Reciprocal modulation of cell functions upon direct interaction of adipose mesenchymal stromal and activated immune cells.

The interaction of adipose mesenchymal stromal cells (ASCs) and allogeneic peripheral blood mononuclear cells (PBMCs) is regulated either through direct or paracrine mechanisms. Here, we examined the impact of direct contact in reciprocal regulation of ASC-PBMC functions. Activated PBMCs in vitro induced ASC immunomodulatory activity, while direct and paracrine intercellular interactions regulated PBMCs themselves: the functional state of the organelles was altered, and activation decreased. Direct contact with immune cells affected the activity of ASC intracellular compartments, in particular, reactive oxygen species (ROS) production, and decreased the growth rate. Some ASC properties, including motility, intercellular adhesion molecule-1 (ICAM-1), and major histocompatibility complex class I and II antigens (HLA-ABC and HLA-DR, respectively) expression, did not depend on contact with PBMCs and were only regulated by paracrine means. Direct ASC and PBMC contact favoured an angiogenesis-supportive microenvironment, possibly due to the greater production of VEGF by ASCs; this microenvironment also contained a higher leukemia inhibitory factor (LIF) level. Thus, a change in the functional activity of ASCs and PBMCs upon interaction promoted the formation of an immunosuppressive, anti-inflammatory, and proangiogenic microenvironment. This environment could help resolve inflammation and further restore damaged tissue. SIGNIFICANCE OF THE STUDY: Numerous studies have demonstrated the beneficial effects of transplanted mesenchymal stromal cells, particularly ASCs, for the treatment of a number of autoimmune diseases as well as various tissue injuries. To improve the efficiency of these methods, it is necessary to understand the principal events that occur when ASCs are introduced, primarily the molecular mechanisms of interaction between ASCs and the recipient immune system. We demonstrated that an anti-inflammatory, immunosuppressive, and angiostimulatory shift in the paracrine profile upon the interaction of activated PBMCs and ASCs changes the functional activity of both cell types, a phenomenon that is potentiated by direct cell-cell contact.

Interaction of multipotent mesenchymal stromal and immune cells: Bidirectional effects.

Adult multipotent mesenchymal stromal cells (MSCs) are considered one of the key players in physiological remodeling and tissue reparation. Elucidation of MSC functions is one of the most intriguing issues in modern cell physiology. In the present review, the interaction of MSCs and immune cells is discussed in terms of reciprocal effects, which modifies the properties of "partner" cells with special focus on the contribution of direct cell-to-cell contacts, soluble mediators and local microenvironmental factors, the most important of which is oxygen tension. The immunosuppressive phenomenon of MSCs is considered as the integral part of the response-to-injury mechanism.

Proinflammatory interleukins' production by adipose tissue-derived mesenchymal stromal cells: the impact of cell culture conditions and cell-to-cell interaction.

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL-6 and IL-8 production by adipose-derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O2 concentrations, some highly up-regulated at hypoxia. IL-6 and IL-8 production was inversely dependent on cell culture density. In early (first-third) passages, IL-6 and IL-8 concentration was higher at 20% O2 and in late (8th-12th) passages under 5% O2. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL-6 and did not result in the elevation of IL-8 concentration. Thereby, the production of proinflammatory interleukins (IL-6 and IL-8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood-borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. SIGNIFICANCE PARAGRAPH: Ex vivo expansion is widely used for increasing the number of adipose-derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.

Adipose tissue-derived stromal cells retain immunosuppressive and angiogenic activity after coculture with cord blood hematopoietic precursors.

Adipose-tissue derived stromal cells (ASCs) are currently considered as a full value alternative source of bone marrow MSCs for prevention of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation due to their immunosuppressive potential. Besides, ASCs are known to support ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). Ex vivo expansion enables to amplify significantly the number of HSPCs of different commitment. Mononuclear cells (MNCs) from cord blood (cb) contain HSPCs and are easily assessed. The rarity of those HSPCs is a serious limitation of its application in cell therapy. Here we expanded cbMNCs in stroma-dependent setting to generate heterocellular associates consisting of ASCs and undifferentiated and low committed hematopoietic cbHSPCs. A part of cbHSPCs in associates demonstrated a primitive phenotype confirmed by formation of "cobblestone areas". ASCs associated with cbHSPCs demonstrated up-regulation of immunosuppressive indoleamine 2,3-dioxygenase (IDO), leukemia inhibitory factor (LIF), cyclooxygenase-2 (PTGS2) genes. ASC-cbHSPCs as well as ASCs provoked the suppression of HLA-DR activation and apoptosis of mitogen-stimulated T cells. VEGF transcription and secretion were elevated providing stimulation of blood vessel formation in ovo. Thus, ASCs retain immunosuppressive and proangiogenic capacities evidencing "third party" potential along with the effective support of ex vivo expansion of cbHSPCs. Above functions expand the relevance of ASCs for needs of regenerative medicine.

Interaction of allogeneic adipose tissue-derived stromal cells and unstimulated immune cells in vitro: the impact of cell-to-cell contact and hypoxia in the local milieu.

Multipotent mesenchymal stem cells (MSCs) are an attractive tool for cell therapy and regenerative medicine. Being applied in vivo, allogeneic MSCs are faced with both activated and unstimulated immune cells. The effects of MSCs on activated immune cells are well described and are mainly suppressive. Less is known about the interaction of MSCs with unstimulated immune cells. We evaluated the contribution of tissue-related O2 level ("physiological" hypoxia-5% O2) and cell-to-cell contact to the interaction between allogeneic adipose tissue-derived MSCs (ASCs) and unstimulated peripheral blood mononuclear cells (PBMCs). Under both O2 levels, ASCs affected the immune response by elevating the proportion of CD69+ T cells and modifying the functional activity of unstimulated PBMCs, providing a significant reduction of ROS level and activation of lysosome compartment. "Physiological" hypoxia partially attenuated the ASC modulation of PBMC function, reducing CD69+ cell activation and more significantly supressing ROS. In direct co-culture, the ASC effects were more pronounced. PBMC viability was preferentially maintained, and the lymphocyte subset ratio was altered in favour of B cells. Our findings demonstrate that allogeneic ASCs do not enhance the activation of unstimulated immune cells and can provide supportive functions. The "hypoxic" phenotype of ASCs may be more "desirable" for the interaction with allogeneic immune cells that may be required in cell therapy protocols.

Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro.

Human adipose tissue-stromal derived cells (ASCs) are considered a perspective tool for regenerative medicine. Depending on the application mode ASC/allogeneic immune cell interaction can occur in the systemic circulation under plenty high concentrations of O2 and in target tissues at lower O2 levels. Here we examined the effects of allogeneic PHA-stimulated peripheral blood mononuclear cells (PBMCs) on ASCs under ambient (20%) oxygen and "physiological" hypoxia (5% O2). As revealed with microarray analysis ASCs under 20% O2 were more affected by activated PBMCs, which was manifested in differential expression of more than 300 genes, whereas under 5% O2 only 140 genes were changed. Altered gene pattern was only partly overlapped at different O2 conditions. Under O2 ASCs retained their proliferative and differentiative capacities, mesenchymal phenotype, and intracellular organelle' state. ASCs were proinflammatory activated on transcription level that was confirmed by their ability to suppress activation and proliferation of mitogen-stimulated PBMCs. ASC/PBMCs interaction resulted in anti-inflammatory shift of paracrine mediators in conditioning medium with significant increase of immunosuppressive LIF level. Our data indicated that under both ambient and tissue-related O2 ASCs possessed immunosuppressive potential and maintained functional activity. Under "physiological" hypoxia ASCs were less susceptible to "priming" by allogeneic mitogen-activated PBMCs.

Human adipose-tissue derived stromal cells in combination with hypoxia effectively support ex vivo expansion of cord blood haematopoietic progenitors.

The optimisation of haematopoietic stem and progenitor cell expansion is on demand in modern cell therapy. In this work, haematopoietic stem/progenitor cells (HSPCs) have been selected from unmanipulated cord blood mononuclear cells (cbMNCs) due to adhesion to human adipose-tissue derived stromal cells (ASCs) under standard (20%) and tissue-related (5%) oxygen. ASCs efficiently maintained viability and supported further HSPC expansion at 20% and 5% O2. During co-culture with ASCs, a new floating population of differently committed HSPCs (HSPCs-1) grew. This suspension was enriched with СD34+ cells up to 6 (20% O2) and 8 (5% O2) times. Functional analysis of HSPCs-1 revealed cobble-stone area forming cells (CAFCs) and lineage-restricted colony-forming cells (CFCs). The number of CFCs was 1.6 times higher at tissue-related O2, than in standard cultivation (20% O2). This increase was related to a rise in the number of multipotent precursors - BFU-E, CFU-GEMM and CFU-GM. These changes were at least partly ensured by the increased concentration of MCP-1 and IL-8 at 5% O2. In summary, our data demonstrated that human ASCs enables the selection of functionally active HSPCs from unfractionated cbMNCs, the further expansion of which without exogenous cytokines provides enrichment with CD34+ cells. ASCs efficiently support the viability and proliferation of cord blood haematopoietic progenitors of different commitment at standard and tissue-related O2 levels at the expense of direct and paracrine cell-to-cell interactions.