Dual Stem Cell Therapy Improves the Myocardial Recovery Post-Infarction through Reciprocal Modulation of Cell Functions.

Mesenchymal stromal cells (MSC) are promising candidates for regenerative therapy of the infarcted heart. However, poor cell retention within the transplantation site limits their potential. We hypothesized that MSC benefits could be enhanced through a dual-cell approach using jointly endothelial colony forming cells (ECFC) and MSC. To assess this, we comparatively evaluated the effects of the therapy with MSC and ECFC versus MSC-only in a mouse model of myocardial infarction. Heart function was assessed by echocardiography, and the molecular crosstalk between MSC and ECFC was evaluated in vitro through direct or indirect co-culture systems. We found that dual-cell therapy improved cardiac function in terms of ejection fraction and stroke volume. In vitro experiments showed that ECFC augmented MSC effector properties by increasing Connexin 43 and Integrin alpha-5 and the secretion of healing-associated molecules. Moreover, MSC prompted the organization of ECFC into vascular networks. This indicated a reciprocal modulation in the functionality of MSC and ECFC. In conclusion, the crosstalk between MSC and ECFC augments the therapeutic properties of MSC and enhances the angiogenic properties of ECFC. Our data consolidate the dual-cell therapy as a step forward for the development of effective treatments for patients affected by myocardial infarction.

Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation.

Subcutaneous transplantation of mesenchymal stromal cells (MSC) emerged as an alternative to intravenous administration because it avoids the pulmonary embolism and prolongs post-transplantation lifetime. The goal of this study was to investigate the mechanisms by which these cells could affect remote organs. To this aim, murine bone marrow-derived MSC were subcutaneously transplanted in different anatomical regions and the survival and behaviour have been followed. The results showed that upon subcutaneous transplantation in mice, MSC formed multicellular aggregates and did not migrate significantly from the site of injection. Our data suggest an important role of hypoxia-inducible signalling pathways in stimulating local angiogenesis and the ensuing modulation of the kinetics of circulating cytokines with putative protective effects at distant sites. These data expand the current understanding of cell behaviour after subcutaneous transplantation and contribute to the development of a non-invasive cell-based therapy for distant organ protection.

A procedure for in vitro evaluation of the immunosuppressive effect of mouse mesenchymal stem cells on activated T cell proliferation.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) represent adult cells with multipotent capacity. Besides their capacity to differentiate into multiple lineages in vitro and in vivo, increasing evidence points towards the immunomodulatory capacity of these cells, as an important feature for their therapeutic power. Although not included in the minimal criteria established by the International Society for Cellular Therapy as a defining MSC attribute, demonstration of the immunomodulatory capacity of MSC can be useful for the characterization of these cells before being considered MSC. METHODS: Here we present a simple and reliable protocol by which the immunosuppressive effect of mouse bone marrow-derived MSC can be evaluated in vitro. It is based on the measuring of the proliferation of activated T cells cultured in direct contact with irradiated MSC. RESULTS: Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation, which can be quantified as a percentage of maximum proliferation. Our data shows that batch-to-batch variability can be determined within one or multiple experiments, by extracting the area under curve of T cell proliferation plotted against the absolute number of MSC in co-culture. CONCLUSIONS: The validation of the immunosupressive capacity of MSC could be added to the characterization of the cells before being used in various MSC-based approaches to treat immunological diseases. Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation. The immunosuppressive properties of MSC vary between batches, but not between different passages of the same batch.

miRNAs generated from Meg3-Mirg locus are downregulated during aging.

Aging determines a multilevel functional decline and increases the risk for cardiovascular pathologies. MicroRNAs are recognized as fine tuners of all cellular functions, being involved in various cardiac diseases. The heart is one of the most affected organs in aged individuals, however little is known about the extent and robustness to which miRNA profiles are modulated in cardiac cells during aging. This paper provides a comprehensive characterization of the aging-associated miRNA profile in the murine cardiac fibroblasts, which are increasingly recognized for their active involvement in the cardiac physiology and pathology. Next-generation sequencing of cardiac fibroblasts isolated from young and old mice revealed that an important fraction of the miRNAs generated by the Meg3-Mirg locus was downregulated during aging. To address the specificity of this repression, four miRNAs selected as representative for this locus were further assessed in other cells and organs isolated from aged mice. The results suggested that the repression of miRNAs generated by the Meg3-Mirg locus was a general feature of aging in multiple organs. Bioinformatic analysis of the predicted target genes identified Integrin Beta-2 as an aged-upregulated gene, which was thereafter confirmed in multiple mouse organs. In conclusion, our study provides new data concerning the mechanisms of natural aging and highlights the robustness of the miRNA modulation during this process.

Identification of a Hematopoietic Cell Population Emerging From Mouse Bone Marrow With Proliferative Potential In Vitro and Immunomodulatory Capacity.

There is continuing interest in therapeutic applications of bone marrow-derived mesenchymal stromal cells (MSC). Unlike human counterparts, mouse MSC are difficult to propagate in vitro due to their contamination with adherent hematopoietic cells that overgrow the cultures. Here we investigated the properties of these contaminating cells, referred to as bone marrow-derived proliferating hematopoietic cells (BM-PHC). The results showed that both BM-PHC and MSC had strong immunomodulatory properties on T cells in vitro, with PGE2 and NO involved in this mechanism. However, BM-PHC were stronger immunomodulators than MSC, with CCL-6 identified as putative molecule responsible for superior effects. In vivo studies showed that, in contrast to BM-PHC, MSC endorsed a more rapid xenograft tumor rejection, thus indicating a particular context in which only MSC therapy would produce positive outcomes. In conclusion, bone marrow contains two cell populations with immunomodulatory properties, which are valuable sources for therapeutic studies in specific disease-relevant contexts.

MiR-29a Increase in Aging May Function as a Compensatory Mechanism Against Cardiac Fibrosis Through SERPINH1 Downregulation.

Deregulation of microRNA (miRNA) profile has been reportedly linked to the aging process, which is a dominant risk factor for many pathologies. Among the miRNAs with documented roles in aging-related cardiac diseases, miR-18a, -21a, -22, and -29a were mainly associated with hypertrophy and/or fibrosis; however, their relationship to aging was not fully addressed before. The purpose of this paper was to evaluate the variations in the expression levels of these miRNAs in the aging process. To this aim, multiple organs were harvested from young (2-3-months-old), old (16-18-months-old), and very old (24-25-months-old) mice, and the abundance of the miRNAs was evaluated by quantitative real-time (RT)-PCR. Our studies demonstrated that miR-21a, miR-22, and miR-29a were upregulated in the aged heart. Among them, miR-29a was highly expressed in many other organs, i.e., the brain, the skeletal muscle, the pancreas, and the kidney, and its expression was further upregulated during the natural aging process. Western blot, immunofluorescence, and xCELLigence analyses concurrently indicated that overexpression of miR-29a in the muscle cells decreased the collagen levels as well as cell migration and proliferation. Computational prediction analysis and overexpression studies identified SERPINH1, a specific chaperone of procollagens, as a potential miR-29a target. Corroborating to this, significantly downregulated SERPINH1 levels were found in the skeletal muscle, the heart, the brain, the kidney, and the pancreas harvested from very old animals, thereby indicating the role of the miR-29a-SERPINH1 axis in the aging process. In vitro analysis of miR-29a effects on fibroblast and cardiac muscle cells pointed toward a protective role of miR-29a on aging-related fibrosis, by reducing cell migration and proliferation. In conclusion, our study indicates an adaptive increase of miR-29 in the natural aging process and suggests its role as a transcriptional repressor of SERPINH1, with a potential therapeutic value against adverse matrix remodeling and aging-associated tissue fibrosis.