Yuji Teramura

Are Therapeutic Human Mesenchymal Stromal Cells Compatible with Human Blood

Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture‐expanded human MSCs elicit an innate immune attack, termed instant blood‐mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell‐passage number. Short‐term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D‐dimer or acute‐phase reactants with the currently applied dose of 1.0–3.0 × 106 cells per kilogram. Culture‐expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose‐dependent and increases after prolonged ex vivo expansion. Currently applied doses of low‐passage clinical‐grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells. Stem Cells2012;30:1565–1574

Innate immunity activation on biomaterial surfaces: A mechanistic model and coping strategies

When an artificial biomaterial (e.g., a stent or implantable pump) is exposed to blood, plasma proteins immediately adhere to the surface, creating a new interface between the biomaterial and the blood. The recognition proteins within the complement and contact activation/coagulation cascade systems of the blood will be bound to, or inserted into, this protein film and generate different mediators that will activate polymorphonuclear leukocytes and monocytes, as well as platelets. Under clinical conditions, the ultimate outcome of these processes may be thrombotic and inflammatory reactions, and consequently the composition and conformation of the proteins in the initial layer formed on the surface will to a large extent determine the outcome of a treatment involving the biomaterial, affecting both the functionality of the material and the patients life quality. This review presents models of biomaterial-induced activation processes and describes various strategies to attenuate potential adverse reactions by conjugating bioactive molecules to surfaces or by introducing nanostructures.

Bioartificial pancreas microencapsulation and conformal coating of islet of Langerhans.

Type 1 diabetes has been successfully treated by transplanting islets of Langerhans (islets), endocrine tissue releasing insulin. Serious issues, however, still remain. The administration of immunosuppressive drugs is required to prolong graft functioning; however, side effects of their long-term use on recipients are not fully understood, and cell transplantation therapy without the use of immunosuppressive drugs is desired. To resolve these issues, the encapsulation of isles with a semi-permeable membrane, or bioartificial pancreas, has been attempted. Many groups have reported that it functions very well in small animal models. Few of the bioartificial pancreases, however, were applied to human patients and their clinical outcome was not clear. In this review, we address obstacles and overview new techniques to overcome these issues, such as conformal coating and islet enclosure with cells.

Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties

We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze‐thawed and freshly harvested cells. We found that freeze‐thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze‐thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti‐inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze‐thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement‐mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation. Stem Cells 2014;32:2430–2442

Behavior of synthetic polymers immobilized on a cell membrane

We used three kinds of polymers that interact with living cells in different modes: poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) and poly(vinyl alcohol) carrying alkyl side chains (PVA-alkyl), expected to anchor to the membrane lipid bilayer through hydrophobic interactions; N-hydroxysuccinimidyl-PEG (PEG-NHS), which covalently bonds with all kinds of membrane proteins having amino groups on cell surfaces; and polyelectrolytes, poly(ethylene imine) (PEI) and carboxylated PVA (PVA-COOH), which interact with cells electrostatically. CCRF-CEM (T-cell like) and HEK293 (adherent cell) cell lines were used. We followed the surface dynamics of fluorescently labeled polymers on living cells over time using confocal laser scanning microscopy and flow cytometry. PEI destroyed cells, while PVA-COOH did not interact with cells. PEG-lipid, PVA-alkyl, and PEG-NHS interacted with cells without cytotoxicity and existed on the cell surface even after cells were washed. PEG-lipid and PEG-NHS were rapidly excluded from the cell surface without cytoplasmic uptake, while PVA-alkyl assembled on the living cell surface was taken into the cytoplasm and then excluded. Most polymers were excluded within 24h although exclusion routes seemed to be different between polymers, suggesting that cell transplant surface modifications are shorter than has been assumed. The short life of modified polymers on the cell surface should be a consideration for cell transplant surface modifications.

Islet encapsulation with living cells for improvement of biocompatibility

Bioartificial pancreas, microencapsulation of islets of Langerhans (islets) within devices has been studied as a safe and simple technique for islet transplantation without the need for immuno-suppressive therapy. Various types of bioartificial pancreas have been proposed and developed such as microcapsule, macrocapsule and diffusion chamber types. However, these materials comprising a bioartificial pancreas are not completely inert and may induce foreign body and inflammatory reactions. The residual materials would be a problem in human body. Here we propose an alternative method for microencapsulation of islets with a layer of living cells. We immobilized HEK293 cells (human endoderm kidney cell line) to the islet surface using amphiphilic poly(ethylene glycol)-conjugated phospholid derivative and biotin/streptavidin reaction and encapsulated islets with a cell layer by culture. No necrosis of islet cells at the center was seen after microencapsulation with a layer of living cells. Insulin secretion ability by glucose stimulation was well maintained on these cell-encapsulated islets.

Islets Surface Modification Prevents Blood-Mediated Inflammatory Responses

Transplantation of islets of Langerhans (islets) is a promising technique for treating insulin-dependent diabetes mellitus (type I). One unresolved issue is early graft loss due to inflammation triggered by blood coagulating on the surface of islets after transplantation into the portal vein. Here, we describe a versatile method for modifying the surface of islets with an ultrathin membrane carrying the fibrinolytic enzyme urokinase or the anticoagulant heparin. The surface of islets was modified with a poly(ethylene glycol)--phospholipid conjugate bearing a biotin group (biotin-PEG-lipids, PEG MW: 5000). Biotin-PEG-lipids were anchored to the cell membranes of islets, and the PEG-lipid layer on the islets was further covered by streptavidin and biotin-bovine serum albumin conjugate using a layer-by-layer method. The surface was further activated with oxidized dextran. Urokinase was anchored to the islets through Schiff base formation. Heparin was anchored to the islets through polyion complex formation between anionic heparin and a cationic protamine coating on the islets. No practical islet volume increase was observed after surface modification, and the modifications did not impair insulin release in response to glucose stimulation. The anchored urokinase retained high fibrinolytic activity, which could help to improve graft survival by preventing thrombosis on the islet surface.

Cell surface modification with polymers for biomedical studies

Surface modification of living cells with natural or synthetic polymers is a powerful and useful tool in biomedical science and engineering. Various functional groups and bioactive substances can be immobilized to the cell surface through covalent conjugation, hydrophobic interaction, or electrostatic interaction. In this review, we provide an overview of the methods and polymers employed in cell surface modification, including: (1) covalent conjugation utilizing amino groups of cell surface proteins, (2) hydrophobic interaction of amphiphilic polymers with a lipid bilayer membrane, and (3) electrostatic interactions between cationic polymers and a negatively charged cell surface. We also discuss their applications in studies on cell therapy, cell–cell interaction analysis, cell arrangement, and lineage determination of stem cells.

Surface modification of islets with PEG-lipid for improvement of graft survival in intraportal transplantation.

Background. Transplantation of islets of Langerhans (islets) is a promising technique for treating insulin-dependent diabetes mellitus (type I). One unsolved issue is the early graft loss due to inflammatory reactions triggered by blood coagulation and complement activation that occurs immediately after transplantation into the liver through the portal vein. Several proposed approaches for improvement of the graft survival include heparin coating and covalent poly(ethylene glycol) (PEG) conjugation. We previously have studied the improvement of graft survival by modification of islet surfaces using amphiphilic PEG-conjugated phospholipid and bioactive molecules. Here, we analyzed the effect of PEG-modification on the improvement of graft survival immediately after intraportal transplantation into streptozotocin-induced diabetic mice. Methods. The surface of hamster islets was modified with PEG-lipid. PEG-lipid modified islets (PEG-islets) were transplanted into the liver through the portal vein of streptozotocin-induced diabetic mice. We measured the graft survival periods and blood insulin levels immediately after intraportal transplantation to determine the cell damage to islets. Histocytochemical analyses of liver were also performed postintraportal transplantation. Results. The graft survival of PEG-islets was significantly prolonged compared with bare islets in livers of diabetic mice. Reduction of blood insulin level within 60 min after transplantation of PEG-islets suggests that the cell damage observed immediately after transplantation could be suppressed by surface modification with PEG in comparison with bare islets. Conclusion. Our approach for the improvement of graft survival will be useful in the clinical setting.

Immobilization of urokinase on the islet surface by amphiphilic poly(vinyl alcohol) that carries alkyl side chains

Transplantation of islets of Langerhans (islets) is a promising method to treat insulin-dependent diabetes mellitus (type I diabetes). However, insulin independence is typically realized for only approximately 30% of transplant recipients, even with sufficient numbers of islets from multiple donors. Innate immunological reactions triggered by blood coagulation play a key role in the loss of islets at the early stage. Here we propose a method to inhibit blood coagulation on the islet surface. A plasminogen activator, urokinase, was immobilized on the islet surface via a poly(vinyl alcohol) (PVA) derivative that carries alkyl chains and thiol groups. When the PVA derivative was added to an islet suspension, the alkyl side chains spontaneously anchored into the lipid bilayer membranes of islet cells. The surfaces of islets were covered with the PVA derivative. Urokinase modified with maleimide groups could be immobilized onto the islet surface by thiol/maleimide bonding with the layer of PVA derivatives. Urokinase-immobilized islets exhibited fibrinolytic properties, indicating that blood coagulation can be controlled on the islet surface. Urokinase immobilization on islets, which does not impair insulin release, represents a promising method to reduce early graft loss after intraportal islet transplantation.