Guido Moll

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study

BACKGROUND Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation†‡§

Mesenchymal stromal cells (MSCs) are explored as a novel treatment for a variety of medical conditions. Their fate after infusion is unclear, and long‐term safety regarding malignant transformation and ectopic tissue formation has not been addressed in patients. We examined autopsy material from 18 patients who had received human leukocyte antigen (HLA)‐mismatched MSCs, and 108 tissue samples from 15 patients were examined by PCR. No signs of ectopic tissue formation or malignant tumors of MSC‐donor origin were found on macroscopic or histological examination. MSC donor DNA was detected in one or several tissues including lungs, lymph nodes, and intestine in eight patients at levels from 1/100 to <1/1,000. Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, as DNA was detected from nine of 13 MSC infusions given within 50 days before sampling but from only two of eight infusions given earlier. There was no correlation between MSC engraftment and treatment response. We conclude that MSCs appear to mediate their function through a “hit and run” mechanism. The lack of sustained engraftment limits the long‐term risks of MSC therapy. STEM CELLS2012;30:1575–1578

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

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

Tissue‐Engineered Airway: A Regenerative Solution

The use of synthetic degradable or permanent polymers and biomaterials has not yet helped to achieve successful clinical whole‐airway replacement. A novel, clinically successful approach involves tissue engineering (TE) replacement using three‐dimensional biologic scaffolds composed of allogeneic extracellular scaffolds derived from nonautologous sources and recellularized with autologous stem cells or differentiated cells. In this paper, we discuss this novel approach and review information that can lead to a better understanding of stem cell recruitment and/or mobilization and site‐specific tissue protection, which can be pharmacologically boosted in humans.

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses.

Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the bodys innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.

Viability and proliferation of rat MSCs on adhesion protein-modified PET and PU scaffolds

In 2011, the first in-man successful transplantation of a tissue engineered trachea-bronchial graft, using a synthetic POSS-PCU nanocomposite construct seeded with autologous stem cells, was performed. To further improve this technology, we investigated the feasibility of using polymers with a three dimensional structure more closely mimicking the morphology and size scale of native extracellular matrix (ECM) fibers. We therefore investigated the in vitro biocompatibility of electrospun polyethylene terephthalate (PET) and polyurethane (PU) scaffolds, and determined the effects on cell attachment by conditioning the fibers with adhesion proteins. Rat mesenchymal stromal cells (MSCs) were seeded on either PET or PU fiber-layered culture plates coated with laminin, collagen I, fibronectin, poly-D-lysine or gelatin. Cell density, proliferation, viability, morphology and mRNA expression were evaluated. MSC cultures on PET and PU resulted in similar cell densities and amounts of proliferating cells, with retained MSC phenotype compared to data obtained from tissue culture plate cultures. Coating the scaffolds with adhesion proteins did not increase cell density or cell proliferation. Our data suggest that both PET and PU mats, matching the dimensions of ECM fibers, are biomimetic scaffolds and, because of their high surface area-to-volume provided by the electrospinning procedure, makes them per se suitable for cell attachment and proliferation without any additional coating.

Crimean-Congo hemorrhagic fever virus activates endothelial cells

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) causes viral hemorrhagic fever with high case-fatality rates and is geographically widely distributed. Due to the requirement for a biosafety level 4 (BSL-4) laboratory and the lack of an animal model, knowledge of the viral pathogenesis is limited. Crimean-Congo hemorrhagic fever (CCHF) is characterized by hemorrhage and vascular permeability, indicating the involvement of endothelial cells (ECs). The interplay between ECs and CCHFV is therefore important for understanding the pathogenesis of CCHF. In a previous study, we found that CCHFV-infected monocyte-derived dendritic cells (moDCs) activated ECs; however, the direct effect of CCHFV on ECs was not investigated. Here, we report that ECs are activated upon infection, as demonstrated by upregulation of mRNA levels for E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Protein levels and cell surface expression of ICAM1 responded in a dose-dependent manner to increasing CCHFV titers with concomitant increase in leukocyte adhesion. Furthermore, we examined vascular endothelial (VE) cadherin in CCHFV-infected ECs by different approaches. Infected ECs released higher levels of interleukin 6 (IL-6) and IL-8; however, stimulation of resting ECs with supernatants derived from infected ECs did not result in increased ICAM1 expression. Interestingly, the moDC-mediated activation of ECs was abrogated by addition of neutralizing tumor necrosis factor alpha (TNF-α) antibody to moDC supernatants, thereby identifying this soluble mediator as the key cytokine causing EC activation. We conclude that CCHFV can exert both direct and indirect effects on ECs.

Do ABO Blood Group Antigens Hamper the Therapeutic Efficacy of Mesenchymal Stromal Cells

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens – genetically governed antigenic determinants – at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.

Different Procoagulant Activity of Therapeutic Mesenchymal Stromal Cells Derived from Bone Marrow and Placental Decidua.

While therapeutic mesenchymal stromal/stem cells (MSCs) have usually been obtained from bone marrow, perinatal tissues have emerged as promising new sources of cells for stromal cell therapy. In this study, we present a first safety follow-up on our clinical experience with placenta-derived decidual stromal cells (DSCs), used as supportive immunomodulatory and regenerative therapy for patients with severe complications after allogeneic hematopoietic stem cell transplantation (HSCT). We found that DSCs are smaller, almost half the volume of MSCs, which may favor microvascular passage. DSCs also show different hemocompatibility, with increased triggering of the clotting cascade after exposure to human blood and plasma in vitro. After infusion of DSCs in HSCT patients, we observed a weak activation of the fibrinolytic system, but the other blood activation markers remained stable, excluding major adverse events. Expression profiling identified differential levels of key factors implicated in regulation of hemostasis, such as a lack of prostacyclin synthase and increased tissue factor expression in DSCs, suggesting that these cells have intrinsic blood-activating properties. The stronger triggering of the clotting cascade by DSCs could be antagonized by optimizing the cell graft reconstitution before infusion, for example, by use of low-dose heparin anticoagulant in the cell infusion buffer. We conclude that DSCs are smaller and have stronger hemostatic properties than MSCs, thus triggering stronger activation of the clotting system, which can be antagonized by optimizing the cell graft preparation before infusion. Our results highlight the importance of hemocompatibility safety testing for every novel cell therapy product before clinical use, when applied using systemic delivery.