TNF-α and IFN-γ Participate in Improving the Immunoregulatory Capacity of Mesenchymal Stem/Stromal Cells: Importance of Cell-Cell Contact and Extracellular Vesicles.

Mesenchymal stem/stromal cells (MSCs) have an immunoregulatory capacity and have been used in different clinical protocols requiring control of the immune response. However, variable results have been obtained, mainly due to the effect of the microenvironment on the induction, increase, and maintenance of MSC immunoregulatory mechanisms. In addition, the importance of cell-cell contact for MSCs to efficiently modulate the immune response has recently been highlighted. Because these interactions would be difficult to achieve in the physiological context, the release of extracellular vesicles (EVs) and their participation as intermediaries of communication between MSCs and immune cells becomes relevant. Therefore, this article focuses on analyzing immunoregulatory mechanisms mediated by cell contact, highlighting the importance of intercellular adhesion molecule-1 (ICAM-1) and the participation of EVs. Moreover, the effects of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), the main cytokines involved in MSC activation, are examined. These cytokines, when used at the appropriate concentrations and times, would promote increases in the expression of immunoregulatory molecules in the cell and allow the acquisition of EVs enriched with these molecules. The establishment of certain in vitro activation guidelines will facilitate the design of conditioning protocols to obtain functional MSCs or EVs in different pathophysiological conditions.

Human Bone Marrow Mesenchymal Stem/Stromal Cells Exposed to an Inflammatory Environment Increase the Expression of ICAM-1 and Release Microvesicles Enriched in This Adhesive Molecule: Analysis of the Participation of TNF-α and IFN-γ.

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory capacity; therefore, they have been used in different clinical protocols in which it is necessary to decrease the immune response. This capacity is mainly regulated by TNF-α and IFN-γ, and it has been observed that cell-cell contact, mainly mediated by ICAM-1, is important for MSCs to carry out efficient immunoregulation. Therefore, in the present work, we analyzed the effect of TNF-α alone or in combination with IFN-γ on the expression of ICAM-1. Besides, given the importance of cell contact in the immunoregulatory function of MSCs, we analyzed whether these cells release ICAM-1+ microvesicles (MVs). Our results show for the first time that TNF-α is capable of increasing the early expression of ICAM-1 in human BM-MSCs. Also, we observed that TNF-α and IFN-γ have a synergistic effect on the increase in the expression of ICAM-1. Furthermore, we found that BM-MSCs exposed to an inflammatory environment release MVs enriched in ICAM-1 (MVs-ICAM-1high). The knowledge generated in this study will contribute to the improvement of in vitro conditioning protocols that favor the therapeutic effect of these cells or their products.

Improved Proliferative Capacity of NP-Like Cells Derived from Human Mesenchymal Stromal Cells and Neuronal Transdifferentiation by Small Molecules.

Neural progenitors (NP), found in fetal and adult brain, differentiate into neurons potentially able to be used in cell replacement therapies. This approach however, raises technical and ethical problems which limit their potential therapeutic use. Alternately, NPs can be obtained by transdifferentiation of non-neural somatic cells evading these difficulties. Human bone marrow mesenchymal stromal cells (MSCs) are suggested to transdifferentiate into NP-like cells, which however, have a low proliferation capacity. The present study demonstrates the requisite of cell adhesion for proliferation and survival of NP-like cells and re-evaluates some neuronal features after differentiation by standard procedures. Mature neuronal markers, though, were not detected by these procedures. A chemical differentiation approach was used in this study to convert MSCs-derived NP-like cells into neurons by using a cocktail of six molecules, CHIR99021, I-BET151, RepSox, DbcAMP, forskolin and Y-27632, defined after screening combinations of 22 small molecules. Direct transdifferentiation of MSCs into neuronal cells was obtained with the small molecule cocktail, without requiring the NP-like intermediate stage.

Bone Marrow Mesenchymal Stromal Cells from Clinical Scale Culture: In Vitro Evaluation of Their Differentiation, Hematopoietic Support, and Immunosuppressive Capacities.

The differentiation capacity, hematopoietic support, and immunomodulatory properties of human bone marrow mesenchymal stromal cells (BM-MSCs) make them attractive therapeutic agents for a wide range of diseases. Clinical scale cultures (CSCs) have been used to expand BM-MSCs for their use in cell therapy protocols; however, little is known about the functionality of the expanded cells. The main goal of the present study was to evaluate the functional characteristics of BM-MSCs expanded from CSCs to determine the quality of the cells for cellular therapy protocols. To address this issue, we analyzed the morphology, immunophenotype, differentiation potential (adipogenic, osteogenic and chondrogenic), hematopoietic support, and immunosuppressive capacity of BM-MSCs from short scale cultures (SSCs) and CSCs in a comparative manner. After 12 days of culture in CSCs (HYPERFlask System), BM-MSCs reached cell numbers of 125.52 × 10(6) ± 25.6 × 10(6) MSCs, which corresponded to the number of cells required for transplantation (∼1.7 × 10(6) MSCs/kg for a 70-kg patient). After expansion, BM-MSCs expressed the characteristic markers CD73, CD90, and CD105; however, expansion decreased their differentiation capacity toward the adipogenic, osteogenic, and chondrogenic lineages and their ability to inhibit T-cell proliferation compared with SSCs-MSCs. Importantly, CSCs-MSCs maintained the ability to support the proliferation and expansion of hematopoietic progenitor cells and the capacity to express the molecules, cytokines, and extracellular matrix proteins involved in the regulation of hematopoiesis. Our study highlights the need to evaluate the functional properties of the expanded BM-MSCs for verification of their quality for cell therapy protocols.

Immunoregulation by mesenchymal stem cells: biological aspects and clinical applications.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into mesenchymal lineages and that can be isolated from various tissues and easily cultivated in vitro. Currently, MSCs are of considerable interest because of the biological characteristics that confer high potential applicability in the clinical treatment of many diseases. Specifically, because of their high immunoregulatory capacity, MSCs are used as tools in cellular therapies for clinical protocols involving immune system alterations. In this review, we discuss the current knowledge about the capacity of MSCs for the immunoregulation of immunocompetent cells and emphasize the effects of MSCs on T cells, principal effectors of the immune response, and the immunosuppressive effects mediated by the secretion of soluble factors and membrane molecules. We also describe the mechanisms of MSC immunoregulatory modulation and the participation of MSCs as immune response regulators in several autoimmune diseases, and we emphasize the clinical application in graft versus host disease (GVHD).

Human mesenchymal stromal cells from adult and neonatal sources: a comparative in vitro analysis of their immunosuppressive properties against T cells.

Bone marrow-mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used in cell therapies as immune regulators for the treatment of graft-versus-host disease. We have previously characterized several biological properties of MSCs from placenta (PL) and umbilical cord blood (UCB), and compared them to those of BM-the gold standard. In the present study, we have compared MSCs from BM, UCB, and PL in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3(+) T cells. Our results confirm the immunosuppressive potential of BM-MSCs, and demonstrate that MSCs from UCB and, to a lesser extent PL, also have immunosuppressive potential. In contrast to PL-MSCs, BM-MSCs and UCB-MSCs significantly inhibited the proliferation of both CD4(+) and CD8(+) activated T cells in a cell-cell contact-dependent manner. Such a reduced proliferation in cell cocultures correlated with upregulation of programmed death ligand 1 on MSCs and cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) on T cells, and increased production of interferon-γ, interleukin-10, and prostaglandin E2. Importantly, and in contrast to PL-MSCs, both BM-MSCs and UCB-MSCs favored the generation of T-cell subsets displaying a regulatory phenotype CD4(+)CD25(+)CTLA-4(+). Our results indicate that, besides BM-MSCs, UCB-MSCs might be a potent and reliable candidate for future therapeutic applications.

In vitro evidence of the presence of mesenchymal stromal cells in cervical cancer and their role in protecting cancer cells from cytotoxic T cell activity.

Mesenchymal stromal cells (MSCs) have been isolated from different tumors and it has been suggested that they support tumor growth through immunosuppression processes that favor tumor cell evasion from the immune system. To date, however, the presence of MSCs in cervical cancer (CeCa) and their possible role in tumor growth remains unknown. Herein we report on the presence of MSCs in cervical tissue, both in normal conditions (NCx-MSCs) and in CeCa (CeCa-MSCs), and described several biological properties of such cells. Our study showed similar patterns of cell surface antigen expression, but distinct differentiation potentials, when we compared both cervical MSC populations to MSCs from normal bone marrow (BM-MSCs, the gold standard). Interestingly, CeCa-MSCs were negative for the presence of human papilloma virus, indicating that these cells are not infected by such a viral agent. Also, interestingly, and in contrast to NCx-MSCs, CeCa-MSCs induced significant downregulation of surface HLA class I molecules (HLA-A*0201) on CaSki cells and other CeCa cell lines. We further observed that CeCa-MSCs inhibited antigen-specific T cell recognition of CaSki cells by cytotoxic T lymphocytes (CTLs). HLA class I downregulation on CeCa cells correlated with the production of IL-10 in cell cocultures. Importantly, this cytokine strongly suppressed recognition of CeCa cells by CTLs. In summary, this study demonstrates the presence of MSCs in CeCa and suggests that tumor-derived MSCs may provide immune protection to tumor cells by inducing downregulation of HLA class I molecules. This mechanism may have important implications in tumor growth.