Antonella Conforti

Multiple infusions of mesenchymal stromal cells induce sustained remission in children with steroid-refractory, grade III-IV acute graft-versus-host disease.

Mesenchymal stromal cell (MSC) infusions have been reported to be effective in patients with steroid‐refractory, acute graft‐versus‐host disease (aGvHD) but comprehensive data on paediatric patients are limited. We retrospectively analysed a cohort of 37 children (aged 3 months‐17 years) treated with MSCs for steroid‐refractory grade III–IV aGvHD. All patients but three received multiple MSC infusions. Complete response (CR) was observed in 24 children (65%), while 13 children had either partial (n = 8) or no response (n = 5). Cumulative incidence of transplantation‐related mortality (TRM) in patients who did or did not achieve CR was 17% and 69%, respectively (P = 0·001). After a median follow‐up of 2·9 years, overall survival (OS) was 37%; it was 65% vs. 0% in patients who did or did not achieve CR, respectively (P = 0·001). The median time from starting steroids for GvHD treatment to first MSC infusion was 13 d (range 5–85). Children treated between 5 and 12 d after steroid initiation showed a trend for better OS (56%) and lower TRM (17%) as compared with patients receiving MSCs 13–85 d after steroids (25% and 53%, respectively; P = 0·22 and 0·06, respectively). Multiple MSC infusions are safe and effective for children with steroid‐refractory aGvHD, especially when employed early in the disease course.

Inhibition of B-Cell Proliferation and Antibody Production by Mesenchymal Stromal Cells Is Mediated by T Cells

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4(+) and CD8(+) cells playing a role. Moreover, cell-cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell-cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired.

Microvescicles derived from mesenchymal stromal cells are not as effective as their cellular counterpart in the ability to modulate immune responses in vitro.

Mesenchymal stromal cells (MSCs) are multipotent cells that possess broad immunomodulatory properties; the mechanisms underlying these properties have not been completely clarified. Aim of this study was to compare in vitro immunomodulatory effects of MSCs with those of microvesicles (MVs) released in supernatants from the same MSCs. MSCs were generated from bone marrow of 12 healthy donors (HDs) and MVs were isolated from their supernatant by serial ultracentrifugation according to two different procedures. Both MSCs and MVs were characterized by flow cytometry and incubated in vitro with peripheral blood mononuclear cells (PBMCs) of 12 HDs after stimulation with PHA and CpG. Growth factors and cytokines were quantified by ELISA. MVs were identified as 0.1-1 μm particles positive for CMFDA, CD107, and CD13. MSCs were significantly more capable to inhibit in vitro PHA-induced T-cell proliferation as compared with the corresponding MVs (P<0.01 and P<0.05 for MSC:PBMC ratio 1:2 and 1:10, respectively). While MVs displayed similar inhibitory activity on B-cell proliferation (P=0.43 as compared with PBMCs/CpG/MSCs; MSC:PBMC ratio 1:10) they induced lower inhibitory effect on plasmacell differentiation and antibody secretion (P<0.05 as compared with PBMCs/CpG/MSCs). For both T and B cells, MSC co-colture induced a statistically significant increase in IL-10 and TGFβ and decrease of GM-CSF and IFNγ, as compared with MV incubation. Our data indicate a lower in vitro immunomodulatory effect of MVs on T-cell proliferation and antibody formation, as compared with their cellular counterpart. The relative clinical benefit of either MSCs or MVs needs to be compared in proper prospective studies.

Biological, functional and genetic characterization of bone marrow-derived mesenchymal stromal cells from pediatric patients affected by acute lymphoblastic leukemia

Alterations in hematopoietic microenvironment of acute lymphoblastic leukemia patients have been claimed to occur, but little is known about the components of marrow stroma in these patients. In this study, we characterized mesenchymal stromal cells (MSCs) isolated from bone marrow (BM) of 45 pediatric patients with acute lymphoblastic leukemia (ALL-MSCs) at diagnosis (day+0) and during chemotherapy treatment (days: +15; +33; +78), the time points being chosen according to the schedule of BM aspirates required by the AIEOP-BFM ALL 2009 treatment protocol. Morphology, proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties and ability to support long-term hematopoiesis of ALL-MSCs were analysed and compared with those from 41 healthy donors (HD-MSCs). ALL-MSCs were also genetically characterized through array-CGH, conventional karyotyping and FISH analysis. Moreover, we compared ALL-MSCs generated at day+0 with those isolated during chemotherapy. Morphology, immunophenotype, differentiation potential and in vitro life-span did not differ between ALL-MSCs and HD-MSCs. ALL-MSCs showed significantly lower proliferative capacity (p<0.001) and ability to support in vitro hematopoiesis (p = 0.04) as compared with HD-MSCs, while they had similar capacity to inhibit in vitro mitogen-induced T-cell proliferation (p = N.S.). ALL-MSCs showed neither the typical translocations carried by the leukemic clone (when present), nor other genetic abnormalities acquired during ex vivo culture. Our findings indicate that ALL-MSCs display reduced ability to proliferate and to support long-term hematopoiesis in vitro. ALL-MSCs isolated at diagnosis do not differ from those obtained during treatment.

Comprehensive characterization of mesenchymal stromal cells from patients with Fanconi anaemia

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony‐forming unit (CFU‐F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long‐term haematopoiesis and immunomodulatory properties of FA‐MSCs were analysed and compared with those of MSCs expanded from 15 age‐matched healthy donors (HD‐MSCs). FA‐MSCs were genetically characterized through conventional karyotyping, diepoxybutane‐test and array‐comparative genomic hybridization. FA‐MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen‐induced T‐cell proliferation and to support long‐term haematopoiesis did not differ between FA‐MSCs and HD‐MSCs. CFU‐F ability and proliferative capacity of FA‐MSCs isolated after HSCT were significantly lower than those of HD‐MSCs. FA‐MSCs reached senescence significantly earlier than HD‐MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA‐MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation.

Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient

BackgroundCystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis.MethodsMorphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs).ResultsDespite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs.ConclusionsOur results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment.

CB2 and TRPV1 receptors oppositely modulate in vitro human osteoblast activity

In the current study, we have investigated the effect of CB2 and TRPV1 receptor ligands on in vitro osteoblasts from bone marrow of human healthy donors. A pivotal role for the endocannabinoid/endovanilloid system in bone metabolism has been highlighted. We have demonstrated a functional cross-talk between CB2 and TRPV1 in human osteoclasts, suggesting these receptors as new pharmacological target for the treatment of bone resorption disease as osteoporosis. Moreover, we have shown the presence of these receptors on human mesenchimal stem cells, hMSCs. Osteoblasts are mononucleated cells originated from hMSCs by the essential transcription factor runt-related transcription factor 2 and involved in bone formation via the synthesis and release of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin. For the first time, we show that CB2 and TRPV1 receptors are both expressed on human osteoblasts together with enzymes synthesizing and degrading endocannabinoids/endovanilloids, and oppositely modulate human osteoblast activity in culture in a way that the CB2 receptor stimulation improves the osteogenesis whereas TRPV1 receptor stimulation inhibits it.

Mesenchymal stromal cells and chronic inflammatory bowel disease

Recent experimental findings have shown the ability of mesenchymal stromal cells (MSCs) to home to damaged tissues and to produce paracrine factors with anti-inflammatory properties, potentially resulting in reduction of inflammation and functional recovery of the damaged tissues. Prompted by these intriguing properties and on the basis of encouraging preclinical data, MSCs are currently being studied in several immune-mediated disorders. Inflammatory bowel diseases (IBD) represent a setting in which MSCs-based therapy has been extensively investigated. Phase I and II studies have documented the safety and feasibility of MSCs. However, efficacy results have so far been conflicting. In this review, we will discuss the biologic rationale that makes MSCs a promising therapeutic tool for IBD, and analyze recent experimental and clinical findings, highlighting current limitations and future perspectives of MSCs-related immunotherapy for IBD.

Human mesenchymal stromal cells primed with paclitaxel, apart from displaying anti-tumor activity, maintain their immune regulatory functions in vitro

Recently, Pessina et al. have shown that human mesenchymal stromal cells (MSCs) loaded ex vivo with the anti-cancer drug paclitaxel (PTX) acquire potent anti-tumor activity that is displayed in vitro and in animal models against both solid tumor and leukemia cell lines (1,2). This phenomenon is mediated through the uptake of PTX by MSCs during ex vivo co-culture and subsequent release of the drug in the culture supernatant. The authors conclude that PTX-primed MSCs (MSCsPTX) might become a new therapeutic tool to deliver chemotherapeutic agents in patients with solid cancers and leukemia. MSCs have already been successfully used in the context of hematopoietic stem cell transplantation (HSCT) for both promoting engraftment of donor cells and for treating acute graft-versus-host disease (3e5). The possibility that, besides their function as immune regulators/anti-inflammatory agents, MSCs can simultaneously release a chemotherapeutic agent that may help prevent re-growth of tumor cells in a patient undergoing HSCT for a malignant disorder is attractive. In this perspective, the question of whether MSCs maintain their peculiar immune regulatory properties after priming with PTX arises as crucial. Here, we report on the ability of MSCsPTX to suppress in vitro peripheral blood mononuclear cell

Biological and functional characterization of bone marrow-derived mesenchymal stromal cells from patients affected by primary immunodeficiency

Mesenchymal stromal cells (MSCs) represent a key component of bone marrow (BM) microenvironment and display immune-regulatory properties. We performed a detailed analysis of biological/functional properties of BM-MSCs derived from 33 pediatric patients affected by primary immune-deficiencies (PID-MSCs): 7 Chronic Granulomatous Disease (CGD), 15 Wiskott-Aldrich Syndrome (WAS), 11 Severe Combined Immunodeficiency (SCID). Results were compared with MSCs from 15 age-matched pediatric healthy-donors (HD-MSCs). Clonogenic and proliferative capacity, differentiation ability, immunophenotype, immunomodulatory properties were analyzed. WB and RT-qPCR for CYBB, WAS and ADA genes were performed. All PID-MSCs displayed clonogenic and proliferative capacity, morphology and immunophenotype comparable with HD-MSCs. PID-MSCs maintained the inhibitory effect on T- and B-lymphocyte proliferation, except for decreased inhibitory ability of SCID-MSCs at MSC:PBMC ratio 1:10. While HD- and CGD-MSCs were able to inhibit monocyte maturation into immature dendritic cells, in SCID- and WAS-MSCs this ability was reduced. After Toll-like Receptor priming, PID-MSCs displayed in vitro an altered gene expression profile of pro- and anti-inflammatory soluble factors. PID-MSCs displayed lower PPARγ levels and WAS- and SCID-MSCs higher levels of key osteogenic markers, as compared with HD-MSCs. Our results indicate that PID-MSCs may be defective in some functional abilities; whether these defects contribute to disease pathophysiology deserves further investigation.