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.

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 × 10(6) 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.

Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease.

BACKGROUND: Mesenchymal stem cells (MSC) have immunomodulatory effects. The aim was to study the effect of MSC infusion on graft-versus-host disease (GVHD). METHODS: We gave MSC to eight patients with steroid-refractory grades III-IV GVHD and one who had extensive chronic GVHD. The MSC dose was median 1.0 (range 0.7 to 9)x10(6)/kg. No acute side-effects occurred after the MSC infusions. Six patients were treated once and three patients twice. Two patients received MSC from HLA-identical siblings, six from haplo-identical family donors and four from unrelated mismatched donors. RESULTS: Acute GVHD disappeared completely in six of eight patients. One of these developed cytomegalovirus gastroenteritis. Complete resolution was seen in gut (6), liver (1) and skin (1). Two died soon after MSC treatment with no obvious response. One of them had MSC donor DNA in the colon and a lymph node. Five patients are still alive between 2 months and 3 years after the transplantation. Their survival rate was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSC during the same period (P = 0.03). One patient treated for extensive chronic GVHD showed a transient response in the liver, but not in the skin and he died of Epstein-Barr virus lymphoma. CONCLUSION: MSC is a very promising treatment for severe steroid-resistant acute GVHD.

Oral mucosal progenitor cells are potently immunosuppressive in a dose-independent manner.

Oral mucosal lamina propria progenitor cells (OMLP-PCs) are a novel, clonally derived PC population of neural crest origin with the potential to differentiate down both mesenchymal and neuronal cell lineages. In this study we aimed to determine the immunological properties of OMLP-PCs and to establish whether they would be suitable candidates for allogeneic tissue engineering and in the treatment of immune-related diseases. OMLP-PCs demonstrated no inherent immunogenicity with insignificant expression of costimulatory molecules (CD40, CD80, CD86, CD154, and CD178) or human leukocyte antigen (HLA) class II. OMLP-PCs required 7 days of stimulation with interferon-γ (IFN-γ) to induce cell surface expression of HLA II. Mixed lymphocyte cultures and mitogen stimulation demonstrated the potent immunosuppressive capability of OMLP-PCs in a contact-independent manner. Complete inhibition of lymphocyte proliferation was seen at doses as low as 0.001% OMLP-PCs to responder lymphocytes, while annexin V staining confirmed that this immunosuppressive effect was not due to the induction of lymphocyte apoptosis. These data demonstrate, for the first time, that OMLP-PC immunomodulation, unlike that for mesenchymal stem cells, occurs via a dose- and HLA II-independent mechanism by the release of immunosuppressive soluble factors and suggests these cells may have wide ranging potential in future immune-related therapies.

Multipotent mesenchymal stromal cells express FoxP3: a marker for the immunosuppressive capacity?

Multipotent mesenchymal stromal cells (MSCs) have immunosuppressive capacity but the exact mechanism by which they suppress proliferation of T lymphocytes is not fully understood. Recently, the characteristics and function of regulatory T lymphocytes (Tregs) have become better defined. Tregs and MSCs have immunosuppressive features in common. Here, we looked for a common basis for immunosuppression in these distinct cell types. Forkhead box P3 (FoxP3) and CD39 expression in MSCs was measured by flow cytometry and real-time quantitative polymerase chain reaction. The importance of FoxP3 in MSC-mediated immunosuppression was investigated by siRNA technology and mixed lymphocyte culture (MLC). The effect of 5-azacytidine and other immunosuppressive drugs on FoxP3 expression and immunosuppression by MSCs was explored by flow cytometry, MLC, and real-time quantitative polymerase chain reaction. MSCs express FoxP3 at variable levels, but they do not express CD39. FoxP3 MSCs suppress MLC to a greater extent than cells with lower FoxP3 expression. However, FoxP3-decreased MSCs were found to retain their immunosuppressive properties. 5-azacytitine had no effect on FoxP3 expression or MLC suppression by MSCs. However, immunosuppressive drugs led to increased FoxP3 levels and MLC inhibition in FoxP3 MSCs. This is the first demonstration of FoxP3 expression by MSCs. Although MSCs share several features with Tregs, and FoxP3 MSCs tend to be more immunosuppressive, MSCs do not require functional FoxP3 for their immunosuppressive activity. The increased MSC-mediated suppression of immune responses by immunosuppressive drugs deserves further investigation.

HSCT recipients have specific tolerance to MSC but not to the MSC donor.

Multipotent mesenchymal stromal cells (MSC) are increasingly used to treat refractory graft-versus-host-disease and other complications after hematopoietic stem cell transplantation (HSCT). We evaluated immunogenicity of HLA-mismatched MSC infused posttransplant to HSCT recipients. Recipient lymphocyte response to MSC and peripheral blood lymphocytes (PBL) from the MSC or third party donors was measured before and after infusion. In vitro primary and rechallenge lymphocyte responses of healthy individuals to MSC and to PBL from the MSC donor were similarly studied. HSCT recipients given MSC responded to third party allostimuli, but showed no response to infused MSC before and upto 6 months after infusion, whereas maintaining an alloresponse to the MSC donor. This indicates immune unresponsiveness restricted to MSC, as the HSCT recipient was not tolerized to the MSC donor. In vitro, we confirmed that MSC failed to prime responder lymphocytes to rechallenge with PBL from the MSC donor, and lymphocytes primed with MSC donor and rechallenged with MSC only showed weak responses at high stimulator-responder ratios. Although MSC up-regulated lymphocyte gene expression of CD25, IFN-gamma, FoxP3, CTLA-4, and IL-10, they failed in both unprimed and primed responders to induce CD25+ (activated) or CD57+ (effector) CD4+ or CD8+ T-lymphocyte subsets and only inconsistently induced FoxP3+ regulatory T-lymphocytes. These results show for the first time that infused MSC are only weakly immunogenic in humans and validate the clinical use of MSC from HLA-mismatched donors.

Extracellular adherence protein from Staphylococcus aureus enhances internalization into eukaryotic cells.

In this study we have shown that Eap (extracellular adherence protein) plays a role in the internalization process of Staphylococcus aureus into eukaryotic cells. Eap is a protein that is mostly extracellularly and to a lesser extent is bound to the bacterial surface as a result of rebinding. Eap is able to bind to several plasma proteins, such as fibronectin, fibrinogen, and prothrombin. It has the capacity to form oligomers and is able to agglutinate S. aureus. A mutant strain, Newman mAH12 (eap:: Ery(r)), with a deficient eap gene was used in the present study. We have demonstrated that (i) strain Newman mAH12 could adhere to and become internalized to a higher extent by eukaryotic cells than the isogenic mutant, (ii) strain Newman mAH12 complemented with the eap gene displayed restoration of the internalization level, (iii) externally added Eap enhanced the internalization of laboratory and clinical S. aureus strains as well as of S. carnosus (a coagulase-negative species devoid of proteins important for internalization), and (iv) antibodies against Eap were able to block the internalization process in strain Newman mAH12 and clinical isolates. Eap, with its broad binding capacity and its surface localization, thus seems to contribute to the internalization of S. aureus into eukaryotic cells. We therefore propose a novel internalization pathway for S. aureus in which Eap plays an enhancing role.