Helena Lönnies

Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells.

Seven patients underwent treatment with mesenchymal stem cells (MSCs), together with allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were given to three patients for graft failure and four patients were included in a pilot study. HSCT donors were three human leukocyte antigen (HLA)-identical siblings, three unrelated donors and one cord blood unit. The conditioning was myeloablative in four patients and reduced in three patients. MSC donors were HLA-identical siblings in three cases and haploidentical in four cases. Neutrophil counts >0.5 × 109/l was reached at a median of 12 (range 10–28) days. Platelet counts >30 × 109/l was achieved at a median of 12 (8–36) days. Acute graft-versus-host disease (GVHD) grade 0–I was seen in five patients. Two patients developed grade II, which in one patient evolved into chronic GVHD. One severe combined immunodeficiency (SCID) patient died of aspergillosis, the others are alive and well. One patient, diagnosed with aplastic anemia had graft failure after her first transplantation and severe Henoch–Schönlein Purpura (HSP). After retransplantation of MSCs and HSCs, she recovered from both the HSP and aplasia. Thus, co-transplantation of MSC resulted in fast engraftment of absolute neutrophil count (ANC) and platelets and 100% donor chimerism, even in three patients regrafted for graft failure/rejection.

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

Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells

BACKGROUND AIMS Mesenchymal stromal cells (MSC) can be expanded in vitro for clinical use and have been evaluated in clinical trials as immunosuppressants and to heal damaged tissues. We investigated the impact of freezing and prolonged storage on cell viability, proliferation and immunosuppression in vitro. METHODS MSC were expanded from bone marrow (BM) of healthy subjects according to standard protocols in the presence of fetal calf serum (FCS). The immunosuppressive potential of MSC was analyzed in mixed lymphocyte cultures (MLC) and after stimulation with phytohemagglutinin (PHA). RESULTS Expansion of frozen mononuclear cells (MNC) diminished MSC yield after expansion compared with plating of fresh MNC. MSC derived from frozen MNC were also less immunosuppressive. MSC harvested at various passages after expansion in vivo suppressed lymphocyte proliferation equally. Pooling of MSC from several donors generated higher and more stable suppression in both MLC and after PHA stimulation. After passage 1, plating at lower densities in 20% FCS increased cell expansion of MSC up to 25-fold compared with standard conditions. CONCLUSIONS MNC should not be frozen prior to MSC expansion. Decreased replating density and increased FCS levels generate higher numbers of MSC. Freezing of ex vivo-expanded MSC for >30 months did not affect cell viability or the ability to suppress lymphocyte proliferation. For effective immunosuppression in vitro MSC should be stored for less than 6 months and pooled from two or three donors.

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-γ, 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.

Effect of four dental varnishes on the colonization of cariogenic bacteria on exposed sound root surfaces.

The aim of this study was to evaluate the effect of four different dental varnishes on the colonization of mutans streptococci, total streptococci and lactobacilli on exposed sound root surfaces. Sixty–five individuals were randomly allotted to one of four groups for treatment with Cervitec® varnish containing 1% chlorhexidine and 1% thymol, a thymol varnish or one of two different fluoride varnishes, Fluor Protector and Duraphat. The varnish was applied to three buccal root surfaces in each patient at baseline and after 1 week. Dental plaque from the root surfaces was collected and analysed on four different occasions: at baseline, after 1 week, 1 month and 6 months. The Cervitec varnish caused a statistically significant reduction in the number of mutans streptococci over time. The reduction was significant at 1 week and 1 month relative to baseline. The numbers of total streptococci and lactobacilli were not significantly affected by treatment with Cervitec. No statistically significant difference over time was found for mutans streptococci, lactobacilli or total streptococci after treatment with the fluoride varnishes or the thymol varnish.

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.

No increased trapping of multipotent mesenchymal stromal cells in bone marrow filters compared with other bone marrow cells

BACKGROUND Multipotent mesenchymal stromal cells (MSC) are candidates for cellular therapy in regenerative medicine and as treatment of graft-versus-host-disease (GvHD) after hematopoietic stem cell (HSC) transplantation. It has been suggested that MSC may be trapped in bone marrow (BM) filters during the stem cell procurement and lost from the HSC graft. METHODS We investigated filtered BM and filters from six HSC donors. MSC were expanded from the two sources and investigated by flow cytometry, doubling capacity, differentiation ability and suppression in mixed lymphocyte cultures. RESULTS A range of 0.3-3.4% cells was trapped in the filters. By flow cytometry, there was no difference in the proportions of different cell types between the filter-retrieved and filtered BM cells. The phenotype, immunosuppressive capacity, differentiation and growth were equal in MSC expanded from the two cell sources. DISCUSSION Given the low number of trapped cells, filters do not appear to be a good source of MSC. When intended for clinical transplantation, MSC need to be expanded ex vivo to achieve sufficient doses for a clinical effect.

Purification and characterization of an aminopeptidase from Streptococcus mitis ATCC 903

An aminopeptidase isolated from the cytoplasmic fraction of a cell extract ofStreptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-ß-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-ß-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0–7.2 and at 37–40°C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co2+ ions. Hg2+ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co2+ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co2+ ions.

Isolation and purification of cell wall-associated arginine carboxypeptidase fromStreptococcus mitis ATCC 15909

An arginine carboxypeptidase was isolated from the cell walls ofStreptococcus mitis ATCC 15909 by mutanolysin extraction of the walls. The enzyme was purified 32-fold by gel filtration on Sephacryl S-300, affinity chromatography on Arginine-Sepharose 4B and by rechromatography on Sephacryl S-300. The molecular mass of the enzyme was calculated to 122 kDa by gel filtration. The enzyme released arginine from the carboxy terminal of hippurylarginine and, at a low rate, lysine from furylacryloylalanyl-lysine and hippuryl-lysine. The carboxypeptidase seemed firmly bound to the cell wall because SDS treatment of the walls did not release measurable amounts of activity.