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The ability of transplantable hemopoietic stem cells (HSC) to maintain their pool was studied using successive bone marrow transplantations with quantitative evaluation of hemopoiesis restoring units (HRU) in each transfer. The number of injected HRU increased (3.6-48.6--fold) upon each transfer; however, the normal level could not be attained. The ability fo HRU for further multiplication was exhausted after five transfers. HRU lost totipotentiality after four transfers. The data obtained support the concertion of Kay (1965) that HSC department is a pool of heterogeneous cells, and the property of "stemness" is inversely related to the number of divisions of ancestral cells. Transplantation, being a proliferative stress for the dormant HSCs, thus lowers the stem potential of the whole pool. The experimental data suggest that while dividing stem cell does not have a choice to self-renew or to differentiate into maturing cells, but it really differentiates into HSCs of lower rank.

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Human B-lymphoblastoid cell lines producing specific anti-D antibodies against Rh0(D) antigen have been established by EBV-transformation. The strong influence of the donor's immune status (serum titer of anti-D antibodies, intravenous booster injection prior to bleeding) is shown. The cell lines cloned by limiting dilution continuously produce anti-D antibodies in culture for 7 months or longer which is sufficient to support large-scale production of anti-D antibodies based on the cultivation of cryopreserved cells. Anti-D IgGl monoclonal antibodies obtained react with Rh-positive and Du cells but not with Rh-negative cells in serological tests. These human monoclonal antibodies could be useful in the prevention of Rh hemolytic disease of newborns.

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The concentration of hemopoiesis restoring units (HRU) in bone marrow of mice was assayed by using the limiting dilution method in transplantation to lethally irradiated mice. 7 to 12.7 HRU were found in 10(6) bone marrow cells of CBF1 mice and 19.2 to 50.6 HRU in BCT6F1 mice when the survival of the recipients was registered in 4 weeks after transplantation. The proportion of not surviving recipients increased with time when marrow doses were low (2.5 X 10(4) to 2 X 10(5) cells or 0.5-2.5 HRU per mouse) and remained stable when middle or high marrow doses (10(6)-10(7) cells) were used.

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In this article serologic characteristics of monoclonal antibodies with anti-N specificity is given. Antibodies are directed to homologous sequence of N-form of A glycophorine (group-specific N-antigen) and B glycophorine, expressed both on N- and on M-erythrocytes. Higher titre and avidity of monoclonal antibodies with anti-N specificity in relation to erythrocytes with N-phenotype make it possible to detect N-antigen in material subjected to expert evaluation.

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Serologic description of murine monoclonal antibodies (H-86/44 and H-86/50) which are capable to detect a membrane-connected form of human H antigen is given. The antibodies are notable for ability (H-86/44) to be absorbed by salivary H substance of ABH secretors i.e. to detect a soluble form of H antigen. Highly specific, highly active and standard anti-H reagents may be produced on the basis of the anti-H monoclonal antibodies obtained.

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The data on the application of monoclonal antibodies (ICO-10) and rabbit complement for working the conditions of allogeneic bone marrow transplantation are presented in the paper. The treatment with monoclonal antibodies and bone marrow complement from BALB/c mice for 2 times prevented the development of transplant versus host reaction and completely protected lethally irradiated (CBA X X C57B1/6)FI mice-recipients from death. Thymus atrophy and the absence of T-cells in the peripheral blood was observed in these mice. The erythrocytes had markers characteristic of BALB/c and (CBA X C57B1/6)FI mice. Mouse splenocytes did not respond to the cells of donors and recipients in mixed lymphocyte culture reaction.

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Long-term cultures from bone marrow of patients with chronic myeloid leukemia were obtained. The cultures were characterized by the reduced period of hemopoiesis. The enrichment of the adherent cell layer with endothelial cells or their extracellular matrix was not stimulative. The Ph'(+) malignant cells persisted in culture during the whole period of hemopoiesis. The data obtained indicate that the long-term bone marrow culture system is not suitable for the elimination of malignant cells from leukemic bone marrow.

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During serial passages of hemopoietic tissue as a whole ectopic hemopoietic site under renal capsule of irradiated mice gradual decrease of proliferative potential of CFUs has been observed. The results suggest that the most probable cause of such decline is random loss of precursor cells with high but limited proliferative potential during serial passages of hemopoietic tissue.

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Lymphoblastoid cell line was obtained from long-term human bone marrow culture. The cell line was characterized using methods of immunological phenotyping, cytochemistry and cytogenetics. This cell line represents different stages of B-cell differentiation, karyotype 46 XX. The cells of this line were able to support their growth during more than 10 months without exogenous stimulation.

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Forty seven individual haemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation, clones appeared at different times, 1 to 12 weeks after explanation, survived during 1-10 weeks and markedly varied in size. Usually, the number of metaphases characteristic of an individual clone rapidly increased, achieved a maximum and declined. The cells of disappeared clones were never seen again. The experimental results provide further evidence for the model of haemopoiesis by clonal succession. The data obtained are discussed with respect to the functioning of haemopoietic stem cell population.

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The influence of the duration of the organ cultivation on the protective potential of mouse fetal liver hemopoietic cells was investigated. The protective potential was evaluated according to a 3-week viability of the lethally irradiated (mean LD88/21) mice following syngeneic transplantation. During 20 days of cultivation the protective potential (calculated per number of injected cells) remained at the initial level. With more prolonged cultivation (24-62 days) the protective potential was retained only in part of cultures and the mean effect was reduced. No parallelism has been revealed between the totipotent hemopoietic precursors capable of replacing the hemopoietic cells of an irradiated recipient and the CFUs in long-cultivated fetal liver cultures.

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Forty-seven individual hemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation clones appeared at different times, from 1 to 12 weeks after explantation, survived during 1-10 more weeks, and were characterized by marked variability in size. Usually, the number of metaphases peculiar to an individual clone rapidly increased, achieved maximum, and then underwent a decline. Cells of reliably disappearing clones were never seen again. The experimental results provide further evidence for the model of hemopoiesis by clonal succession.

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Long-term haemopoiesis in bone marrow culture is sustained by the progeny of haemopoietic progenitor cells (HPC), which differ from CFUs by very low sensitivity to repeated hydroxyurea (HU) injections. The transit time of a haemopoietic clone from HPC to cells proliferating in culture is 6-7 weeks. The results suggest that the stem cell continuum is an expansion type compartment, members of which gradually lose their proliferative potential during differentiation.

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The CFU-s proliferative potential varied greatly during long-term cultivation. Most of the CFU-s in the cultures were represented by cells with low renewal capacity. Pre-CFU-s cells capable of producing multipotential colonies in methylcellulose, which contained CFU-s with a high proliferative potential, were identified in the culture. In cultivation of a mixture of cells of different karyotype their ratio changed rapidly from week to week. The findings were consistent with the hypothesis that haemopoietic stem cells are maintained in the culture by the products of a small number of clones which arise and decline in succession, and that pre-CFU-s, but not the CFU-s themselves, are clonogenic progenitors.

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In long-term cultures of murine bone marrow, clonal succession of hemopoietic cells was observed as measured by karyologic analysis. There were high oscillations in self-renewal of CFUs in the cultures. A close correlation between the CFUmix karyotype and mitotic non-adherent cells in culture (but not between these cell types and CFUs) was revealed.

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The adherent cell layer of long-term Dexter type bone marrow culture contains hemopoietic stromal precursors capable of transferring the hemopoietic microenvironment on transplantation in vivo. The foci of ectopic hemopoiesis that are created correspond to normal bone marrow in the character of the maintained hemopoiesis. Radiosensitivity of the cultured stromal precursors does not differ from preculture sensitivity. Stromal precursors lose osteogenic potential after 5-6 wk in culture, but are capable of creating stable foci of ectopic hemopoiesis composed of up to 10(8) nucleated hemopoietic cells. These results prove that bone is not an essential component of the hemopoietic environment in ectopic foci. In chimeras exposed to equal doses of radiation but reconstituted by freshly isolated or repeatedly passaged bone marrow, the content of stromal precursors is lower when hemopoietic cells of diminished self-maintenance are used. These data suggest the existence of a hemopoietic stromal precursor hierarchy with at least two levels of precursor differentiation.

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