; 3Department of Women's and Children's Health, Uppsala University hospital, Uppsala, Sweden; 4Linnæus Center of Biomaterials Chemistry, Linnæus University, Kalmar, Sweden

Introduction: Cell transplantation is an attractive alternative to whole organ transplantation. However, the infusion of cells into human body is associated with large loss of transplanted cells, which was caused by immune responses termed as an instant blood mediated inflammatory reaction (IBMIR). IBMIR is an innate immunity attack triggered by the activation of complement and coagulation systems, followed by rapid binding of platelets and infiltration of leukocytes into the clot resulting in early graft loss of transplanted cells.  Therefore it is important to protect the cell surfaces from the attack of IBMIR. In order to address these issues, heparinzation of the cell surface was achieved by a cell surface modification technique using poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) derivatives. To this construct several heparin-binding peptides were conjugated to PEG-lipid for the immobilization of heparin conjugates on cell surfaces such as on human MSC and human hepatocyte. The heparinized cells were incubated in human whole blood to evaluate the hemocompatibility by measuring blood parameters such as platelet count, coagulation marker (thrombin-antithrombin complex (TAT)), complement markers (C3a and sC5b-9), Factor Xa activity.
Methods: Three heparin-binding peptides (peptide I: WQPPRARIC, peptide II:  CNSAHRTRGRQRS, peptide III: CWGGRARARARARARARA, control: GPQGPGGGWC) were used for the immobilization of heparin conjugates (Corline AB, Uppsala, Sweden). The maleimide group of PEG-lipid reacts with SH group of cysteine in those peptides. SPR and QCM-D were utilized for the binding assay of the peptides with heparin conjugates and antithrombin III. Human MSC (from Katarina Le Blanc, Karolinska Institute) and human hepatocyte were used in the whole blood experiments. Both cells were incubated with peptide-PEG-lipid (1mg/mL, in PBS) for 30min at RT, and then incubated with heparin conjugates (100 µg/mL in PBS). Control and heparinized cells were then exposed to fresh human whole blood in a whole blood loop model for 60 min. Blood was analyzed for platelet count, TAT, C3a, sC5b-9, Factor Xa activity.
Results: Peptide-PEG-lipid was used for the binding assay for heparin-conjugate and antithrombin III, indicating that peptide II and III have larger binding of heparin-conjugates compared to peptide I and control peptide. When cells were treated with peptide III-PEG-lipid and heparin conjugates, the cell viability was much lower than the control and also depended on the ratio of peptide III, indicating the cytotoxicity due to the positive charge. On the other hand, there was no cytotoxicity after coating with peptide II-PEG-lipid and heparin conjugates. The binding of heparin conjugates on the cell surface depends on the ratio of peptide II-PEG-lipid, which was analyzed by FACS. In the whole blood experiments with peptide II-PEG-lipid, there was no platelet aggregation in the heparin-conjugate-immobilized cells group in comparison to the control (non-coated cells) for both human MSCs and hepatocytes. Also, TAT, C3a, and sC5b-9 levels were significantly lower in heparin-conjugate-immobilized cells group compared to the control, indicating a lower activation of coagulation and complement. Factor Xa analysis indicated that the activity of heparin conjugate was still detected on the cell surface during 24hr.
Conclusion: Heparinzation of MSC and hepatocyte employing a PEG-lipid construct attenuate thromboinflammatory reactions in whole blood. 

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