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.

Mesenchymal stromal cell therapy: a revolution in Regenerative Medicine?

Because of their immunomodulatory and engraftment-promoting properties, mesenchymal stromal cells (MSCs) have been tested in the clinical setting both to facilitate haematopoietic recovery and to treat steroid-resistant acute GVHD. More recently, experimental findings and clinical trials have focused on the ability of MSCs to home to damaged tissue and to produce paracrine factors with anti-inflammatory properties, resulting in functional recovery of the damaged tissue. The mechanisms through which MSCs exert their therapeutic potential rely on some key properties of the cells: the ability to secrete soluble factors capable of stimulating survival and recovery of injured cells; the capacity to home to sites of damage and the ability to blunt exaggerated immune responses. These fundamental properties are being tested within a novel therapeutic field defined as Regenerative Medicine. This review deals with recent research on the anti-inflammatory/reparative properties of MSCs and considers the possible mechanisms of function responsible for these effects. Moreover, current and potential clinical applications of MSC-based treatment strategies in the context of Regenerative Medicine are being discussed. Key issues such as optimal timing of MSC administration, cell dose and schedule of administration, advantages and disadvantages of using autologous or allogeneic cells are still open. Nonetheless, MSCs promise to represent a revolution for many severe or presently untreatable disorders.

Mesenchymal stromal cells: a novel and effective strategy for facilitating engraftment and accelerating hematopoietic recovery after transplantation?

MSCs are multipotent cells that can be isolated from several human tissues and expanded ex vivo for clinical use. They comprise a heterogeneous population of cells, which, through production of growth factors, cell-to-cell interactions and secretion of matrix proteins, has a role in the regulation of hematopoiesis. In recent years, several experimental studies have shown that MSCs are endowed with immunomodulatory properties and with the capacity to promote graft survival in animal models. In view of these properties, MSCs have been tested in pilot studies aimed at preventing/treating graft rejection and at accelerating recovery after hematopoietic cell transplantation (HCT). The available clinical evidence deriving from these studies indicates that MSC infusion is safe and promising in terms of capacity of preventing graft failure. More debated is the effect of MSCs for what concerns their capacity of accelerating hematopoietic reconstitution after HCT. Whether the favorable effect of MSCs largely depends on the type of transplantation remains also a field of future investigation. Moreover, future researches should be oriented to gain more insights on MSC biological and functional mechanisms relevant for exploiting their use in the modulation of alloreactivity and in the promotion of hematopoietic reconstitution.

Immunomodulatory properties of porcine, bone marrow-derived multipotent mesenchymal stromal cells and comparison with their human counterpart.

Thanks to their immunonodulatory properties, multipotent mesenchymal stromal cells (MSCs) are a promising strategy for preventing/reducing the risk of graft rejection after hematopoietic cell and solid organ transplantation. We have previously demonstrated that porcine MSCs (pMSCs) can be isolated from bone marrow and display similar morphology and differentiative capacity as compared to human MSC (hMSCs). In this study, we investigated the in vitro immunomodulatory properties (namely the ability to suppress lymphocyte proliferation in response to phytohemagglutinin and the cytokine production in the culture supernatants) of pMSCs from six Large White 6-month old piglets. Similarly to hMSCs, pMSCs reduced the phytohemagglutinin-induced lymphocyte proliferation. High levels of IL-6 were found in culture supernatants, whereas IL-10 and TGF-ß were not detectable. In conclusion, ex vivo expanded pMSCs share selected biological/functional properties with hMSCs. pMSCs may be used in in vivo models to investigate novel approaches of prevention of graft rejection in solid organ transplantation.

Co-infusion of ex vivo-expanded, parental MSCs prevents life-threatening acute GVHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation.

When compared with BMT, umbilical cord blood transplantation (UCBT) is associated with a lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of TRM in the early post transplantation period due to infection. Acute GVHD, although occurring less frequently in UCBT compared with BMT, is also significantly associated with increased rate of early TRM. BM MSCs are known to support normal in vivo hematopoiesis, and co-transplantation of MSCs has been shown to enhance engraftment of human cord blood hematopoietic cells in nonobese diabetic/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA-disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared with 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III and IV acute GVHD was significantly decreased in the study cohort when compared with controls (P=0.05), thus resulting in reduced early TRM. Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly because of their immunosuppressive effect, may abrogate life-threatening acute GVHD and reduce early TRM.

Isolation and ex vivo expansion of bone marrow-derived porcine mesenchymal stromal cells: potential for application in an experimental model of solid organ transplantation in large animals.

Pharmacological aspecific immunosuppression, despite being widely used in solid organ transplantation recipients, is unable to completely prevent allograft rejection. It promotes the occurrence of sometimes life-threatening infections. Due to their immunosuppressive and anti- inflammatory properties, there is great interest in the therapeutic use of bone marrow (BM)-derived mesenchymal stromal cells (MSC). Large animal models play a crucial role to investigate the biological and functional properties of MSCs as novel cellular therapy. In the current study we sought to isolate expand ex vivo, and phenotypically characterize MSC derived from BM of 4 Large White 6-month-old piglets. Porcine MSC (pMSC) were characterized for their in vitro differentiation capacity. pMSC were successfully isolated from all BM samples. They showed spindle-shaped morphology and a stable doubling time on culture. They were positive for CD90, CD29, CD105, and negative for CD45 and CD11b. Furthermore, they differentiated, upon specific in vitro conditions toward adipogenic and osteogenic lineages. The optimization of methods for the isolation and characterization of pMSC may be useful to elucidate their biological and functional properties. The anatomy and physiology of the pig, which is similar to humans, make this animal model more attractive than small animals to test the safety and efficacy of MSC in the context of solid organ transplantation.

Assessment of human herpesvirus-6 infection in mesenchymal stromal cells ex vivo expanded for clinical use.

Infection or reactivation of human herpesvirus (HHV)-6 represents a potentially serious complication (often involving the central nervous system) in patients receiving either solid organ or hematopoietic stem cell transplantation. The objective of this study was to assess the risk of HHV-6 infection/reactivation in mesenchymal stromal cells (MSCs). MSCs are multipotent cells displaying immunomodulatory properties that have been already successfully used in the clinical setting to enhance hematopoietic stem cell engraftment and to treat steroid-refractory acute graft-versus-host disease. We analyzed 20 samples of ex vivo expanded MSCs, at different passages of culture, isolated both from bone marrow and from umbilical cord blood. Through Western blotting and immunocytochemistry techniques, we investigated the presence of the HHV-6 receptor (CD46) on cell surface, whereas the presence of HHV-6 DNA was evaluated by nested polymerase chain reaction assay. All of the MSC samples tested were positive for the virus receptor (CD46), suggesting their potential susceptibility to HHV-6. However, none of the MSC samples derived from cultures, performed in the perspective of clinical use, was found to harbor HHV-6. This preliminary observation on a consistent number of MSC samples, some of them tested at late in vitro passages, indicates a good safety profile of the product in terms of HHV-6 contamination. Nevertheless, it remains important to set up in vitro experimental models to study MSCs' susceptibility to HHV-6 (and HHV-7) infection, to verify their capacity to integrate the virus into cellular DNA, and to investigate which experimental conditions are able to induce virus reactivation.

Potential role of mesenchymal stromal cells in pediatric hematopoietic SCT.

Mesenchymal stromal cells (MSCs) can be isolated from several human tissues and expanded for clinical use. MSCs are identified by phenotypic and functional characteristics, and are poor Ag-presenting cells not expressing MHC class II or co-stimulatory molecules. MSCs have potent immune-modulatory effects and in vitro induce a more anti-inflammatory or tolerant phenotype. Clinical studies have exploited both the immune-modulatory properties of MSCs as well as their hematopoietic supportive role. MSCs have been safely administered for the treatment of severe steroid refractory GVHD. A phase I/II multicenter study included 25 children in whom 80% responded to either one or two infusions of MSCs derived mainly from third party donors. Twenty children have undergone co-transplantation of haploidentical MSCs with PBSC in a phase I/II study, which has overcome the problems of graft failure in HLA-disparate grafts. Similarly, co-transplantation of MSCs and cord blood stem cells is under investigation. MSCs may have important future potential for the treatment of pediatric autoimmune disease as well as inborn errors such as osteogenesis imperfecta. Currently, much needed randomized studies under the auspices of the EBMT are ongoing to determine the optimal use of these exciting new modalities of treatment.

Human mesenchymal stem cells derived from bone marrow display a better chondrogenic differentiation compared with other sources.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into several mesodermal lineages. These cells have been isolated from various tissues, such as adult bone marrow, placenta, and fetal tissues. The comparative potential of these cells originating from different tissues to differentiate into the chondrogenic lineage is still not fully defined. The aim of our study was to investigate the chondrogenic potential of MSCs isolated from different sources. MSCs from fetal and adult tissues were phenotypically characterized and examined for their differentiation capacity, based on morphological criteria and expression of extracellular matrix components. Our results show that both fetal and adult MSCs have chondrogenic potential under appropriate conditions. The capacity of bone marrow-derived MSCs to differentiate into chondrocytes was reduced on passaging of cells. MSCs of bone marrow origin, either fetal or adult, exhibit a better chondrogenesis than fetal lung- and placenta-derived MSCs, as demonstrated by the appearance of typical morphological features of cartilage, the intensity of toluidine blue staining, and the expression of collagen type II, IX, and X after culture under chondrogenic conditions. As MSCs represent an attractive tool for cartilage tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for these therapeutic approaches.

Preemptive therapy of EBV-related lymphoproliferative disease after pediatric haploidentical stem cell transplantation.

The treatment of Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell transplantation (HSCT) is still unsatisfactory. We conducted a prospective trial to evaluate the impact of routine EBV surveillance and preemptive treatment with the anti-CD20 monoclonal antibody rituximab on the development of PTLD in pediatric recipients of extensively T-cell depleted HSCT from an HLA-haploidentical relative. Twenty-seven patients were included in the surveillance program, 12 developed EBV DNA positivity, with 8 of 12 presenting with sustained viral DNA levels requiring treatment with rituximab. Treatment was well tolerated, and induced clearance of EBV DNA in all patients. However, 4/8 patients showed a new increase in EBV load, coincident with the emergence of CD20(-)/CD19(+) B cells in peripheral blood, accompanied by overt PTLD in 3 patients. The latter cleared PTLD after receiving donor EBV-specific cytotoxic T-lymphocytes (CTLs), and persist in remission at a median 30-month follow-up. EBV-specific T-cell frequency, undetectable at time of EBV DNA positivity, was restored by T-cell therapy to levels comparable with controls. We conclude that preemptive therapy with rituximab is safe, but only partly effective in haplo-HSCT recipients. Patients who progress to PTLD under rituximab treatment can be rescued permanently by infusion of EBV-specific CTLs.

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.

Rapid control of two outbreaks of Serratia marcescens in a Northern Italian neonatal intensive care unit.

Serratia marcescens is a recognized cause of outbreaks in neonatal intensive care units (NICUs). The aim of the present study was to investigate two nosocomial outbreaks of S. marcescens that occurred in an NICU in Northern Italy. In order to determine the origin of the outbreaks and the associated morbidity and mortality of S. marcescens infections and epidemiological investigation was established including molecular typing of the isolates. Containment of the outbreaks was achieved by means of strict hygienic measure and cohort nursing of the infected and/or colonized infants. We experimented with the use of probiotics as an infection control measure.

CSF electrophoresis: an adaptation using cellulose acetate for the identification of oligoclonal banding.

An adaptation of cellulose acetate electrophoresis for studying concentrated cerebrospinal fluid is described. Two hundred and twenty-one patients have been studied, and the specificity for multiple sclerosis and sub-acute sclerosing panencephalitis is discussed. This has been positive for oligoclonal banding (OB) in 79% of patients with clinically definite multiple sclerosis.