Roberta Tasso

Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway.

Bone marrow mesenchymal progenitor cells (BMSC) are used for regenerating tissues of mesodermal origin, as well as tissues of different embryological derivation. Experimental evidence shows that BMSC are able to suppress the activation of the immune response by mechanisms that are still not completely understood. Thus far, in vitro studies carried using human or mouse cells indicate that autologous or allogeneic BMSC strongly suppress proliferation of T lymphocytes, triggered by cellular stimuli, nonspecific mitogenic stimuli, or antigenic peptides. Using cell proliferation and blocking assays, we demonstrated that BMSC inhibited the activation of murine splenocytes, T, and B lymphocytes. Direct contact of BMSC and target cells in a cognate fashion determined the inhibition of cell proliferation via engagement of the inhibitory molecule programmed death 1 (PD‐1) to its ligands PD‐L1 and PD‐L2, leading the target cells to modulate the expression of different cytokine receptors and transduction molecules for cytokine signaling. Soluble factors present on supernatants of BMSC cultures were effective in suppressing proliferation of B cells to a mitogenic stimulus. Taken together, these results highlight the complexity of the role of BMSC in regulating the immune response, asserting the possibility of their therapeutic application in transplantation and autoimmune diseases.

The development of tissue-engineered bone of different origin through endochondral and intramembranous ossification following the implantation of mesenchymal stem cells and osteoblasts in a murine model

The study of host cell recruitment by implanted exogenous cells is one of the novel challenges in tissue engineering. We previously reported the development of tissue-engineered bone deposited by host cells in porous ceramic scaffolds seeded with murine mesenchymal stem cells (MSC) and implanted in immunocompromised mice. To better highlight the contribution of host cells to the development of the engineered tissue and to investigate whether the capacity to recruit host cells was dependent on the donor cell commitment, we implanted ceramic scaffolds seeded with either murine GFP labeled MSC or GFP labeled osteoblasts (OB) into immunocompromised mice. Although we observed formation of bone in all scaffolds, the origin of bone cells and the ossification type were strictly dependent on the nature and commitment of the seeded cells. MSC implants led to formation of bone of host origin through the activation of an endochondral ossification process while an intramembranous ossification directly performed by the seeded cells was observed in OB implants. Moreover, we observed an increased vascularization in MSC implants due to the higher capacity of MSC to recruit host CD31+ endothelial cells. The relationship between this enhanced vascularization and the type of ossification is discussed.

Development of sarcomas in mice implanted with mesenchymal stem cells seeded onto bioscaffolds

Bone marrow-derived mesenchymal stem cells (MSCs) are precursors of bone, cartilage and fat tissue. MSC can also regulate the immune response. For these properties, they are tested in clinical trials for tissue repair in combination with bioscaffolds or injected as cell suspension for immunosuppressant therapy. Experimental data, however, indicate that MSC can undergo or induce a tumorigenic process in determined circumstances. We used a modified model of ectopic bone formation in mice by subcutaneously implanting porous ceramic seeded with murine MSC. In this new model, host-derived sarcomas developed when we implanted MSC/bioscaffold constructs into syngeneic and immunodeficient recipients, but not in allogeneic hosts or when MSCs were injected as cell suspensions. The bioscaffold provided a tridimensional support for MSC to aggregate, thus producing the stimulus for triggering the process eventually leading to the transformation of surrounding cells and creating a surrogate tumor stroma. The chemical and physical characteristics of the bioscaffold did not affect tumor formation; sarcomas developed either when a stiff porous ceramic was used or when the scaffold was a smooth collagen sponge. The immunoregulatory function of MSC contributed to tumor development. Implanted MSC expanded clones of CD4+CD25+ T regulatory lymphocytes that suppressed hosts antitumor immune response.

The recruitment of two consecutive and different waves of host stem/progenitor cells during the development of tissue-engineered bone in a murine model.

Angiogenesis plays a central role in bone regeneration, not only for the transport of nutrients, but also for locally directing skeletal stem/progenitor cells. Following ectopic implantation of porous ceramic cubes seeded with mouse GFP-labeled mesenchymal stem cells (MSC) into syngenic mice, we investigated the cascade of events leading to bone formation. Implants harvested at different times were enzymatically digested to generate single-cell suspensions. Recovered cells were sorted to separate GFP+implanted MSC and host recruited GFP- cells. We isolated and characterized two different waves of cells, migrating from the host to the MSC-seeded ceramic. The first migrated cell population, recovered 7 days after implantation, was enriched in CD31+endothelial progenitors, while the second one, recruited at day 11, was enriched in CD146+pericyte-like cells. Both populations were not recruited into the scaffold following implantation of a non-MSC seeded ceramic. Pericyte-like cell mobilization was dependent on the first migrated endothelial cell population. Pericyte-like cells retained properties distinctive of stem cells, such as capacity of performing a high number of in vitro cell divisions and showed an osteogenic potential. Studies on the cross talk between implanted exogenous MSC and resident stem/progenitor cells could open new perspectives for future clinical applications.

Recruitment of a Host's Osteoprogenitor Cells Using Exogenous Mesenchymal Stem Cells Seeded on Porous Ceramic

The contribution of the hosts circulating progenitor cells after implantation of mesenchymal stem cells (MSC)/bioscaffold combinations for repairing bone defects has not been elucidated, although this issue affects the clinical application of the tissue engineering approach. We implanted blocks of hydroxyapatite loaded with murine MSCs into syngenic, allogenic, and immunocompromised recipients. After 8 weeks, we found that bone tissue was formed in syngenic and immunocompromised animals. The implanted cells appeared pivotal in the early stages of tissue development, but cells of the recipients origin finally made bone. In this system, osteoprogenitors migrated from the recipient to the implant, whereas the implanted cells left the scaffold and entered the circulatory flow. We observed rapid destruction of implanted cells when allogenic MSC/bioscaffold combinations were grafted onto immunocompetent recipients without immunosuppressant therapy. This destruction blocked the recruitment process and did not allow the formation of new bone tissue. The possibility that the implanted exogenous MSCs could engage the hosts osteoprogenitor cells to form new bone tissue could open new perspectives for the tissue engineering approach to bone repair, including the opportunity of using allogenic cells combined with a temporary immunosuppressant therapy, stimulating the replacement of the exogenous cells with autologous cells.

Mesenchymal Stem Cells Induce Functionally Active T-Regulatory Lymphocytes in a Paracrine Fashion and Ameliorate Experimental Autoimmune Uveitis

PURPOSE Mesenchymal stem/progenitor cells (MSCs) have regenerative and immunomodulatory properties, exerted by cell-cell contact and in a paracrine fashion. Part of their immunosuppressive activity has been ascribed to their ability to promote the induction of CD4+CD25+FoxP3+ T lymphocytes with regulatory functions (Treg). Here the authors studied the effect of MSCs on the induction of Treg and on the development of autoimmunity, and they examined the possibility that MSC-mediated Treg induction could be attributed to the secretion of soluble factors. METHODS The authors induced experimental autoimmune uveitis (EAU) in mice by immunization with the 1-20 peptide of the intraphotoreceptor binding protein. At the same time, some of the animals were treated intraperitoneally with syngeneic MSCs. The authors checked T-cell responses and in vitro Treg conversion by cell proliferation and blocking assays, in cell-cell contact and transwell settings. TGFβ and TGFβ receptor gene expression analyses were performed by real-time PCR. RESULTS The authors found that a single intraperitoneal injection of MSCs was able to significantly attenuate EAU and that a significantly higher percentage of adaptive Treg was present in MSC-treated mice than in MSC-untreated animals. In vitro blocking of antigen presentation by major histocompatibility complex class II precluded priming and clonal expansion of antigen-specific Treg, whereas blockade of TGFβ impaired the expression of FoxP3, preventing the conversion of CD4+ T cells into functionally active Treg. CONCLUSIONS The authors demonstrated that MSCs can inhibit EAU and that their immunomodulatory function is due at least in part to the induction of antigen-specific Treg in a paracrine fashion by secreting TGFβ.

The role of bFGF on the ability of MSC to activate endogenous regenerative mechanisms in an ectopic bone formation model

The view depicting bone marrow (BM)-derived mesenchymal stem cells (MSC) as a uniform population differentiating into new-tissue builder cells is evolving toward the concept of a heterogeneous population of stem/progenitor cells secreting bioactive molecules, and contributing to establish an on-site regenerative microenvironment. We report that in an ectopic bone formation model the intrinsic MSC capability to activate endogenous regenerative mechanisms is critically dependent on the commitment level of implanted MSC. We demonstrate that the presence of bFGF in the culture medium during mouse MSC expansion in vitro is the key factor for the selection of subpopulations inducing host regenerative responses. We developed a novel strategy combining SILAC-LC-MS/MS quantitative proteomics of conditioned culture media and gene expression profiling to disentangle the major role of MSC in modulating the microenvironment toward the damage resolution. The correspondence between results provided by the applied techniques proved that the most statistically significant biological processes favored by the bFGF treatment were carried out by secreted factors. In particular, the immune response, the inflammatory response, the response to wounding and chemotaxis were all upregulated in bFGF-selected MSC. We propose these processes as majorly involved in activating the endogenous responses triggered by trophic effects of implanted bFGF-selected MSC.

An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III‐transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.—Castelnuovo, M., Massone, S., Tasso, R., Fiorino, G., Gatti, M., Robello, M., Gatta, E., Berger, A., Strub, K., Florio, T., Dieci, G., Cancedda, R., Pagano, A. An Alu‐like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells. FASEB J. 24, 4033–4046 (2010). www.fasebj.org

Bone Turnover in Wild Type and Pleiotrophin-Transgenic Mice Housed for Three Months in the International Space Station (ISS)

Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity’s negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.

Mesenchymal stem cell-derived extracellular vesicles as mediators of anti-inflammatory effects: Endorsement of macrophage polarization

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived‐MSCs to investigate their involvement as modulators of MSC anti‐inflammatory effects inducing macrophage polarization. The EV‐isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo). Both types of EVs were efficiently internalized by responding bone marrow‐derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin‐induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV‐treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC‐EVs possess effective anti‐inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. Stem Cells Translational Medicine 2017 Stem Cells Translational Medicine 2017;6:1018–1028