title: Heparinization of cell surfaces with short peptide-conjugated PEG-lipid regulates thromboinflammation in transplantation of human MSCs and hepatocytes.
abstract: Infusion of therapeutic cells into humans is associated with immune responses, including thromboinflammation, which result in a large loss of transplanted cells. To address these problems, heparinization of the cell surfaces was achieved by a cell-surface modification technique using polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives. A short heparin-binding peptide was conjugated to the PEG-lipid for immobilization of heparin conjugates on the surface of human mesenchymal stem cells (hMSCs) and human hepatocytes. Here three kinds of heparin-binding peptides were used for immobilizing heparin conjugates and examined for the antithrombogenic effects on the cell surface. The heparinized cells were incubated in human whole blood to evaluate their hemocompatibility by measuring blood parameters such as platelet count, coagulation markers, complement markers, and Factor Xa activity. We found that one of the heparin-binding peptides did not show cytotoxicity after the immobilization with heparin conjugates. The degree of binding of the heparin conjugates on the cell surface (analyzed by flow cytometer) depended on the ratio of the active peptide to control peptide. For both human MSCs and hepatocytes in whole-blood experiments, no platelet aggregation was seen in the heparin conjugate-immobilized cell group vs. the controls (non-coated cells or control peptide). Also, the levels of thrombin-antithrombin complex (TAT), C3a, and sC5b-9 were significantly lower than those of the controls, indicating a lower activation of coagulation and complement. Factor Xa analysis indicated that the heparin conjugate was still active on the cell surface at 24h post-coating. It is possible to immobilize heparin conjugates onto hMSC and human hepatocyte surfaces and thereby protect the cell surfaces from damaging thromboinflammation.
  
  
  CONCLUSIONS
  We present a promising approach to enhance the biocompatibility of therapeutic cells. Here we used short peptide-conjugated PEG-lipid for cell surface modification and heparin conjugates for the coating of human hepatocytes and MSCs. We screened the short peptides to find higher affinity for heparinization of cell surface and performed hemocompatibility assay of heparinized human hepatocytes and human MSCs in human whole blood. Using heparin-binding peptide with higher affinity, not only coagulation activation but also complement activation was significantly suppressed. Thus, it was possible to protect human hepatocytes and human MSCs from the attack of thromboinflammatory activation, which can contribute to the improvement graft survival.
authors.affiliation: Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden.
authors.forename: Sana
authors.initials: S
authors.lastname: Asif
authors.affiliation: Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden; Linnæus Center of Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden.
authors.forename: Kristina N
authors.initials: KN
authors.lastname: Ekdahl
authors.affiliation: Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Fromell
authors.affiliation: Department of Women's and Children's Health, Uppsala University Hospital, Sweden.
authors.forename: Elisabet
authors.initials: E
authors.lastname: Gustafson
authors.affiliation: Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden.
authors.forename: Andreea
authors.initials: A
authors.lastname: Barbu
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institute, and Hematology and Regenerative Medicine Centre at Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
authors.forename: Katarina
authors.initials: K
authors.lastname: Le Blanc
authors.affiliation: Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden.
authors.forename: Bo
authors.initials: B
authors.lastname: Nilsson
authors.affiliation: Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden; Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Electronic address: teramura@bioeng.t.u-tokyo.ac.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Teramura
mesh_terms: D000595:Amino Acid Sequence; D000991:Antithrombins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D006493:Heparin / Q000494:pharmacology / Q000627:therapeutic use*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000637:transplantation*; D006801:Humans; D007249:Inflammation / Q000188:drug therapy* / Q000473:pathology; D008055:Lipids / Q000737:chemistry*; D008297:Male; D008422:Materials Testing; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D011092:Polyethylene Glycols / Q000737:chemistry*; D013927:Thrombosis / Q000150:complications / Q000188:drug therapy*
chemical_list: D000991:Antithrombins; D008055:Lipids; D010455:Peptides; D011092:Polyethylene Glycols; D006493:Heparin
keywords: Cell surface modification; Heparinization; Hepatocyte; MSCs; Polyethylene glycol-conjugated phospholipid (PEG-lipid); Thromboinflammation