Delayed effects of long-term administration of granulocyte colony-stimulating factor to mice.

We studied the effects of chronic administration of granulocyte colony-stimulating factor in nonmobilizing doses to mice. Over 18 months of the study, 55% animals of the treatment group died of unknown cause, blood diseases and tumors were found in 20% mice, and in 5% animals pathological changes were absent. Control mice had no diseases (normal values of total and differential leukocyte count). The diagnoses made over the first 7 months mainly included myeloproliferative diseases. Solid tumors were found at later terms. Suppurative inflammation at the site of injection was observed in all mice after 3-month treatment with granulocyte colony-stimulating factor. Our results indicate that chronic administration of granulocyte colony-stimulating factor in low doses leads to the development of etiologically different tumors and sharply reduced animal life span. The use of granulocyte colony-stimulating factor during allogeneic transplantation of hemopoietic stem cells can be hazardous for donors.

[Effect of parathyroid hormone (PTH 1-34) on hemopoiesis in long-term cultures of human bone marrow].

AIM: To study effects of parathyroid hormone (PTH) used in therapy of osteoporosis on hemopoiesis in long-term culture of human bone marrow (LCBM) in terms of its potential influence on stem hemopoietic and stromal cells. MATERIAL AND METHODS: For a long time LCBM was treated with PTH (1-34) and compared for cell production, concentration of late and early hemopoietic precursors. Maintenance of hemopoiesis and adhesion at early hemopoiesis precursors on the stromal sublayers treated with PTH (1-34) was used as a functional test. A relative level of expression of genes participating in regulating proliferation and self-support of stem hemopoietic cells was studied. RESULTS: PTH (1-34) in the above concentrations did not affect hemopoiesis in LCBM. Stromal sublayer treated with PTH (1-34) for a long time supports cell precursors better, their adhesion to such sublayers enhances. CONCLUSION: PTH (1-34) in pharmacological but small concentrations had no irreversible effects on hemopoiesis, i.e. contraindications for its use in the treatment of osteoporosis were not revealed.

Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization.

Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

Changed homing of hemopoietic precursor cells after long-term treatment with parathyroid hormone.

Changes in the capacity of hemopoietic stromal microenvironment to promote homing of hemopoietic stem cells from different hierarchical compartments were evaluated in mice treated with parathyroid hormone by determining their 24-h precipitation factor. This parameter did not change for splenic short-living hemopoietic stem cells (splenic CFU) and considerably decreased for the bone marrow of mice treated with parathyroid hormone. For earlier long-living hemopoietic stem cells (cells forming the cobblestone area on day 28) the precipitation factor after injections of parathyroid hormone did not change in the bone marrow and decreased in the spleen. These data suggest that parathyroid hormone decreases the efficiency of homing of short-living hemopoietic stem cells in the bone marrow.

[The differentiation potential of stem cells (the problem of plasticity)].

Numerous publications on the ability of adult stem cells to differentiate into the cells of various tissues, not always homodermic (stem cell flexibility), to contain serious methodic errors. The main flexibility phenomena, such as "transdifferentiation" of hemopoietic stem cells into hepatocytes, cardiomyocytes, beta-cells of islets of Langerhans, neurons etc., are caused not by a shift of the differentiation path, but by cell merging, resulting in appearance of hybrids with unusual markers of cells of non-hemopoietic origin. The second most frequent error is wrong identification of macrophages and lymphocytes, which are present in any tissue and have the donor's genotype in chimeras. Even when the cause of the error is unknown, the phenomenon of unusual cell formation is exclusively rare and never bears therapeutic potential. In general, it is at least too early to revise the main tenets of the stem cell doctrine. Embryonic stem cells are totipotent indeed; however, the time of their clinical use has not come yet. Attempts to induce their ordered differentiation keep on failing; they very often lead to formation of teratomas and, even if necessary cells such as hemopoietic stem cells are formed, they do not work after administration into an organism that has been exposed to radiation. Clinical use of embryonic stem cells do not seem possible in this decade.

Ex vivo expansion of hemopoietic precursor cells on a sublayer treated with parathyroid hormone.

The kinetics of hemopoietic precursor cells was studied in cultures treated with parathyroid hormone in a concentration of 10(-7) M. Long-term culturing of bone marrow with parathyroid hormone did not change the number of mature cells, while the number of precursors forming colonies in semisolid media increased 7-fold and the number of cells forming cobblestone areas on day 28 increased 9-10-fold. After 24 h culturing of bone marrow cells on an irradiated sublayer pretreated with parathyroid hormone for 8 and 12 weeks, the number of early hemopoietic precursor cells forming cobblestone areas on day 28 of culturing increased 2-and 5.5-fold, respectively. The expression of Bmi-1 gene responsible for self-maintenance of stem hemopoietic cells increased in cultures treated with parathyroid hormone. It seems that parathyroid hormone can be used for expansion of hemopoietic stem cells ex vivo, which is essential for their transplantation to patients.

Regeneration of the spleen in intact animals and radiation chimeras.

Regeneration of the splenic tissue after partial splenectomy is incomplete in adult non-irradiated mice and lethally irradiated animals reconstituted with donor syngeneic bone marrow. Transplantation of the splenic tissue to intact adult animals after partial splenectomy resulted in virtually complete regeneration of the spleen. In chimeras recovery of the splenic tissue was decreased; autotransplantation of the whole spleen or its part did not lead to appreciable changes in the weight and cellularity of this organ. No more than 30% splenic tissue is restored after complete splenectomy and transplantation of the splenic tissue in intact and chimeric mice.

Lentivirus vector can integrate in the genome and exist and replicate in the cell as an episome.

Lethally irradiated mice were reconstituted with few purified primitive hemopoietic stem cells containing sequences of a gene encoding green fluorescent protein. The gene was transferred using a lentivirus vector. The presence of the marker gene in splenocyte colonies derived from the bone marrow of reconstituted mice and in cells of other hemopoietic and non-hemopoietic organs was studied during the life. It was shown that the lentivirus vector can persist for a long time and replicate in hemopoietic cells as an episome.

Long-term self-maintenance of hemopoietic precursors in bone marrow culture derived from tumor necrosis factor-deficient mice is not a result of neoplastic transformation.

In long-term bone marrow cultures derived from tumor necrosis factor-deficient mice the total cell production and the total duration of hemopoiesis are increased (the latter is comparable with mouse life span). Telomerase activity in cells of nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice increases with time and peaks after 1-year culturing. Karyotyping of nonadherent and adherent cells of long-term bone marrow cultures revealed instability of nonadherent cells and hyperploidy of the stromal sublayer cells, which attested to the presence of a neoplastic transformation. However, cell differentiation is not blocked in long-term bone marrow cultures. The nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be cultured without exogenous growth factors; in the presence of growth factors the cells proliferate, but cannot be passaged; stromal sublayer cells cannot be passaged as well. Intraperitoneal and intravenous injections of nonadherent cells to recipients with normal and radiation-attenuated immunity induced no tumor growth. Hence, peculiar dynamics of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be explained by neoplastic transformation.

Abnormal hemopoiesis in long-term bone marrow culture from tumor necrosis factor-deficient mice.

We studied hemopoiesis in mice deficient by the tumor necrosis factor gene. The total number of cells in long-term bone marrow cultures from these mice 2-fold surpassed that in wild-type and tumor necrosis factor p55 receptor-deficient animals. Increased cell production was related to the absence of tumor necrosis factor expression by hemopoietic precursors. The total cell production by explanted hemopoietic cells from tumor necrosis factor-deficient mice did not depend on the genotype of irradiated stromal sublayer in long-term cell cultures from wild-type mice and animals deficient by tumor necrosis factor or p55 receptor. These results suggest that tumor necrosis factor, but not its p55 receptor, is involved in transduction of signals regulating production of cultured cells. Tumor necrosis factor probably regulates hemopoiesis in long-term bone marrow cultures by initiating apoptosis of hemopoietic cells or inhibiting cell proliferation. Increased cell production probably attests to the absence of one or both effects.

Radiation-induced hemopoietic cell growth factor: detection in a culture.

A simple, rapid, and easily reproducible method was developed for testing activity stimulating the growth of hemopoietic microenvironment in long-term bone marrow culture. It was found that in irradiated mice this activity is produced by bones.

Changes in the hemopoietic system of mice deficient for tumor necrosis factor or lymphotoxin-alpha.

Hemopoietic and stromal precursor cells were studied in mice deficient for tumor necrosis factor or lymphotoxin-alpha. In normal hemopoiesis the main characteristics of hemopoiesis in knockout mice did not differ from those in wild-type mice. Implantation of bone marrow cells from mice deficient for tumor necrosis factor onto irradiated sublayer of a long-living bone marrow culture led to a notable increase in the number of mature cells and granulocytic-macrophage precursor cells. This can be due to the fact that tumor necrosis factor inhibits proliferation of hemopoietic precursor cells, while in the absence of this factor precursor cells actively proliferate. On the other hand, cell composition and number of colony-forming units of granulocytes-macrophages are significantly decreased in cultures onto which bone marrow cells from lymphotoxin-alpha-deficient mice were implanted. This can be explained by impaired expression of adhesion molecules in these animals. In addition, the number of stromal precursor cells was changed in mice deficient by genes of the tumor necrosis factor cluster.

Clone-forming activity of embryonal stem hemopoietic cells after transplantation to newborn or adult sublethally irradiated mice.

Hemopoietic activity of stem hemopoietic cells from the liver of embryos was studied at different terms of intrauterine development. The fate of individual clones of hemopoietic cells marked by human adenosine deaminase gene was followed up in sublethally irradiated or newborn recipients. The efficiency of marker gene incorporation in primitive stem hemopoietic cells from the liver of 12-, 13-, and 17-day embryos was not high. Gene transfer was performed without cell prestimulation to division, and hence, these data show that primitive stem cells proliferate even in 17-day embryos. Cells from embryonal liver in all terms maintain hemopoiesis both in newborn and adult microenvironment, hemopoiesis being realized according to the clonal succession model, i. e. in the some way after transplantation of the bone marrow from adult mice.