HEMATOPOIETIC PROGENITORS CELLS IN PERIPHERAL BLOOD OF BALB/C MICE UNDER IONIZING RADIATION ACTION IN SUBLETHAL DOSE. / ГЕМОПОЕТИЧНІ КЛІТИНИ/ПОПЕРЕДНИКИ У ПЕРИФЕРИЧНІЙ КРОВІ МИШЕЙ BALB/C ЗА ДІЇ ІОНІЗУЮЧОЇ РАДІАЦІЇ У СУБЛЕТАЛЬНІЙ ДОЗІ.

OBJECTIVE: determination of the content of hematopoietic progenitor cells circulating in peripheral blood of Balb/Cmice, under ionizing radiation action in sublethal dose, at different periods after the irradiation, using cell culturein diffusion chambers in vivo. METHODS: Peripheral blood smears of Balb/C mice were prepared and studied, its cellular composition was determined, as well as by cultivation of peripheral blood cells in diffusion chambers in vivo their colony-forming efficien-cy was determined on the 0th, 5th, and 30th day after external irradiation in sublethal dose 5.85 Gy. RESULTS: The content of myelocytes and metamyelocytes among blood nucleated cells of the irradiated animals wasincreased, compared to control, during the whole investigated period. In particular, on the 30th day after irradiationthe content of myelocytes in peripheral blood was 3.3 ± 0.7 % compared to (0.8 ± 0.4) % in control, and the content of metamyelocytes - (3.4 ± 0.7) % compared to (0.9 ± 0.3) % in control. A significant increase in the amountof circulating progenitor cells in the peripheral blood was observed in the early stages after irradiation (12.5 ± 1.6colony-forming units per 100,000 explanted cells, compared to 5.1 ± 0.8 in control). However, on the 5th day theircontent was slightly reduced compared to control (1.3 ± 0.9), and only to the 30th day a normalization of the amountof progenitor cells occurred in the peripheral blood (6.8 ± 0.7 colony-forming units per 100,000 explanted cells). CONCLUSIONS: The analysis of the obtained results revealed an increased level of immature forms of cells in theperipheral blood of irradiated animals, compared to control, in the early stages after irradiation, includinghematopoietic progenitor cells, which are able to colony forming in cell culture. Therefore, the action of ionizingradiation in sublethal dose had a critical effect on the proliferation of hematopoietic cells in bone marrow and provoked their increased migration into the bloodstream. Determination of the content of hematopoietic cells' immature forms in peripheral blood allowed assessing the degree of hematopoietic damage due to the action of ionizing radiation.

ANALYSIS OF THE INFLUENCE OF PROLONGED IRRADIATION ON HEMATOPOIETIC PROGENITOR CELLS IN GEL DIFFUSION CHAMBERS USING MATHEMATICAL MODELLING. / ANALIZ VPLYVU PROLONGOVANOGO OPROMINENNIa NA GEMOPOETYChNI KLITYNY-POPEREDNYKY U GELEVYKh DYFUZIINYKh KAMERAKh ZA DOPOMOGOIu MATEMATYChNOGO MODELIuVANNIa.

OBJECTIVE: determining of the functional activity of mice bone marrow hematopoietic progenitor cells, cultivated in gel diffusion chambers, on the stages of hematopoiesis recovery after their prolonged irradiation in the lethal dose in a comparative aspect with the method of colony forming in spleen using mathematical model. MATERIALS AND METHODS: The method of cell cultivation in gel diffusion chambers, cytological methods, mathematical modeling, and statistical methods of research were used. Bone marrow samples extracted from the femur of mice irradiated with a total dose of 8 Gy with a power 0.0028 Gy/min were cultivated in diffusion chambers with semi solid agar in the abdominal cavity of CBA recipient mice. RESULTS: Comparative analysis of the colonyforming efficiency of progenitor cells (CFU) was carried out during cultivation in gel diffusion chambers in the process of hematopoiesis recovery for 30 days, as well as in the spleen of lethally irradiated animals, in accordance with the mathematical model. Analysis of colony forming kinetics in gel diffusion chambers after prolonged exposure to ionizing radiation indicated the biphasic nature of hematopoiesis recovery. Thus, in the first few days after the irradiation a drop in the number of CFU is observed compared to the control, which continues until the 9th day. Subsequently there is a sharp increase in the number of CFU in cell culture, which continues until the complete recovery of hematopoiesis. The obtained data, recalculated per mouse femur, correspond to the results of colony forming in the spleen of irradiated animals, described by K. S. Chertkov and taken as a basis while developing our mathematical model, as well as to its parameters, which describe the process of hematopoiesis recovery. CONCLUSIONS: Conformity of the indices obtained during the cultivation using the method of gel diffusion chambers of mice bone marrow prolongedly irradiated at a total dose of 8 Gy with a power 0.0028 Gy/min, to the results of colony forming in spleen of lethally irradiated mice, which were the basis for mathematical model development, is the evidence of the feasibility of using a mathematical model to assess the process of hematopoiesis recovery by progenitor cells of different maturation levels, and the experimental approach of CFU growing in gel diffusion chambers can be considered as an additional method of researching the hematopoiesis recovery along with the spleen colony method.

HUMANIZED MODEL OF ISOLATED SUSPENSION CULTIVATION OF HEMATOPOIETIC PROGENITOR CELLS FOR THE INVESTIGATION OF IONIZING RADIATION INFLUENCE IN VIVO. / GUMANIZOVANA MODEL' KUL'TYVUVANNIa IZOL'OVANOÏ SUSPENZIÏ GEMOPOETYChNYKh KLITYN/POPEREDNYTs' DLIa VYVChENNIa VPLYVU IONIZUIuChOÏ RADIATsIÏ IN VIVO.

OBJECTIVE: development of the humanized system for cells cultivation outside the human organism (human-mouse)and investigation of the influence of ionizing radiation in increasing doses on the colony-forming ability ofhematopoietic progenitor cells. MATERIALS AND METHODS: Bone marrow samples of individuals without blood system diseases were cultivated in geldiffusion chambers with semi-solid agar in the abdominal cavity of CBA mice exposed to ionizing radiation action.Cell aggregates, which were obtained in the culture of diffusion chambers in vivo, were counted and colony-formingefficiency of bone marrow cells was determined. RESULTS: We revealed the stimulation of colony forming under the action of ionizing radiation in increasing doseson the animals-recipients of the chambers, which indirectly indicates the synthesis of colony-stimulating factor inthe mice organism and its permeation into the diffusion chambers with human bone marrow cells. The effect of cyto-statics action on the mice organism was investigated, which in experimentally selected dose cause stimulation ofcolony forming in cell cultures, both 24 hours and 2 hours after administration. CONCLUSIONS: The ability of hematopoietic progenitor cells of bone marrow to form colonies and clusters was eval-uated during the cultivation in semi-solid agar in gel diffusion chambers in vivo, as well as the association with thenumber of explanted cells in the appropriate range was established, which indicates the clonal nature of cell aggre-gates growth in culture. It was shown that the treatment of animals the day prior to experiment with administra-tion of cytostatics is comparable to the action of ionizing radiation and can be used to study hematopoiesis in«human-mouse¼ system.

COMPARATIVE MATHEMATICAL ANALYSIS OF COLONY-FORMING ABILITYOF THE BONE MARROW OF MICE LETHALLY AND NON-LETHALLY IRRADIATED WITH EQUAL DOSE RATE INTENSITY. / PORIVNIaL'NYI MATEMATYChNYI ANALIZ KOLONIIeUTVORIuIuChOÏ ZDATNOSTI KISTKOVOGO MOZKU MYShEI, OPROMINENYKh Z ODNAKOVOIu POTUZhNISTIu U LETAL'NII I NELETAL'NII DOZAKh.

OBJECTIVE: To perform comparative analysis of the characteristics of population functioning process of mice bonemarrow colony-forming units after their prolonged irradiation in lethal and non-lethal doses with equal dose rateintensity with the aid of mathematical model. MATERIALS AND METHODS: Assigned task is solved by means of mathematical model of alterations in the number ofbone marrow colony-forming units after continuous irradiation, described in previous works, with the use of experimental results of K. S. Chertkov works (1972, 1973). Mathematical model is developed basing on the hematopoiesisscheme introduced by I. L. Chertkov (1984, 1991). RESULTS AND CONCLUSIONS: By applying original mathematical model, new scheme of hematopoiesis [6], with theuse of experimental results of γ-irradiation influence in the doses of 4 Gy and 8 Gy with the dose rate intensity of0.0028 Gy/min on the number of mice bone marrow colony-forming units, as well as experimental data concerningthe processes of their number recovery, obtained from literature references, we determined the parameters, whichcharacterize hematopoietic system reaction on the different stages of recovery processes of mice bone marrowcolony-forming units after the termination of ionizing radiation action; comparative analysis of obtained resultswas performed.

ASSESSMENT OF RADIOPROTECTIVE ACTION OF BASIDIOMYCOTIC MELANIN PIGMENTS ON THE HEMATOPOIETIC SYSTEM OF Balb/C MICE UNDER EXPOSURE TO IONIZING RADIATION IN SUBLETHAL DOSE. / OTsINKA RADIOPROTEKTORNOÏ DIÏ MELANINOVYKh PIGMENTIV BAZYDIOMITsETIV NA GEMOPOETYChNU SYSTEMU MYShEY̆ Balb/C PRY OPROMINENNI IONIZUIuChOIu RADIATsIIeIu U SUBLETAL'NIY̆ DOZI.

OBJECTIVE: Assessment of radioprotective action of basidiomycotic melanin pigments on hematopoietic stem and progenitor bone marrow cells of Balb/C mice in case of exposure to ionizing radiation in sublethal dose. MATERIALS AND METHODS: Using original method of cultivation in gel diffusion chambers in vivo of bone marrow cells of Balb/C mice we investigated the colony-forming efficiency of hematopoietic progenitor cells of the ani- mals, which were exposed to ionizing radiation action in sublethal dose, in case of treatment with melanin pig- ments solution of basidiomycotic fungi as radioprotector. RESULTS AND CONCLUSIONS: Investigation of functional activity of bone marrow progenitor cells of Balb/C mice allowed assessing their hematopoiesis state in case of ionizing radiation action, as well as in case of previous treat- ment of the animals with the solution of melanin pigments as radioprotector. It was determined that under the influence of ionizing radiation the colony-forming activity of mice bone marrow has decreased comparing to con- trol. Solution of melanin pigments was able to enhance the functional activity of bone marrow of irradiated ani- mals. Obtained results of radioprotective action of basidiomycotic melanin pigments solution on irradiated stem cells and their descendants (progenitor cells) may become the evidence for development of the protective means for human organism from the injuring action of ionizing radiation.

MATHEMATICAL ANALYSIS OF FUNCTIONAL PROPERTIES OF MICE BONE MARROW IN THE INITIAL PHASE OF ACUTE FRACTIONATED IRRADIATION. / MATEMATYChNYI ANALIZ FUNKTsIONAL'NYKh VLASTYVOSTEI KISTKOVOGO MOZKU MYShEI U POChATKOVII FAZI GOSTROGO FRAKTsIONOVANOGO OPROMINENNIa.

OBJECTIVE: to determine the quantitative characteristics of population functioning of mice bone marrow colony-forming units during seven days of acute fractionated irradiation. MATERIALS AND METHODS: Assigned task is solved by means of described in works R. V. Boiko et al. (2015, 2016) math-ematical model of alterations in the number of bone marrow colony-forming units using the experimental results ofwork W.Chu-Tse, L. G.Lajtha (1975). Mathematical model is developed basing on the new hematopoiesis scheme,which was introduced by I. Chertkov(1984, 1991). RESULTS: By applying original mathematical model of hematopoiesis scheme using results concerning the change innumber of bone marrow colony-forming units of mice femur we determined quantitative characteristics of theirfunctioning during seven days of fractionated irradiation under daily acute γ-radiation in the dose of 0.7 Gy. CONCLUSIONS: Mathematical model is introduced, which describes changes in the relative number of colony-formingunits in mice bone marrow in the process of their acute fractionated irradiation.

Mesenchymal stem and progenitor cells of rats' bone marrow under chronic action of ionizing radiation. / MEZENKhIMAL'NI STOVBUROVI KLITYNY TA KLITYNY POPEREDNYKY KISTKOVOGO MOZKU ShchURIV V UMOVAKh KhRONIChNOÏ DIÏ IONIZUIuChOÏ RADIATsIÏ.

Under the influence of ionizing radiation on hematopoietic system, the level of its injury is determined not only by the radiosensitivity of hematopoietic stem cells, but also by radiation induced changes in microenvironment func tioning, in particular, mesenchymal stem cells as its components. OBJECTIVE: to define functioning characteristics of mesenchymal stem and progenitor cells of rats' bone marrow under prolonged action of ionizing radiation as a result of 90Sr incorporation. MATERIALS AND METHODS: We applied the model of Wistar rats' internal irradiation with 90Sr radionuclide and per formed the in vitro cultivation of their bone marrow mesenchymal cells. Colony forming efficiency in the in vitro cell culture was determined, as well as the possibility of these cells to form feeder layers and to support rat bone mar row hematopoietic cells in the culture of diffusion chambers in vitro. RESULTS AND CONCLUSIONS: We established that chronic action of incorporated 90Sr radionuclide induced considerable decrease in proliferative activity of mesenchymal stem cells comparing to control, as well as the inhibition of the capability to prolonged support of hematopoietic processes in vitro by their feeder layers.Thus, bone marrow mesenchymal stem cells and their closest progeny - progenitor cells were characterized by rather high radiosensitivity under the influence of ionizing radiation, which was revealed in considerable decline of their functional activity in cell culture in vitro comparing to control indices as a result of irradiation.

Circulating hematopoietic progenitor cells in patients affected by Chornobyl accident.

High radiation sensitivity of stem cells and their ability to accumulate sublethal radiation damage provides the basis for investigation of hematopoietic progenitors using in vivo culture methodology. Unique samples of peripheral blood and bone marrow were derived from the patients affected by Chornobyl accident during liquidation campaign. AIM: To investigate functional activity of circulating hematopoietic progenitor cells from peripheral blood and bone marrow of cleanup workers in early and remote periods after the accident at Chornobyl nuclear power plant (CNPP). MATERIALS AND METHODS: The assessment of the functional activity of circulating hematopoietic progenitor cells was performed in samples of peripheral blood and bone marrow of 46 cleanup workers, who were treated in the National Scientific Center for Radiation Medicine of the Academy of Medical Sciences of Ukraine alongside with 35 non radiated patients, who served as a control. Work was performed by culturing peripheral blood and bone marrow mononuclear cells in the original gel diffusion capsules, implanted into the peritoneal cavity of CBA mice. RESULTS: It was shown that hematopoietic progenitor cells could be identified in the peripheral blood of liquidators of CNPP accident. At the same time the number of functionally active progenitor cells of the bone marrow was significantly decreased and during the next 10 years after the accident, counts of circulating progenitor cells in the peripheral blood as well as functionally active hematopoietic cells in bone marrow returned to normal levels. CONCLUSION: It was shown that hematopoietic progenitor cells are detected not only in the bone marrow but also in the peripheral blood of liquidators as a consequence of radiation exposure associated with CNPP accident. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

Pattern changes in quantitative and qualitative markers of hematopoietic stem cells during acute and chronic exposure to 90Sr isotope in cell culture. / ZAKONOMIRNOSTI ZMIN KIL'KISNYKh I IaKISNYKh POKAZNYKIV GEMOPOETYChNYKh STOVBUROVYKh KLITYN U KUL'TURI KLITYN V UMOVAKh DIi RADIONUKLIDU STRONTsIIu-90.

OBJECTIVE: The objective of our research was investigation of the hematopoietic system of laboratory rats under the influence of acute and chronic internal exposure to 90Sr isotope. MATERIALS AND METHODS: To study the condition of stem cells and their immediate progenitors we implemented cell culture methodology in vivo in gel diffusion capsules with subsequent analysis of the colonies and clusters. RESULTS: On the basis of experiments it was established that long-term effects of incorporated 90Sr isotope leads to significant disturbances in the hematopoietic system and in particular, revealing changes in hematological parameters of irradiated animals such as the appearance of circulating progenitor cells in peripheral blood, reducing the colony-forming efficiency of the bone marrow derived progenitor cells, as well as quantitative and qualitative changes in the clones. CONCLUSIONS: Indices confirm the connection of the detected effects in individuals exposed to ionizing radiation described in the earlier publications and can serve as basis for developing criteria for the formation of risk groups among people exposed to 90Sr.

[PREIMPLANTATION DEVELOPMENT OF HUMAN EMBRYOS WITH NUMERICAL CHROMOSOME ABNORMALITIES IN VITRO].

The study was focused on morphokynetic characteristics of in vitro cultured human embryos that were considered to be aneuploid or euploid according to the preimplantation genetic screening results. Among all the embryos examined only 34.2% were chromosomally balanced, while others possessed isolated or combined chromosome abnormalities. Although morphological features of cleaving pathologic and euploid embryos did not differ significantly, on the fifth day of culture chromosomally balanced specimen formed "expanded" blastocyst twice as frequently as abnormal ones. Moreover, development of 38.4% of aneuploid embryos was compromised before the initiation of cavitation. Thus, prolonged embryo culture advances selection of samples with the highest implantation potential for the transfer on the basis of the morphokynetic characteristics and helps to avoid additional genetic testing.

Determination of the optimal chemotherapy drugs pretreatment time through cultivation of hemopoietic cells in CML-patients treated with tyrosine kinase inhibitors.

BACKGROUND: Targeted therapy drugs, including imatinib, are used for inhibiting the marker oncoprotein of chronic myeloid leukemia - BCR-ABL tyrosine kinase. However, in some patients the drug resistance can emerge too rapidly and a previous treatment with chemotherapy drugs can lead to formation of resistance. AIM: To evaluate the influence of drugs that were used prior to the imatinib on the performance of the functional activity of bone marrow cells from chronic myeloid leukemia patients and their individual responses to therapy. METHODS: Bone marrow aspirate from 57 patients, who were getting busulfan (19 patients) or hydroxycarbamide (38 patients) prior to imatinib was studied with cytogenetic and tissue culture methods in vitro. RESULTS: Obtained data suggested that pretreatment with busulfan, regardless of duration, negatively affects the response to further therapy with imatinib. Instead, after using hydroxycarbamide as a previous therapy for six month, there was optimal response to imatinib. In those cases when duration of pretreatment with hydroxycarbamide was increased to a year or more, there was a suboptimal response and a resistance to imatinib therapy. In addition, there was a positive correlation between the number of cell aggregates (colonies and clusters) in semisolid agar and the duration of a prior treatment with hydroxycarbamide, if previous therapy did not exceed 20 months. With an increase of pretreatment terms to 21 months or more, such a correlation was not observed. CONCLUSIONS: These results suggest that chemotherapeutic agents (busulfan and hydroxycarbamide) may additionally contribute to the accumulation of mutations in the genome of leukemic cell clone affecting the behavior of these cells in vitro.

Character of interaction between irradiated and non-irradiated cells in culture in diffusion chambers in vivo.

OBJECTIVE: Current investigation is devoted to determination of interaction type between the non-irradiated murine bone-marrow cells and irradiated microenvironment in cell culture in diffusion chambers in vivo. MATERIALS AND METHODS: To study the characteristics of interaction between the cells we implemented an original cultivation model with bone-marrow cells of intact Balb/c mice inserted in the diffusion chambers in peritoneum of irradiated animals (it provided the access of nutrients and signaling molecules from outside). RESULTS: As a result of investigations performed, we have determined the direct influence of microenvironment factors of irradiated mice organism on the non-irradiated bone-marrow cells. Thus, cultivation of the cells in the organism of non-irradiated mice revealed a low colony-forming activity due to the absence of additional growth factors release. At the same time an irradiation of the recipient mice and, to a lesser extent, a treatment by cytostatic agent cyclophosphamide increased the proliferative activity of bone-marrow cells in chambers, which indicated the pronounced impact of factors released by the cells as an effect of ionizing radiation. Influence of signaling molecules was realized through the wall of diffusion chamber, causing the stimulation of colony-forming activity. Moreover, we should mention that a direct contact between cells was not necessary for the delivery of a signal. CONCLUSIONS: Stimulating influence on the colony-forming activity of non-irradiated hematopoietic progenitor cells in diffusion chambers, which is performed by cellular and extra-cellular structures forming external microenvironment, is associated with the action of growth factors produced by irradiated cells and affecting non-irradiated cells through the chamber wall, which is impermeable for the cells but freely transmits diffusible molecules.

Characterization of the interactions between stromal and haematopoietic progenitor cells in expansion cell culture models.

Development of the long-term culture models of haematopoietic stem cells (HSCs) is one of the important tasks in modern biotechnology. It has been suggested that stromal presence is important for haematopoiesis in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive and definitive haematopoietic cells, or direct cell-to-cell contacts of the cultured material with underlying stromal base would be required. During present studies, influence of various feeder layers and feeder layer conditioned media on proliferative, differentiative and clonogenic activity of human AC133+ derived from human umbilical cord blood was investigated. Cell extracts for feeder layers were prepared from 4-6 weeks old human embryos and co-cultured feeder cells. Effects of the conditioned media were also determined. Culture and feeder layer media were additionally supplemented with commonly implemented factors such as GM-CSF, IL-3 and LIF. Estimation of morpho-functional properties of AC133+ cultivated suspension cultures was performed in subculture experiments using semisolid agar culture conditions. Multipotential CFU-MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were observed and analyzed. Our data suggest that haematopoiesis can be sustained for prolonged cultivation periods in the presence of feeder layer cells or conditioned media supported culture models. Prolonged support of primitive haematopoietic cells and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient for haematopoiesis and suggests that direct cell-to-cell contacts may not be exclusively required for successful long-term in vitro haematopoiesis.