Molecular Changes in Immunological Characteristics of Bone Marrow Multipotent Mesenchymal Stromal Cells in Lymphoid Neoplasia.

Immune system and bone marrow stromal cells play an important role in maintaining normal hematopoiesis. Lymphoid neoplasia disturbs not only development of immune cells, but other immune response mechanisms as well. Multipotent mesenchymal stromal cells (MSCs) of the bone marrow are involved in immune response regulation through both intercellular interactions and secretion of various cytokines. In hematological malignancies, the bone marrow stromal microenvironment, including MSCs, is altered. Aim of this study was to describe the differences of MSCs' immunological function in the patients with acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). In ALL, malignant cells arise from the early precursor cells localized in bone marrow, while in DLBCL they arise from more differentiated B-cells. In this study, only the DLBCL patients without bone marrow involvement were included. Growth parameters, surface marker expression, genes of interest expression, and secretion pattern of bone marrow MSCs from the patients with ALL and DLBCL at the onset of the disease and in remission were studied. MSCs from the healthy donors of corresponding ages were used as controls. It has been shown that concentration of MSCs in the bone marrow of the patients with ALL is reduced at the onset of the disease and is restored upon reaching remission; in the patients with DLBCL this parameter does not change. Proliferative capacity of MSCs did not change in the patients with ALL; however, the cells of the DLBCL patients both at the onset and in remission proliferated significantly faster than those from the donors. Expression of the membrane surface markers and expression of the genes important for differentiation, immunological status maintenance, and cytokine secretion differed significantly in the MSCs of the patients from those of the healthy donors and depended on nosology of the disease. Secretomes of the MSCs varied greatly; a number of proteins associated with immune response regulation, differentiation, and maintenance of hematopoietic stem cells were depleted in the secretomes of the cells from the patients. Lymphoid neoplasia leads to dramatic changes in the functional immunological status of MSCs.

Humoral Effect of a B-Cell Tumor on the Bone Marrow Multipotent Mesenchymal Stromal Cells.

The properties of bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) are altered in the patients with the diffuse large B cell lymphoma (DLBCL) without BM involvement. It was suggested that plasma from the patients contains soluble factors that affect MSCs. Plasma and BM-derived MSCs from the DLBCL patients at the onset of the disease and one month after the end of treatment were studied. Concentration of the plasma cytokines and gene expression in the MSCs were evaluated by the Bio-Plex Pro Human Cytokine Panel kit to measure 27 analytes and real-time PCR. Plasma and MSCs from the healthy donors were used as controls. Analysis of cytokines in the plasma from healthy donors and patients before and one month after the end of treatment revealed significant differences in the concentration of 14 out of 27 cytokines. Correlations between the levels of secreted cytokines were altered in the plasma from patients indicating that the immune response regulation was disturbed. Cultivation of the MSCs from the healthy donors in the medium supplemented with the plasma from patients led to the changes in the MSC properties, similar to those observed in the MSCs from patients. The BM-derived MSCs were shown to participate in the humoral changes occurring in the DLBCL patients. For the first time, it was shown that the precursors of the stromal microenvironment - multipotent mesenchymal stromal cells - are altered in the patients with DLBCL without bone marrow involvement due to the humoral effect of the tumor and the response of organism to it. Comprehensive analysis of the results shows that, when remission is achieved in the patients with DLBCL, composition of the plasma cytokines normalizes, but does not reach the level observed in the healthy donors. The discovery of a new aspect of the effect of the tumor B-cells on the organism could help to reveal general regularities of the humoral effect of various tumors on the bone marrow stromal cells.

Clonal composition of human multipotent mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MMSCs) are a heterogeneous population consisting of cells with a distinct proliferative potential. The aim of this study was to define clonal composition in MMSCs and trace the dynamics of individual clones in MMSC subpopulations with different proliferative potentials during the process of cultivation. The investigation was performed at single-cell level using genetically marked cells. Specifically, human bone marrow MMSCs were infected with a lentiviral vector-bearing marker gene. Integration site analysis was performed for clones at each passage by ligation-mediated polymerase chain reaction and Southern blot hybridization. Sibling connections between clones and clonal composition of MMSC culture at each passage were revealed. The MMSC population contained multiple, different, mainly small, clones. It was found that large long-living clones with a high, but limited proliferative potential could be detected rarely in MMSCs population. These data suggest that the human MMSC population does not fit the "stem cell" criteria, however, MMSCs may contain a subpopulation of large clones with a high proliferative potential.

Alterations in hematopoietic microenvironment in patients with aplastic anemia.

Mechanisms of hematopoietic failure in patients with aplastic anemia (AA) are obscure. We investigate alterations in the hematopoietic microenvironment in AA patients. We present the results of studying mesenchymal stromal cells (MSC), fibroblastic colony-forming units (CFU-F), and adherent cell layers (ACL) of long-term bone marrow cultures (LTBMC) from bone marrow (BM) samples of AA patients. MSC of AA patients proliferated longer than those of donors. In half of the patients' MSC cultures, adipogenesis was impaired. Osteogenic differentiation was not achieved in 36% of AA MSC. CFU-F formed enlarged colonies, and their concentration in the BM of AA patients was significantly increased. Our data suggest that the physiological activation of the stromal microenvironment is characteristic of AA. We detected a decrease in the expression of the angiopoetin-1 (ANG-1) and vascular cell adhesion molecule-1 (VCAM-1) genes, together with an increase in the expression of vascular endothelial growth factor (VEGF) in ACL of AA patients. This indicates abnormal regulatory patterns in both osteoblastic and vascular contexts. Addition of AA patients' serum to donors' LTBMC for 3 weeks induced similar gene expression alterations. The addition of parathyroid hormone (PTH) resulted in the expression levels of analyzed genes returning to normal, in both AA LTBMC and donor cultures treated with AA serum. The physiologic status of the BM stromal microenvironment (MSC, CFU-F, and ACL of LTBMC) of AA patients was altered.