Analysis of expression of genes involved in immune response modulation in silent multipotent mesenchymal stromal cells.

The expression of some genes modulating the immune response was studied in multipotent mesenchymal stromal cells (MMSC) from the bone marrow of a healthy donor. Non-activated MMSC expressed IL-6 and IL-10, complement H factor, macrophage growth factor, prostaglandin E2 synthase, and indoleamine-2,3-dioxygenase. The expression of all these genes was higher in female MMSC. A close inverse relationship between IL-6 expression in MMSC and male donor age, close relationship between body weight index and fibroblast CFU concentration in female donor bone marrow and between indoleamine-2,3-dioxygenase and macrophage growth factor in MMSC from these donors were detected. The expression of the analyzed genes was higher in MMSC of donors who had no antibodies to cytomegalovirus, herpes simplex virus, and Epstein-Barr virus in the blood. The results demonstrate the MMSC regulation of immune reactions by MMSC at the cell and organism levels.

VCAM-1 expression on bone marrow stromal cells from patients with myelodysplastic syndromes.

We studied the expression of VCAM-1 adhesion molecules on stromal cells from the bone marrow of patients with myelodysplastic syndromes, healthy donors, and patients with chronic myeloproliferative diseases and acute leukemias. Expression of adhesion molecule on mesenchymal stromal cells from the bone marrow of patients and healthy donors was evaluated after 2-4 passages by the methods of immunoprecipitation and electrophoresis. VCAM-1 expression in the majority of patients with myelodysplastic syndromes was lower than in healthy donors. At the same time, VCAM-1 expression was not identified on mesenchymal cells from acute leukemia patients. VCAM-1 expression on cells from patients with chronic myeloproliferative diseases did not differ from that in healthy donors. We conclude that VCAM-1 synthesis in bone marrow stromal cells is impaired in patients with myelodysplastic syndromes and acute leukemias. These changes can be followed by the loss of relationships between hemopoietic cells and stromal microenvironment in bone marrow niches. Hemopoietic cells gain the ability for uncontrolled growth, which results in progression of the disease.

Early and late hemopoietic precursor cells in patients with chronic myeloproliferative diseases.

We evaluated the content of early and late cobblestone area-forming cells, immediate progeny of hemopoietic stem cells, and committed precursor cells in the bone marrow and peripheral blood of patients with chronic myeloproliferative diseases and healthy donors. In patients with essential thrombocythemia, the number of late cobblestone area-forming cells in the peripheral blood decreased, while other parameters did not differ from those in healthy donors. In patients with idiopathic myelofibrosis, we found a decreased number of late and early cobblestone area-forming cells in the bone marrow and late cobblestone area-forming cells in the peripheral blood, while the count of early cobblestone area-forming cells in the peripheral blood increased. In patients with chronic myeloid leukemia, the number of early cobblestone area-forming cells in the bone marrow decreased, but the count of late and early cobblestone area-forming cells in the peripheral blood increased. The number of endogenous committed precursor cells in the peripheral blood increased in all groups of patients with chronic myeloproliferative diseases and, particularly, in patients with idiopathic myelofibrosis and chronic myeloid leukemia. Functional characteristics of immediate descendants of hemopoietic stem cells probably reflect the level of damage and attest to the existence of various mechanisms underlying the defect of the hemopoietic stem cell during chronic myeloproliferative diseases.

Stromal regulation of hemopoietic stem cells in long-term human bone marrow tissue cultures under the effect of parathyroid hormone.

We studied the interaction between different categories of hemopoietic precursors with parathyroid hormone-activated stromal microenvironment. Improved survival of early precursors capable long-term hemopoiesis maintenance and increased number of later short-term repopulating precursors was demonstrated on the model of co-culturing of human bone marrow cells on a layer of adherent cells of long-term bone marrow cultures treated with parathyroid hormone. These changes correlate with increased expression of genes involved in the maintenance of the hemopoietic stem cells in the sublayer activated by parathyroid hormone. Simultaneously, the expression of some stromal differentiation genes, adhesion molecules for hemopoietic stem cells, and growth factors increased in adherent cell layers treated with parathyroid hormone. These findings attest to activating effect of parathyroid hormone on cells forming the niches for both early and later hemopoietic precursors, and hence parathyroid hormone can be used as a potential agent promoting expansion of early hemopoietic stem cells ex vivo.

Defective adhesion and homing of hemopoietic precursors from the bone marrow of patients with myelodysplasia to stromal cells of normal bone marrow cultures.

Hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome were characterized by lower adhesion to normal stromal sublayer compared to bone marrow precursors from healthy donors, while adhesion to fibroblast monolayer and fibronectin was similar in bone marrow cells from patients and donors. In vitro experiments showed that the percentage of adherent hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome in normal stromal sublayer and fibroblasts was lower compared to healthy donors. The decrease in adhesive activity of hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome probably contributes to impairment of cell-cell interactions in the bone marrow of these patients.

In vivo production of cytokines by bone marrow stromal cells and macrophages from patients with myelodysplastic syndrome.

We studied functional disturbances in hemopoietic microenvironment and cytokine production by stromal sublayer in long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome. Production of factors stimulating the growth of normal erythroid and granulocytic precursors by cells of the stromal sublayer from patients with refractory sideroblast anemia and refractory anemia with excess blasts is impaired compared to cells from healthy donors. The medium conditioned by macrophages from patients with chronic myelomonocytic leukemia displayed a higher ability to stimulate the growth of granulocytes and macrophages compared to media conditioned by cells from donors and patients with refractory sideroblast anemia and refractory anemia with excess blasts. Cultured stromal cells and macrophages produced tumor necrosis factor-alpha and interleukin-6. Their content in media conditioned by cells from patients with myelodysplastic syndrome surpassed that in healthy donors. Our results suggest that production of cytokines by stromal microenvironmental cells is impaired in patients with various forms of myelodysplastic syndrome.