Ultralow-dose irradiation enables engraftment and intravital tracking of disease initiating niches in clonal hematopoiesis.

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion, governing progression, and leukemic transformation of hematological clonal disorders. However, our ability to interrogate the niche in pre-malignant conditions has been limited, as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here, we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity, cell viability, and HSC-supportive functions of the stromal cells, suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2+/- and its healthy counterpart, showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably, stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context.

Single-Cell Transcriptomics Reveals Early Effects of Ionizing Radiation on Bone Marrow Mononuclear Cells in Mice.

Ionizing radiation exposure can cause damage to diverse tissues and organs, with the hematopoietic system being the most sensitive. However, limited information is available regarding the radiosensitivity of various hematopoietic cell populations in the bone marrow due to the high heterogeneity of the hematopoietic system. In this study, we observed that granulocyte-macrophage progenitors, hematopoietic stem/progenitor cells, and B cells within the bone marrow showed the highest sensitivity, exhibiting a rapid decrease in cell numbers following irradiation. Nonetheless, neutrophils, natural killer (NK) cells, T cells, and dendritic cells demonstrated a certain degree of radioresistance, with neutrophils exhibiting the most pronounced resistance. By employing single-cell transcriptome sequencing, we investigated the early responsive genes in various cell types following irradiation, revealing that distinct gene expression profiles emerged between radiosensitive and radioresistant cells. In B cells, radiation exposure led to a specific upregulation of genes associated with mitochondrial respiratory chain complexes, suggesting a connection between these complexes and cell radiosensitivity. In neutrophils, radiation exposure resulted in fewer gene alterations, indicating their potential for distinct mechanisms in radiation resistance. Collectively, this study provides insights into the molecular mechanism for the heterogeneity of radiosensitivity among the various bone marrow hematopoietic cell populations.

Biomarkers for Radiation Biodosimetry and Correlation with Hematopoietic Injury in a Humanized Mouse Model.

After a large-scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation and require subsequent medical management. Acute exposure to moderate doses (2-6 Gy) of radiation can lead to the hematopoietic acute radiation syndrome, in which the bone marrow (BM) is severely compromised, and severe hemorrhage and infection are common. Previously, we have developed a panel of intracellular protein markers (FDXR, ACTN1, DDB2, BAX, p53 and TSPYL2), designed to reconstruct absorbed radiation dose from human peripheral blood (PB) leukocyte samples in humanized mice up to 3 days after exposure. The objective of this work was to continue to use the humanized mouse model to evaluate biomarker dose-/time- kinetics in human PB leukocytes in vivo, at an earlier (day 2) and later (day 7) time point, after exposure to total-body irradiation (TBI) doses of 0 to 2 Gy of X rays. In addition, to assess hematological sensitivity and radiation-induced injury, PB leukocyte cell counts, human BM hematopoietic stem cell (HSC) and progenitor cell [multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte myeloid progenitor (GMP), megakaryocyte/erythrocyte progenitor (MEP) and multi-lymphoid progenitor (MLP)] levels were measured, and their correlation was also examined as the BM damages are difficult to assess by routine tests. Peripheral blood B-cells were significantly lower after TBI doses of 0.5 Gy on day 2 and 2 Gy on days 2 and 7; T-cells were significantly reduced only on day 2 after 2 Gy TBI. Bone marrow HSCs and MPP cells showed a dose-dependent depletion after irradiation with 0.5 Gy and 2 Gy on day 2, and after 1 Gy and 2 Gy on day 7. Circulating B cells correlated with HSCs, MPP and MLP cells on day 2, whereas T cells correlated with MPP, and myeloid cells correlated with MLP cells. On day 7, B cells correlated with MPP, CMP, GMP and MEP, while myeloid cells correlated with CMP, GMP and MEP. The intracellular leukocyte biomarkers were able to discriminate unirradiated and irradiated samples at different time points calculated by receiver operating characteristic (ROC) curve. Using machine learning algorithm methods, combining ACTN1, p53, TSPYL2 and PB-T cell and PB-B cell counts served as a strong predictor (area under the ROC >0.8) to distinguish unirradiated and irradiated samples independent of the days after TBI. The results further validated our biomarker-based triage assay and additionally evaluated the radiation sensitivity of the hematopoietic system after TBI exposures.

Insights into ionizing radiation-induced bone marrow hematopoietic stem cell injury.

With the widespread application of nuclear technology across various fields, ionizing radiation-induced injuries are becoming increasingly common. The bone marrow (BM) hematopoietic tissue is a primary target organ of radiation injury. Recent researches have confirmed that ionizing radiation-induced hematopoietic dysfunction mainly results from BM hematopoietic stem cells (HSCs) injury. Additionally, disrupting and reshaping BM microenvironment is a critical factor impacting both the injury and regeneration of HSCs post radiation. However, the regulatory mechanisms of ionizing radiation injury to BM HSCs and their microenvironment remain poorly understood, and prevention and treatment of radiation injury remain the focus and difficulty in radiation medicine research. In this review, we aim to summarize the effects and mechanisms of ionizing radiation-induced injury to BM HSCs and microenvironment, thereby enhancing our understanding of ionizing radiation-induced hematopoietic injury and providing insights for its prevention and treatment in the future.

Ultraviolet Light-Emitting Diode Radiation Kills Leukemia Cells Without Affecting Hematopoiesis of Hematopoietic Stem Cells in Mice.

OBJECTIVES: The eradication of leukemia cells while sparing hematopoietic stem cells in the graft before autologous hematopoietic stem cell transplant is critical to prevention of leukemia relapse. Proliferating cells have been shown to be more prone to apoptosis than differentiated cells in response to ultraviolet radiation; however, whether leukemia cells are more sensitive to ultraviolet LED radiation than hematopoietic stem cells remains unclear. MATERIALS AND METHODS: We compared the in vitro responses between murine leukemia L1210 cells and murine hematopoietic stem cells to 280-nm ultraviolet LED radiation. We also investigated the effects of ultraviolet LED radiation on the tumorigenic and metastatic capacity of L1210 cells and hematopoietic stem cell hematopoiesis in a mouse model of hematopoietic stem cell transplant. RESULTS: L1210 cells were more sensitive to ultraviolet LED radiation than hematopoietic stem cells in vitro, as evidenced by significantly reduced colony formation rates and cell proliferation rates, along with remarkably increased apoptosis rates in L1210 cells. Compared with corresponding unirradiated cells, ultraviolet LED-irradiated L1210 cells failed to generate palpable tumors in mice, whereas ultraviolet LED-irradiated bone marrow cells restored hematopoiesis in vivo. Furthermore, transplant with an irradiated mixture of L1210 cells and bone marrow cells showed later onset of leukemia, milder leukemic infiltration, and prolonged survival in mice, compared with unirradiated cell transplant. CONCLUSIONS: Our results suggest that ultraviolet LED radiation can suppress the proliferative and tumorigenic abilities of leukemia cells without reducing the hematopoietic reconstitution capacity of hematopoietic stem cells, serving as a promising approach to kill leukemia cells in autograft before autologous hematopoietic stem cell transplant.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

BACKGROUND: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has yet to be elucidated. METHODS: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 h post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. RESULTS: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. CONCLUSIONS: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

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.

COMPARISON OF THE PROLIFERATIVE RESPONSES OF HEMATOPOIETIC STEM CELLS EXPOSED TO LOW DOSE RATE RADIATION IN VIVO AND EX VIVO.

The hematopoietic stem cells (HSCs) are sensitive to radiation. Chronic exposure to low dose rate (LDR) radiation at 20 mGy/day results in a decrease in the number of HSCs and an increase of leukemia. In this study, the proliferative capacities of ex vivo HSCs, exposed to 20 mGy/day of gamma-rays for 20 days, were compared with those of in vivo HSCs from similarly whole-body-irradiated mice. Radiation suppressed the growth of the ex vivo HSCs after Day 16 of irradiation and until Day 7 post-exposure. Almost all types of cells, particularly multipotent progenitors, common myeloid progenitors, granulocytes and macrophages, were significantly reduced in number at Day 20 of irradiation and Day 7 post-exposure in culture. HSCs and multipotent progenitors irradiated in vivo, however, decreased transiently and recovered by Day 7 post-exposure. These findings suggest that the microenvironment in vivo protects HSCs from the effects of LDR radiation.

Age and Sex Divergence in Hematopoietic Radiosensitivity in Aged Mouse Models of the Hematopoietic Acute Radiation Syndrome.

The hematopoietic system is highly sensitive to stress from both aging and radiation exposure, and the hematopoietic acute radiation syndrome (H-ARS) should be modeled in the geriatric context separately from young for development of age-appropriate medical countermeasures (MCMs). Here we developed aging murine H-ARS models, defining radiation dose response relationships (DRRs) in 12-month-old middle-aged and 24-month-old geriatric male and female C57BL/6J mice, and characterized diverse factors affecting geriatric MCM testing. Groups of approximately 20 mice were exposed to ∼10 different doses of radiation to establish radiation DRRs for estimation of the LD50/30. Radioresistance increased with age and diverged dramatically between sexes. The LD50/30 in young adult mice averaged 853 cGy and was similar between sexes, but increased in middle age to 1,005 cGy in males and 920 cGy in females, with further sex divergence in geriatric mice to 1,008 cGy in males but 842 cGy in females. Correspondingly, neutrophils, platelets, and functional hematopoietic progenitor cells were all increased with age and rebounded faster after irradiation. These effects were higher in aged males, and neutrophil dysfunction was observed in aged females. Upstream of blood production, hematopoietic stem cell (HSC) markers associated with age and myeloid bias (CD61 and CD150) were higher in geriatric males vs. females, and sex-divergent gene signatures were found in HSCs relating to cholesterol metabolism, interferon signaling, and GIMAP family members. Fluid intake per gram body weight decreased with age in males, and decreased after irradiation in all mice. Geriatric mice of substrain C57BL/6JN sourced from the National Institute on Aging were significantly more radiosensitive than C57BL/6J mice from Jackson Labs aged at our institution, indicating mouse source and substrain should be considered in geriatric radiation studies. This work highlights the importance of sex, vendor, and other considerations in studies relating to hematopoiesis and aging, identifies novel sex-specific functional and molecular changes in aging hematopoietic cells at steady state and after irradiation, and presents well-characterized aging mouse models poised for MCM efficacy testing for treatment of acute radiation effects in the elderly.

Upregulation of SIRT1 Contributes to dmPGE2-dependent Radioprotection of Hematopoietic Stem Cells.

Exposure to potentially lethal high-dose ionizing radiation results in bone marrow suppression, known as the hematopoietic acute radiation syndrome (H-ARS), which can lead to pancytopenia and possible death from hemorrhage or infection. Medical countermeasures to protect from or mitigate the effects of radiation exposure are an ongoing medical need. We recently reported that 16,16 dimethyl prostaglandin E2 (dmPGE2) given prior to lethal irradiation protects hematopoietic stem (HSCs) and progenitor (HPCs) cells and accelerates hematopoietic recovery by attenuating mitochondrial compromise, DNA damage, apoptosis, and senescence. However, molecular mechanisms responsible for the radioprotective effects of dmPGE2 on HSCs are not well understood. In this report, we identify a crucial role for the NAD+-dependent histone deacetylase Sirtuin 1 (Sirt1) downstream of PKA and CREB in dmPGE2-dependent radioprotection of hematopoietic cells. We found that dmPGE2 increases Sirt1 expression and activity in hematopoietic cells including HSCs and pharmacologic and genetic suppression of Sirt1 attenuates the radioprotective effects of dmPGE2 on HSC and HPC function and its ability to reduce DNA damage, apoptosis, and senescence and stimulate autophagy in HSCs. DmPGE2-mediated enhancement of Sirt1 activity in irradiated mice is accompanied by epigenetic downregulation of p53 activation and inhibition of H3K9 and H4K16 acetylation at the promoters of the genes involved in DNA repair, apoptosis, and autophagy, including p53, Ku70, Ku80, LC3b, ATG7, and NF-κB. These studies expand our understanding of intracellular events that are induced by IR but prevented/attenuated by dmPGE2 and suggest that modulation of Sirt1 activity may facilitate hematopoietic recovery following hematopoietic stress. Graphical Abstract.

Identifying serum miRNA biomarkers for radiation exposure in hematopoietic humanized NSG-SGM3 mice.

BACKGROUND: Humans are commonly exposed to ionizing radiation. The conventional approach for estimating radiation exposure is to integrate physical and clinical measurements for optimizing the dose calculation. However, these methods have several limitations. The present study attempted to identify candidate microRNA (miRNA) biomarkers for radiation exposure in a hematopoietic humanized NSGS (hu-NSGS) mouse model. METHODS: We grafted human CD34+ hematopoietic stem cells into NSG-SGM3 (NSGS) mice. The hu-NSGS mice underwent total body irradiation at doses of 2, 3, and 4 Gy. Tissues from the spleen, thymus, and lymph nodes of hu-NSGS mice were prepared to analyze levels of CD45+ and CD3+ T cells and CD 20+ B cells using flow cytometry and immunohistochemistry. Serum miRNAs were profiled using a digital multiplexed NanoString n-Counter. RESULTS: The expression of 45 miRNAs was upregulated/downregulated hu-NSGS mice. The miRNAs hsa-mir-188-5p, hsa-let-7a-5p, hsa-mir-612, hsa-mir-671-5p, and hsa-mir-675-5p were highly radiation-responsive in irradiated hu-NSGS mice. When compared with control mice, radiation-exposed mice exhibited significant upregulated of hsa-let-7a-5p expression and significant downregulation of hsa-mir-188-5p expression. CONCLUSIONS: Single miRNAs or combinations of hsa-mir-188-5p, hsa-let-7a-5p, hsa-mir-675-5p, hsa-mir-612, and hsa-mir-671-5p can be used as biomarkers for predicting the impact of radiation exposure. The current findings suggest the usefulness of hu-NSGS models for investigating radiation biomarkers.

Environmental radiation on large Japanese field mice in Fukushima reduced colony forming potential in hematopoietic progenitor cells without inducing genomic instability.

PURPOSE: To study the environmental radiation effects of wild animals after the Fukushima Dai-ichi nuclear power plant accident, we assessed effects on hematopoietic progenitor cells (HPCs) in large Japanese field mice (Apodemus speciosus). MATERIALS AND METHODS: A. speciosus were collected from three contaminated sites and control area. The air dose-rates at the control and contaminated areas were 0.96 ± 0.05 µGy/d (Hirosaki), 14.4 ± 2.4 µGy/d (Tanashio), 208.8 ± 31.2 µGy/d (Ide), 470.4 ± 93.6 µGy/d (Omaru), respectively. We investigated possible DNA damage and pro-inflammatory markers in the bone marrow (BM) cells. The colony-forming potential of BM cells was estimated by the number of HPC colony-forming cells. Radiation-induced genomic instability (RIGI) in HPCs was also analyzed by quantifying delayed DNA damage in CFU-GM clones. RESULTS: Although no significant differences in DNA damage and inflammation markers in BM cells from control and contaminated areas, the number of HPC colonies exhibited an inverse correlation with air dose-rate. With regard to RIGI, no significant differences in DNA damage of CFU-GM clones between the mice from the control and the three contaminated areas. CONCLUSIONS: Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

Residual effects of busulfan and irradiation on murine hematopoietic stem and progenitor cells.

Exposure of young C57BL/6 (B6) mice to two courses of busulfan (BSF) injections or two rounds of sublethal total-body irradiation (TBI) induced significant damage to the function of hematopoietic stem and progenitor cells (HSPCs). Fifteen weeks after treatment, BSF- and TBI-treated mice had reduced white blood cells without significant change in red blood cells or platelets, indicating that BSF and TBI hematotoxicity was chronic, with leukocytes being the major targets. Hematopoietic damage induced by BSF or TBI persisted long term. Residual adverse effects were reflected by significantly decreased CD45R B cells and reduced recovery of total bone marrow cells, especially HSPCs carrying markers for KSL (Kit+Sca-1+Lin-) cells, multipotent progenitor (MPP) cells (KSLCD34+CD135+), myeloid progenitor (MP) cells (Kit+Sca-1-Lin-), and common lymphoid progenitor (CLP) cells 62 wk posttreatment. Transplantation of bone marrow (BM) cells from BSF and TBI donors at 49 weeks after treatment into lethally irradiated hosts resulted in decreased engraftment of CD45R B cells in blood and reduced reconstitution of BM HSPCs including KSL cells, short-term hematopoietic stem cells (KSLCD34+CD135-), MPP cells, and MP cell subsets. TBI donor had better reconstitution of CLP cells in recipients posttransplantation than did BSF donor, suggesting an impact of TBI and BSF on B cells at different development stages. In summary, BSF and TBI exposure produced long-lasting adverse effects on hematopoiesis with pronounced effects on mature B cells, immature ST-HSCs, and hematopoietic progenitor cells. Our results may have implications for therapy of human diseases.

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.

Caffeic acid attenuates irradiation-induced hematopoietic stem cell apoptosis through inhibiting mitochondrial damage.

Hematopoietic stem cells (HSCs) are sensitive to ionizing radiation (IR) damage, and its injury is the primary cause of bone marrow (BM) hematopoietic failure and even death after exposure to a certain dose of IR. However, the underlying mechanisms remain incompletely understood. Here we show that mitochondrial oxidative damage, which is characterized by mitochondrial reactive oxygen species overproduction, mitochondrial membrane potential reduction and mitochondrial permeability transition pore opening, is rapidly induced in both human and mouse HSCs and directly accelerates HSC apoptosis after IR exposure. Mechanistically, 5-lipoxygenase (5-LOX) is induced by IR exposure and contributes to IR-induced mitochondrial oxidative damage through inducing lipid peroxidation. Intriguingly, a natural antioxidant, caffeic acid (CA), can attenuate IR-induced HSC apoptosis through suppressing 5-LOX-mediated mitochondrial oxidative damage, thus protecting against BM hematopoietic failure after IR exposure. These findings uncover a critical role for mitochondria in IR-induced HSC injury and highlight the therapeutic potential of CA in BM hematopoietic failure induced by IR.

DNA damage response of haematopoietic stem and progenitor cells to high-LET neutron irradiation.

The radiosensitivity of haematopoietic stem and progenitor cells (HSPCs) to neutron radiation remains largely underexplored, notwithstanding their potential role as target cells for radiation-induced leukemogenesis. New insights are required for radiation protection purposes, particularly for aviation, space missions, nuclear accidents and even particle therapy. In this study, HSPCs (CD34+CD38+ cells) were isolated from umbilical cord blood and irradiated with 60Co γ-rays (photons) and high energy p(66)/Be(40) neutrons. At 2 h post-irradiation, a significantly higher number of 1.28 ± 0.12 γ-H2AX foci/cell was observed after 0.5 Gy neutrons compared to 0.84 ± 0.14 foci/cell for photons, but this decreased to similar levels for both radiation qualities after 18 h. However, a significant difference in late apoptosis was observed with Annexin-V+/PI+ assay between photon and neutron irradiation at 18 h, 43.17 ± 6.10% versus 55.55 ± 4.87%, respectively. A significant increase in MN frequency was observed after both 0.5 and 1 Gy neutron irradiation compared to photons illustrating higher levels of neutron-induced cytogenetic damage, while there was no difference in the nuclear division index between both radiation qualities. The results point towards a higher induction of DNA damage after neutron irradiation in HSPCs followed by error-prone DNA repair, which contributes to genomic instability and a higher risk of leukemogenesis.

Thrombopoietin from hepatocytes promotes hematopoietic stem cell regeneration after myeloablation.

The bone marrow niche plays critical roles in hematopoietic recovery and hematopoietic stem cell (HSC) regeneration after myeloablative stress. However, it is not clear whether systemic factors beyond the local niche are required for these essential processes in vivo. Thrombopoietin (THPO) is a key cytokine promoting hematopoietic rebound after myeloablation and its transcripts are expressed by multiple cellular sources. The upregulation of bone marrow-derived THPO has been proposed to be crucial for hematopoietic recovery and HSC regeneration after stress. Nonetheless, the cellular source of THPO in myeloablative stress has never been investigated genetically. We assessed the functional sources of THPO following two common myeloablative perturbations: 5-fluorouracil (5-FU) administration and irradiation. Using a Thpo translational reporter, we found that the liver but not the bone marrow is the major source of THPO protein after myeloablation. Mice with conditional Thpo deletion from osteoblasts and/or bone marrow stromal cells showed normal recovery of HSCs and hematopoiesis after myeloablation. In contrast, mice with conditional Thpo deletion from hepatocytes showed significant defects in HSC regeneration and hematopoietic rebound after myeloablation. Thus, systemic THPO from the liver is necessary for HSC regeneration and hematopoietic recovery in myeloablative stress conditions.

Altered Induction of Reactive Oxygen Species by X-rays in Hematopoietic Cells of C57BL/6-Tg (CAG-EGFP) Mice.

Previous work pointed to a critical role of excessive production of reactive oxygen species (ROS) in increased radiation hematopoietic death in GFP mice. Meanwhile, enhanced antioxidant capability was not demonstrated in the mouse model of radio-induced adaptive response (RAR) using rescue of radiation hematopoietic death as the endpoint. ROS induction by ex vivo X-irradiation at a dose ranging from 0.1 to 7.5 Gy in the nucleated bone marrow cells was comparatively studied using GFP and wild type (WT) mice. ROS induction was also investigated in the cells collected from mice receiving a priming dose (0.5 Gy) efficient for RAR induction in WT mice. Significantly elevated background and increased induction of ROS in the cells from GFP mice were observed compared to those from WT mice. Markedly lower background and decreased induction of ROS were observed in the cells collected from WT mice but not GFP mice, both receiving the priming dose. GFP overexpression could alter background and induction of ROS by X-irradiation in hematopoietic cells. The results provide a reasonable explanation to the previous study on the fate of cells and mice after X-irradiation and confirm enhanced antioxidant capability in RAR. Investigations involving GFP overexpression should be carefully interpreted.

Radiation-Induced Senescence in p16+/LUC Mouse Lung Compared to Bone Marrow Multilineage Hematopoietic Progenitor Cells.

We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-ß. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and ß-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis.