Federico Mosna

Human Bone Marrow and Adipose Tissue Mesenchymal Stem Cells: A User's Guide

Mesenchymal stem cells (MSCs) are adult stem cells that hold great promise in the field of regenerative medicine. They can be isolated from almost any tissue of the body and display, after expansion, very similar properties and minor differences, probably due to their microenvironment of origin. Expansion in vitro can be obtained in cytokine-free, serum-enriched media, as well as in serum-free, basic fibroblast growth factor-enriched media. A detailed immunophenotypic analysis is required to test the purity of the preparation, but no unique distinguishing marker has been described as yet. Functional assays, that is, differentiation studies in vitro, are needed to prove multilineage differentiation of expanded cells, and demonstration of pluripotency is necessary to identify most immature precursors. MSCs show powerful immunomodulative properties toward most of the cells of the immune system: this strengthens the theoretical rationale for their use also in an allogeneic setting across the major histocompatibility complex (MHC) immunological barriers. Systemic intravenous injection and local use have been tried: after systemic injection, MSCs show a high degree of chemotaxis based on pro-inflammatory cytokines, and localize at inflamed and neoplastic tissues; local regeneration has been improved using synthetic, as well as organic scaffolds. On the other hand, inadequate heterotopic in vivo differentiation and neoplastic transformation are potential risks of this form of cell therapy, even if evidence of this sort has been collected only from studies in mice, and generally after prolonged in vitro expansion. This review tries to provide a detailed technical overview of the methods used for human bone-marrow (BM)-derived and adipose-tissue (AT)-derived MSC isolation, in vitro expansion, and characterization for tissue repair. We chose to use BM-MSCs as a model to describe techniques that have been used for MSC isolation and expansion from very different sources, and AT-MSCs as an example of a reliable and increasingly common alternative source.

Toll‐Like Receptor‐3‐Activated Human Mesenchymal Stromal Cells Significantly Prolong the Survival and Function of Neutrophils

Bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) are stromal precursors endowed with extensive immunomodulative properties. In this study, we aimed to assess whether Toll‐like receptor‐3 (TLR3)‐ and TLR4‐activated BM‐MSC influence human neutrophil (PMN) responses under coculture conditions. We show that TLR3 triggering by polyinosinic:polycytidylic acid dramatically amplifies, in a more significant manner than TLR4 triggering by lipopolysaccharide, the antiapoptotic effects that resting BM‐MSC constitutively exert on PMN under coculture conditions, preserving a significant fraction of viable and functional PMN up to 72 hours. In addition, TLR3‐ and TLR4‐activated BM‐MSC enhance respiratory burst ability and CD11b expression by PMN. The coculture in the absence of cell contact and the incubation of PMN in supernatants harvested from TLR3‐ and TLR4‐activated BM‐MSC yield comparable results in terms of increased survival and immunophenotypic changes, thus suggesting the involvement of endogenous soluble factors. Neutralizing experiments reveal that the biological effects exerted on PMN by TLR3‐activated BM‐MSC are mediated by the combined action of interleukin 6, interferon‐β (IFN‐β), and granulocyte macrophage colony‐stimulating factor (GM‐CSF), while those exerted by TLR4‐activated BM‐MSC mostly depend on GM‐CSF. MSC isolated from thymus, spleen, and subcutaneous adipose tissue behaves similarly. Finally, the effects exerted by TLR3‐ or TLR4‐stimulated BM‐MSC on PMN are conserved even after the previous priming of BM‐MSC with IFN‐γ and tumor necrosis factor‐α. Our data highlight a novel mechanism by which MSC sustain and amplify the functions of PMN in response to TLR3‐ and TLR4‐triggering and may consequently contribute to inflammatory disorders. STEM CELLS 2011;29:1001–1011

Notch-3 and Notch-4 signaling rescue from apoptosis human B-ALL cells in contact with human bone marrow―derived mesenchymal stromal cells

Although many literature data are available on the role of Notch signaling in T-cell acute lymphoblastic leukemia (ALL) biology, the importance of this molecular pathway in the development of B-lineage ALL (B-ALL) cells in the BM microenvironment is unknown so far. In this study, we used anti-Notch molecules neutralizing Abs and γ-secretase inhibitor (GSI) XII to investigate the role of the Notch signaling pathway in the promotion of human B-ALL cell survival in presence of stromal cell support. The treatment with combinations of anti-Notch molecule neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of stromal cells from normal donors and B-ALL patients. Interestingly, the inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. Our data suggest that the stromal cell-mediated antiapoptotic effect on B- ALL cells is mediated by Notch-3 and -4 or Jagged-1/-2 and DLL-1 in a synergistic manner.

IFN-γ-mediated upmodulation of MHC class I expression activates tumor-specific immune response in a mouse model of prostate cancer

De novo expression of B7-1 impaired tumorigenicity of TRAMP-C2 mouse prostate adenocarcinoma (TRAMP-C2/B7), but it did not elicit a protective response against TRAMP-C2 parental tumor, unless after in vitro treatment with IFN-gamma. TRAMP-C2 cells secrete TGF-beta and show low MHC-I expression. Treatment with IFN-gamma increased MHC-I expression by induction of some APM components and antagonizing the immunosuppressant activity of TGF-beta. Thus, immunization with TRAMP-C2/B7 conferred protection against TRAMP-C2-derived tumors in function of the IFN-gamma-mediated fine-tuned modulation of either APM expression or TGF-beta signaling. To explore possible clinical translation, we delivered IFN-gamma to TRAMP-C2 tumor site by means of genetically engineered MSCs secreting IFN-gamma.

Complex karyotype, older age, and reduced first-line dose intensity determine poor survival in core binding factor acute myeloid leukemia patients with long-term follow-up

Approximately 40% of patients affected by core binding factor (CBF) acute myeloid leukemia (AML) ultimately die from the disease. Few prognostic markers have been identified. We reviewed 192 patients with CBF AML, treated with curative intent (age, 15–79 years) in 11 Italian institutions. Overall, 10‐year overall survival (OS), disease‐free survival (DFS), and event‐free survival were 63.9%, 54.8%, and 49.9%, respectively; patients with the t(8;21) and inv(16) chromosomal rearrangements exhibited significant differences at diagnosis. Despite similar high complete remission (CR) rate, patients with inv(16) experienced superior DFS and a high chance of achieving a second CR, often leading to prolonged OS also after relapse. We found that a complex karyotype (i.e., ≥4 cytogenetic anomalies) affected survival, even if only in univariate analysis; the KIT D816 mutation predicted worse prognosis, but only in patients with the t(8;21) rearrangement, whereas FLT3 mutations had no prognostic impact. We then observed increasingly better survival with more intense first‐line therapy, in some high‐risk patients including autologous or allogeneic hematopoietic stem cell transplantation. In multivariate analysis, age, severe thrombocytopenia, elevated lactate dehydrogenase levels, and failure to achieve CR after induction independently predicted longer OS, whereas complex karyotype predicted shorter OS only in univariate analysis. The achievement of minimal residual disease negativity predicted better OS and DFS. Long‐term survival was observed also in a minority of elderly patients who received intensive consolidation. All considered, we identified among CBF AML patients a subgroup with poorer prognosis who might benefit from more intense first‐line treatment. Am. J. Hematol. 90:515–523, 2015.

Minimal Residual Disease in Acute Myeloid Leukemia: Still a Work in Progress?

Minimal residual disease evaluation refers to a series of molecular and immunophenotypical techniques aimed at detecting submicroscopic disease after therapy. As such, its application in acute myeloid leukemia has greatly increased our ability to quantify treatment response, and to determine the chemosensitivity of the disease, as the final product of the drug schedule, dose intensity, biodistribution, and the pharmakogenetic profile of the patient. There is now consistent evidence for the prognostic power of minimal residual disease evaluation in acute myeloid leukemia, which is complementary to the baseline prognostic assessment of the disease. The focus for its use is therefore shifting to individualize treatment based on a deeper evaluation of chemosensitivity and residual tumor burden. In this review, we will summarize the results of the major clinical studies evaluating minimal residual disease in acute myeloid leukemia in adults in recent years and address the technical and practical issues still hampering the spread of these techniques outside controlled clinical trials. We will also briefly speculate on future developments and offer our point of view, and a word of caution, on the present use of minimal residual disease measurements in “real-life” practice. Still, as final standardization and diffusion of the methods are sorted out, we believe that minimal residual disease will soon become the new standard for evaluating response in the treatment of acute myeloid leukemia.

Efficacy Assessment of Interferon-Alpha–Engineered Mesenchymal Stromal Cells in a Mouse Plasmacytoma Model

Bone marrow mesenchymal stromal cells (BM-MSCs) may survive and proliferate in the presence of cycling neoplastic cells. Exogenously administered MSCs are actively incorporated in the tumor as stromal fibroblasts, thus competing with the local mesenchymal cell precursors. For this reason, MSCs have been suggested as a suitable carrier for gene therapy strategies, as they can be genetically engineered with genes encoding for biologically active molecules that can inhibit tumor cell proliferation and enhance the antitumor immune response. We used BM-MSCs engineered with the murine interferon-alpha (IFN-α) gene (BM-MSCs/IFN-α) to assess in a mouse plasmacytoma model the efficacy of this approach toward neoplastic plasma cells. We found that IFN-α can be efficiently produced and delivered inside the tumor microenvironment. Subcutaneous multiple administration of BM-MSCs/IFN-α significantly hampered the tumor growth in vivo and prolonged the overall survival of mice. The antitumor effect was associated with enhanced apoptosis of tumor cells, reduction in microvessel density, and ischemic necrosis. By contrast, intravenous administration of BM-MSCs/IFN-α did not significantly modify the survival of mice, mainly as a consequence of an excessive entrapment of injected cells in the pulmonary vessels. In conclusion, BM-MSCs/IFN-α are effective in inhibiting neoplastic plasma cell growth; however, systemic administration of engineered MSCs needs to be improved to make this approach potentially suitable for the treatment of multiple myeloma.

Cell therapy for cardiac regeneration after myocardial infarct: which cell is the best?

In the last decade several attempts have been made to achieve the goal of cardiac regeneration after myocardial infarction. To date, two cell types have completed phase-III clinical trials: Skeletal Myoblasts and Bone-Marrow Mononuclear Cells (BM-MNCs). In the first case, all benefits have been limited by an increased risk of arrhythmia. In the case of BM-cells, most studies showed a significant, although limited, advantage in the cell-treated group. This may be due to the choice of the wrong BM cell type: other candidates would be e.g. CD34(+) HSCs, or non-hematopoietic Mesenchymal Stem Cells. After positive results from the experimental studies, phase I/II clinical trials are currently on-going for both. Ideally, the best cell to use to regenerate the heart would be a precursor of all cardiac lineages; until the isolation and expansion of Cardiac Stem Cells (CSCs), such a cell was thought to exist only during embryogenesis. Using CSCs researchers managed to generate electrically-coupled contractile tissue within the infarct of animal models. Still, some doubts persist over the possibility to translate such results in real-life patients. Another approach, therefore, involves the use of induced Pluripotent Stem Cells (iPS) obtained from fibroblasts after genetic reprogramming. This new type of cell would combine the pluripotency of embryonal stem cells with the advantages of an autologous use. Nevertheless, iPS cells form teratomas, and their effective differentiation in vivo is largely unknown. This review will critically compare the data from the Literature concerning cell therapy after myocardial infarction. Can we name the best cell?

Clinical outcome of myeloid sarcoma in adult patients and effect of allogeneic stem cell transplantation. Results from a multicenter survey

INTRODUCTION Myeloid Sarcoma (MS) is a rare hematologic myeloid neoplasm that can involve any site of the body. It can occur as an exclusively extramedullary form or it can be associated with an acute myeloid leukemia (AML), a chronic myeloproliferative neoplasm (MPN) or a myelodysplastic syndrome (MDS) at onset or at relapse. The rarity of MS does not enable prospective clinical trials and therefore a specific multicenter register can be useful for the clinical and biological studies of this rare disease. PATIENTS AND RESULTS we report the clinical characteristics and outcome of 48 histologically confirmed MS, diagnosed and treated in 9 Italian Hematological Centers in the last 10 years. The patients median age was 46 years. There were 9/48 de novo extramedullary MS, 24/48 de novo AML-related MS and 15/48 were secondary AML-related MS. The most common extramedullary anatomic sites of disease were: skin, lymph nodes and soft tissues. Forty-three patients (90%) underwent a program of intensive chemotherapy including FLAI, HDAC-IDA, HyperCVAD and MEC schemes, with a DDI of 5% and a CR Rate of 45%. Twenty-two (46%) patients underwent Allogeneic SCT, 13 from a MUD, 8 from an HLA-identical sibling donor and 1 from an haploidentical donor. The median OS of the whole population (48 pts) was 16.7 months. The OS probability at 1, 2 and 5 years was 64%, 39% and 33%, respectively. The OS was better in patients that underwent an intensive therapeutic program (median OS: 18 months vs 5 months). Among the intensively treated patients, in univariate analysis, the OS was better in young patients (P=0,008), in patients that underwent Allo-SCT (P=0,009) and in patients that achieved a CR during treatment (P=0,001), and was worse in pts with secondary AML-related MS (P=0,007). Age, response to intensive chemotherapy and Allo-SCT were the only three variables that significantly influenced DFS (P=0,02, P=0,01 and P=0,04, respectively). In multivariable analysis, Allo-SCT and response to intensive chemotherapy remained significant in predicting a better OS (P=0,04 and P=0,001, respectively), and response to intensive chemotherapy was the only significant variable in predicting DFS (P=0,01). After Allo-SCT we observe a survival advantage in patients who achieved a pre-transplant CR (P=0,008) and in those who developed a chronic GvHD (P=0,05). CONCLUSIONS Patients with MS, both with de novo and secondary forms, still have a very unfavorable outcome and require an intensive therapeutic program, that includes Allo-SCT whenever possible. The outcome after Allo-SCT is positively influenced by the development of chronic GvHD suggesting a Graft versus MS effect.

A proliferation-inducing ligand (APRIL) serum levels predict time to first treatment in patients affected by B-cell chronic lymphocytic leukemia.

Purpose:  A proliferation‐inducing ligand (APRIL), a tumor necrosis factor superfamily member involved in B‐lymphocytes differentiation and survival, plays a role in protecting B‐Cell Chronic lymphocytic leukemia (B‐CLL) cells from apoptosis. Having observed that APRIL serum (sAPRIL) levels were higher in B‐CLL patients with CLL at diagnosis as compared to healthy donors (14.61 ± 32.65 vs. 4.19 ± 3.42 ng/mL; P < 0.001), we tested the correlation existing in these patients between sAPRIL, clinical–biological parameters and disease progression. Experimental design: sAPRIL levels were measured by ELISA in 130 patients with B‐CLL at diagnosis and in 25 healthy donors. Results: sAPRIL levels did not correlate with gender, age, clinical stage, blood cell counts, β2‐microglobulin (β2M) levels, ZAP‐70 and CD38 expression. Using median sAPRIL natural logarithm (ln) as cutoff, we distinguished two groups of patients (APRILLOW and APRILHIGH) who were comparable with regard to clinical–biological parameters and overall survival, but different with regard to time to the first treatment (TTFT; P = 0.035). According to univariate analysis, high lymphocyte count, high β2M, Binet stage B–C, ZAP‐70 expression and ln(sAPRIL) above median were associated with earlier TTFT. Advanced clinical stage, high β2M, ZAP‐70 expression and ln(sAPRIL) above median remained independently predictive of shorter TTFT at multivariate analysis. Moreover, sAPRIL increased its prognostic significance when patients were stratified according to independent favorable clinical–biological characteristics (low β2M, stage A and lack of ZAP‐70 expression). Conclusions: sAPRIL is a novel indicator of shorter TTFT in B‐CLL and a predictor of progression especially in patients otherwise considered at low risk according to validated prognostic factors.