[Screening for Biological Marker of Dose-optimization in Cancer Radiotherapy].

PURPOSE AND METHODS: External radiotherapy of target regions using high-energy beams leads to excessive exposure along with individual variation in therapeutic and adverse effects. However, high-precision radiotherapy utilizes 3D-multi detector computed tomography to confirm both target position and administer radiation dose. To install the individual bioinformation in the radiotherapy plan (particularly, radiosensitivity into the target region and/or the around normal tissue), the investigation of biomarkers, which are able to estimate their radiosensitivity was performed. The aim of this investigation is to screen for suitable radiosensitivity biomarkers using the human colorectal cancer-derived HCT 116 cell line. RESULTS: We found that cell damage and micronucleus frequency significantly increased dose dependently after exposure to 6 Gy X-irradiation (1 Gy/min). In contrast, total RNA concentration (69.8-85.2 ng/ml) remained stable in the cell culture supernatant despite radiation dose variation. Additionally, 52 specific micro RNAs were detected after exposure to 6 Gy X-irradiation. CONCLUSION: These results suggest that radiosensitivity, including extent of cellular damage in target or normal tissue, can be indirectly estimated by monitoring the expression of micro RNAs.

Characteristics of sphingomyelin metabolism in the MCF7 and BT474 radiotherapy­resistant HER2­positive breast cancer cell lines.

Breast cancer is the most common cancer globally in terms of incidence. This cancer is classified into subtypes based on histological or immunological characteristics. HER2-positive cases account for 15-25% of breast cancer cases, and one of the first events in breast carcinogenesis is HER2 upregulation. Furthermore, HER2 expression increases the detection rate of metastatic or recurrent breast cancers by 50-80%. The epidermal growth factor receptor family includes HER2, which is a transmembrane receptor protein. In our previous case report, patients who were resistant to anti-HER2 monoclonal antibody therapy, chemotherapy and radiotherapy had higher concentrations of phospholipid metabolites such as phosphatidylcholine and sphingomyelin (SM), which was associated with cancer recurrence progression. To better understand the relationship between radiotherapy resistance and SM expression, breast cancer cell lines with and without HER2 expression (MCF7 and BT474) after exposure to ionizing radiation (IR) were examined. In the cell culture supernatant, similar levels of SM in MCF7 cells were identified after 1-4 Gy exposure. However, SM levels in BT474 cells were upregulated compared with those of in the control group. Intracellular SM levels were upregulated in BT474 cells exposed to 1 and 4 Gy compared with the non-irradiated control group. Furthermore, significantly increased mRNA expression levels of sphingomyelin synthase 2 (SGMS2) in BT474 cells exposed to IR were observed compared with those in nonirradiated cells; however, the SGMS2 levels in MCF7 cells did not differ significantly among the 0, 2 and 4 Gy groups. These findings suggested that a higher dose of IR induced the secretion of SM and its associated gene expression in HER2-positive breast cancer cells.

Additive antitumor effect of arsenic trioxide with exposure to ionizing radiation to human acute promyelocytic leukemia HL­60 cells.

Arsenic trioxide (ATO) is expected to be a chemical drug with antitumor activity against acute promyelocytic leukemia (APL), a type of acute myeloid leukemia. In Japan, its antitumor effects were confirmed in clinical trials for APL, and it has been approved in various countries around the world. However, there have been no reports on ATO's antitumor effects on radioresistant leukemia cells, which can be developed during radiotherapy and in combination with therapeutic radiation beams. The present study sought to clarify the antitumor effect of ATO on APL cells with radiation resistance and determine its efficacy when combined with ionizing radiation (IR). The radiation­resistant HL60 (Res­HL60) cell line was generated by subjecting the native cells to 4­Gy irradiation every week for 4 weeks. The half­maximal inhibitory concentration (IC50) for cell proliferation by ATO on native cell was 0.87 µM (R2=0.67), while the IC50 for cell proliferation by ATO on Res­HL60 was 2.24 µM (R2=0.91). IR exposure increased the sub­G1 and G2/M phase ratios in both cell lines. The addition of ATO resulted in a higher population of G2/M after 24 h rather than 48 h. When the rate of change in the sub­G1 phase was examined in greater detail, the sub­G1 phase in both control cells without ATO significantly increased by exposure to IR at 24 h, but only under the condition of 2 Gy irradiation, it had continued to increase at 48 h. Res­HL60 supplemented with ATO showed a higher rate of sub­G1 change at 24 h; however, 2 Gy irradiation resulted in a decrease compared with the control. There was a significant increase in the ratio of the G2/M phase in cells after incubation with ATO for 24 h, and exposure to 2 Gy irradiation caused an even greater increase. To determine whether the inhibition of cell proliferation and cell cycle disruptions is related to reactive oxygen species (ROS) activity, intracellular ROS levels were measured with a flow cytometric assay. Although the ROS levels of Res­HL60 were higher than those of native cells in the absence of irradiation, they did not change after 0.5 or 2 Gy irradiation. Furthermore, adding ATO to Res­HL60 reduced intracellular ROS levels. These findings provide important information that radioresistant leukemia cells respond differently to the antitumor effect of ATO and the combined effect of IR.

Exploring predictive molecules of acute adverse events in response to volumetric­modulated arc therapy for prostate cancer using urinary metabolites.

Volumetric-modulated arc therapy (VMAT) is a radiotherapy technique used to treat patients with localized prostate cancer, which is frequently associated with acute adverse events (AEs) that can affect subsequent treatment. Notably, the radiation dose of VMAT can be tailored to each patient. In the present study, a retrospective analysis was performed to predict acute AEs in response to a therapeutic high radiation dose rate based on urinary metabolomic molecules, which are easily collected as noninvasive biosamples. Urine samples from 11 patients with prostate cancer who were treated with VMAT (76 Gy/38 fractions) were collected. The study found that seven patients (~64%) exhibited genitourinary toxicity (Grade 1) and four patients had no AEs. A total of 630 urinary metabolites were then analyzed using a mass spectrometer (QTRAP6500+; AB SCIEX), and 234 relevant molecules for biological and clinical applications were extracted from the absolute quantified metabolite values using the MetaboINDICATOR tool. In the Grade 1 acute AE group, there was a significant negative correlation (rs=-0.297, P<0.05) between the number of VMAT fractions and total phospholipase A2 activity in the urine. Additionally, patients with Grade 1 AEs exhibited a decrease in PC aa C40:1, a phospholipid. These findings suggested that specific lipids found in urinary metabolites may serve as predictive biomarkers for acute AEs in response to external radiotherapy.

[A continuous 4-year evaluation of medical informatics education in a graduate school of health sciences using a questionnaire survey].

The purpose of this study was to identify the level of awareness among undergraduate students regarding medical informatics and to ascertain whether educational training has progressed with time in the Department of Health Sciences at Hirosaki University, Japan, which is a co-medical staff training institution that conducts a 4-year university course in medical informatics. The university accepts students who have completed the 3rd grade of medical licensing tests and who have attended the medical informatics lectures for 4 years (2007-2010). The ratio of first sight terminology percentage in any given fiscal year in all the 30 terminology categories varied widely from 0% to 80%, but the trend in various categories did not vary between fiscal years. The terminology of informatics under medical technology students obtained high scores of 52.5-77.3% after attending courses, which was higher compared with students from other classes. On the other hand, student nurses and occupational therapy students obtained 0-44.2%. Each class scored a high percentage of correct answers in the medical information-related terminology. Among the radiology students who attended the classes, the percentage of correct answers in categories of "digital imaging and communication in medicine" and "picture archiving and communication system" were lower than other medical terminology categories. These results reflect the gaps in educational curriculum of 1st and 2nd grades of medical licensing tests.

Proteomics and secreted lipidomics of mouse-derived bone marrow cells exposed to a lethal level of ionizing radiation.

High doses of ionizing radiation (IR) exposure can lead to the development of severe acute radiation syndrome with bone marrow failure. Defining risk factors that predict adverse events is a critical mission to guide patient selection for personalized treatment protocols. Since non-hematopoietic stem cells act as feeder cells in the niche and their secreted lipids may regulate hematopoietic stem cells, we focused on non-hematopoietic stem cells and aimed to discover biomarkers that can assess radiation exposure from their secreted lipids. Bone marrow stromal cells (BMSCs) and osteoblast differentiation-inducing cells (ODICs) isolated from mouse femurs were exposed to lethal doses of IR and the proteomic differences between BMSC and ODIC cell layers were compared. We observed an increased Nrf2-mediated oxidative stress response and IL6 expression in ODICs and decreased expression of mitochondrial proteins in BMSCs. To elucidate secreted factors, lipidomics of the cultures were profiled; the relevant lipids distinguishing IR-exposed and control groups of BMSC were acyl-acyl phosphatidylcholine (PC aa C34:1 and PC aa C34:4), lysophosphatidylcholine (lyso-PC a C18:0 and lyso PC a C17:0) and sphingomyelin (SM C20:2). These analyses suggest that certain lipids are candidate markers for the toxic effects of IR.

Prediction of hub genes and key pathways associated with the radiation response of human hematopoietic stem/progenitor cells using integrated bioinformatics methods.

Hematopoietic stem cells (HSCs) are indispensable for the maintenance of the entire blood program through cytokine response. However, HSCs have high radiosensitivity, which is often a problem during radiation therapy and nuclear accidents. Although our previous study has reported that the combination cytokine treatment (interleukin-3, stem cell factor, and thrombopoietin) improves the survival of human hematopoietic stem/progenitor cells (HSPCs) after radiation, the mechanism by which cytokines contribute to the survival of HSPCs is largely unclear. To address this issue, the present study characterized the effect of cytokines on the radiation-induced gene expression profile of human CD34+ HSPCs and explored the hub genes that play key pathways associated with the radiation response using a cDNA microarray, a protein-protein interaction-MCODE module analysis and Cytohubba plugin tool in Cytoscape. This study identified 2,733 differentially expressed genes (DEGs) and five hub genes (TOP2A, EZH2, HSPA8, GART, HDAC1) in response to radiation in only the presence of cytokines. Furthermore, functional enrichment analysis found that hub genes and top DEGs based on fold change were enriched in the chromosome organization and organelle organization. The present findings may help predict the radiation response and improve our understanding of this response of human HSPCs.

Dose-dependent expression of extracellular microRNAs in HCT116 colorectal cancer cells exposed to high-dose-rate ionising radiation.

Biomarkers of tumour response to radiotherapy may help optimise cancer treatment. The aim of the present study was to identify changes in extracellular microRNAs (miRNAs) as a biomarker of radiation-induced damage to human colorectal cancer cells. HCT116 cells were exposed to increasing doses of X-rays, and extracellular miRNAs were analysed by microarray. The results were correlated with the frequency of micronuclei. A total of 59 miRNAs with a positive correlation and 4 with a negative correlation between dose (up to 6 Gy) and extracellular miRNA expression were identified. In addition, for doses between 0 and 10 Gy, 12 miRNAs among those 59 miRNAs with a positive correlation were identified; for these extracellular miRNAs, a significantly positive correlation was observed between their expression and the frequency of micronuclei for doses up to 10 Gy. These results suggest that specific miRNAs may be considered as cell damage markers and may serve as secreted radiotherapy response biomarkers for colorectal cancer; however, the results must be further validated in serum samples collected from patients undergoing radiotherapy.

Hepatic gene expression and functional changes associated with nonalcoholic steatohepatitis.

Non­alcoholic steatohepatitis (NASH) is a pathological condition of the liver in which hepatocyte steatosis, invasion of inflammatory cells and hepatic injury occur without alcohol abuse. Despite the known risk of liver cancer and liver fibrosis that may progress to liver cirrhosis that exists with NASH, an understanding of related gene expression and associated functional changes remains insufficient. The present study used a mouse model of NASH induced by a high­fat diet to examine gene expression in the liver and to search for transcripts that could predict early liver fibrosis in the future. Mice fed a high­fat diet for 2 weeks showed typical NASH liver histology by hematoxylin and eosin staining, and increased fibrosis was confirmed by Sirius red staining after 6 weeks. Functional changes associated with liver damage, liver inflammation, liver steatosis and liver fibrosis were predicted by toxicological ontology analysis using Ingenuity Pathways Analysis. Downregulated microRNA (miR)­21 and upregulated collagen type III α1 mRNA in the liver and upregulated exosomal miR­21 in serum of mice fed a high­fat diet for 1 and/or 2 weeks were confirmed by reverse transcription­quantitative PCR, suggesting that these changes occur prior to histological confirmation of fibrosis. Therefore, it may be possible to predict future liver fibrosis by analyzing fibrosis­related genes that shift prior to pathological findings.

Effects of proteoglycan extracted from nasal cartilage of salmon heads on maturation of dendritic cells derived from human peripheral blood monocytes.

Dendritic cells (DCs) play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires appropriate stimuli such as pro-inflammatory cytokines. Proteoglycans (PGs) are one of the main components of extracellular matrix, and some types of PGs are known to induce maturation of murine DCs. Recent studies have investigated the potential benefits of PG from nasal cartilage of salmon head (S-PG). This study investigated the effects of S-PG on maturation of human monocyte-derived DCs. iDCs were prepared from human monocytes using the appropriate cytokines and then stimulated by S-PG alone. In another experiment, iDCs were stimulated by a combination of pro-inflammatory cytokines (MIX) plus S-PG. Although the stimulation of S-PG alone did not induce phenotypic maturation from iDCs, CD40 expression on DCs stimulated by S-PG alone was lower than that of iDCs. In contrast, the phenotypic and functional characteristics of DCs stimulated by MIX+S-PG were similar to those of DCs stimulated by MIX alone. As a result, S-PG did not demonstrate a significant effect with regard to maturation of human monocyte-derived DCs.

Proteoglycans regulate the chemotaxis of dendritic cells derived from human peripheral blood monocytes.

Dendritic cells (DCs) are a type of antigen-presenting cell which play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires appropriate maturation stimuli, such as pro-inflammatory cytokines or pathogen-derived components. Proteoglycans (PGs), which are composed of core proteins and the glycosaminoglycans that bind to them, are one of the main components of the extracellular matrix around pathogens such as bacteria. This study investigated the effects of PG extracted from the nasal septum cartilage of whale (W-PG) on the maturation of DCs derived from human peripheral blood monocytes. iDCs were prepared from human monocytes using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The iDCs were stimulated by W-PG alone. In another type of experiment, the iDCs were stimulated by MIX (tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and prostaglandin E(2) (PGE(2))) or a combination of MIX plus W-PG. The stimulation of W-PG alone did not induce the phenotypic maturation from iDCs. However, W-PG promoted the up-regulation of chemokine receptor CCR7-surface expression and the chemotactic responsiveness to CCR7 ligand macrophage inflammatory protein-3beta on MIX-stimulated mDCs although W-PG did not influence matrix metalloproteinase-9 activity which is an important factor in DC migration through the extracellular matrix. The findings that W-PG can selectively regulate the chemotactic activity of DCs in vitro under inflammatory conditions therefore indicate that the interaction of PGs with immune cells including DCs plays an important role in the immune response under the milieu of innate immunity.

Changes in miRNA expressions in the injured small intestine of mice following high­dose radiation exposure.

The small intestine is one of the most highly regenerative and radiosensitive tissues in mammals, including humans. Exposure to high doses of ionizing radiation causes serious intestinal damage. Recently, several investigations have been conducted using radioprotective agents to determine ways for reducing intestinal damage caused by radiation exposure. However, a thorough understanding of functional changes occurring in the small intestine of mice exposed to high­dose radiation is necessary for developing novel and more potent radioprotective agents. In this study, we examined changes in microRNA (miRNA/miR) expressions in the small intestine of mice at 72 h after X­ray exposure (10 Gy). We identified seven upregulated miRNAs and six downregulated miRNAs in the small intestine of mice following radiation exposure using miRNA microarray analysis. Particularly, miR­34a­5p was highly expressed, which was confirmed by reverse transcription-quantitative PCR. Forkhead box P1 (Foxp1) was predicted to be a target of the mRNA of miR­34a­5p using OmicsNet. Decreased Foxp1 expression in the small intestine following radiation exposure was confirmed, suggesting that Foxp1 expression recovery may induce the suppression of radiation­induced enteritis. Therefore, miR­34a­5p is a potential target molecule for developing novel radioprotective agents.

HER2-positive breast cancer that resists therapeutic drugs and ionizing radiation releases sphingomyelin-based molecules to circulating blood serum.

Breast cancer is the second most common cancer in the world based on incidence, reaching more than 2 million new cases in 2018, while continuing to increase. Invasive ductal carcinoma is the most common type of this cancer, making up approximately 70-80% of all breast cancer diagnoses. In particular, the type of breast cancer overexpressing human epidermal growth factor receptor 2 (HER2) has potential of strong proliferation, migration and invasion and early treatment is necessary. The authors identified and studied a single patient displaying complete therapeutic resistance to monoclonal anti-HER2 antibody therapy, chemotherapy and radiotherapy. A patient who exhibited resistance to postoperative adjuvant therapy after mastectomy was selected from HER2-positive breast cancer, and this patient had the grade of T4bN2aM0, Stage IIIB. The patient samples, blood serum and cancer tissue, were analyzed by metabolome and immunostaining technique, respectively. The characteristics of peripheral blood serum and solid tumor were investigated, aiming to find new serum biomarker(s) using the metabolomics technique. A correlation between the appearance of HER2-positive cancer tissue and serum concentration of the sphingomyelin family was found. In addition, HER2-positive tumor tissue in both the primary and recurrent cancer express the sphingomyelinase. These results suggest that sphingomyelins from this cancer tissue leads to therapy resistance, induction of invasion and strong proliferation.

Time-related study on external exposure dose of 2-deoxy-2-[F-18]fluoro-D-glucose PET for workers' safety.

Time-course study of individual dose equivalents of 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography (18F-FDG PET) was conducted in different hospital workers, and the daily work duties were analyzed. For the measurements, a semiconductor dosimeter was used. The values at intervals of 1 min and 1 h, the monthly cumulative and daily cumulative doses, and trend graphs were acquired with dedicated software and displayed on the reader. The following radiation workers with duties involving maximum external exposure work were included: doctors making diagnoses (4.8 µSv/procedure), nurses removing injection needles (3.1 µSv/procedure), pharmacists performing quality control tests (2.9 µSv/procedure), nuclear medicine technologists assisting patient positioning (6.5 µSv/procedure), and cyclotron engineers performing daily checks (13.4 µSv/procedure). The results of analysis of daily work duties revealed the influencing factors of external exposure dose. To reduce the external exposure dose, investigators should shorten the patient's contact time with the 18F-FDG source or patient tracer.

Radiation sensitivities in the terminal stages of megakaryocytic maturation and platelet production.

These studies examined the effects of X radiation and interleukin 3 (IL-3), which is an effective cytokine for the generation of megakaryocytopoiesis from X-irradiated hematopoietic stem/progenitor cells, on the terminal process of human megakaryocytopoiesis and thrombopoiesis. Mature megakaryocytes were induced by culturing CD34(+) cells from normal human peripheral blood in a serum-free liquid culture stimulated with thrombopoietin. The experiments contained the following groups: control cultures with nonirradiated cells incubated for 15 days; cultures treated with IL-3 on day 7 or day 11, cultures irradiated with 2 Gy on day 7 or day 11, and cultures treated with IL-3 immediately after X irradiation. The nonirradiated control cultures produced megakaryocytes from day 7, and both the megakaryocyte and platelet generation reached a peak on day 12-13. When X irradiation was performed on day 7, both the megakaryocyte and platelet numbers decreased remarkably, while no significant effect was observed on those numbers when cultures were X-irradiated on day 11. IL-3 showed neither protective nor promoting effects on the terminal stages of megakaryocytic maturation and platelet production. The results demonstrated that mature megakaryocytes are radiosensitive but that the radiosensitivity decreased with the terminal stages of megakaryocytic maturation, especially for the megakaryocytes entering into proplatelet formation.

Heavy ion beam irradiation regulates the mRNA expression in megakaryocytopoiesis from human hematopoietic stem/progenitor cells.

Heavy ion beams are a high-LET radiation that has greater biological effect than electron beams or X-rays. However, little is known about the effect of heavy ion beams on the proliferation and differentiation of human hematopoietic stem/progenitor cells (HSPCs). The present study examined the effect of heavy ion beams on gene expression in human HSPCs, especially during early stage of megakaryocytopoiesis. Human CD34+ cells were exposed to monoenergetic carbon-ion beams (290 MeV/nucleon, LET = 50 KeV/m) that were generated by an accelerator (Heavy Ion Medical Accelerator in Chiba). The expression of various genes related to early hematopoiesis, megakaryocytopoiesis/erythropoiesis, cytokine receptors and oxidative stress were analyzed by real-time RT-PCR. Friend leukemia virus integration 1, an early hematopoiesis-related gene, showed significantly higher mRNA expression than the control at 6 hr after irradiation. In contrast, no significant differences were observed in almost all of the other early hematopoiesis-related genes, cytokine receptor-coded genes and megakaryocytopoiesis/erythropoiesis-differentiation pathway-related genes, respectively. An analysis of the response of the genes to oxidative stress revealed the expression of heme oxygenase 1 to show a 1.5-fold and 11.9-fold increase from the day 0 control at 24 hr after 0.5 Gy and 2 Gy irradiation, respectively. Similarly, the NAD(P)H dehydrogenase-quinone 1 expression also showed a 22.0-fold and a 21.8-fold increase at 6 hr in comparison to the initial control. These results showed that the heavy ion beams affect megakaryocytopoiesis/ erythropoiesis differentiation of human HSPCs on the gene expression level.

Exposure to a MRI-type high-strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood.

The biological response after exposure to a high-strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34+ cells from human placental and umbilical cord blood were exposed under conditions of high-strength SMF in vitro. The high-strength SMF exposure system was comprised of a magnetic field generator with a helium-free superconducting magnet with built-in CO2 incubator. Freshly prepared CD34 cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst-forming unit erythroid- and megakaryocytic progenitor cells-derived colony formation was observed, thus producing 1.72- and 1.77-fold higher than the control, respectively. Furthermore, early hematopoiesis-related and cell cycle-related genes were found to be significantly up-regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP.

Understanding the mechanism underlying the acquisition of radioresistance in human prostate cancer cells.

Acquisition of radioresistance (RR) has been reported during cancer treatment with fractionated irradiation. However, RR is poorly understood in the prognosis of radiotherapy. Although radiotherapy is important in the treatment of prostate cancer (PCa), acquisition of RR has been reported in PCa with an increased number of cancer stem cells (CSCs), neuroendocrine differentiation (NED) and epithelial-mesenchymal transition. However, to the best of our knowledge, the mechanism underlying RR acquisition during fractionated irradiation remains unclear. In the present study, human PCa cell lines were subjected to fractionated irradiation according to a fixed schedule as follows: Irradiation (IR)1, 2 Gy/day with a total of 20 Gy; IR2, 4 Gy/day with a total of 20 Gy; and IR3, 4 Gy/day with a total of 56 Gy. The expression of cluster of differentiation (CD)44, a CSC marker, was identified to be increased by fractionated irradiation, particularly in DU145 cells. The expression levels of CD133 and CD138 were increased compared with those in parental cells following a single irradiation or multiple irradiations; however, the expression levels decreased with subsequent irradiation. RR was evidently acquired by exposure to 56 Gy radiation, which resulted in increased expression of the NED markers CD133 and CD138, and increased mRNA expression levels of the pluripotency-associated genes octamer-binding transcription factor 4 and Nanog homeobox. These data indicate that radiation-induced CSCs emerge due to the exposure of cells to fractionated irradiation. In addition, the consequent increase in the expression of NED markers is possibly induced by the increased expression of pluripotency-associated genes. Therefore, it can be suggested that cancer cells acquire RR due to increased expression of pluripotency-associated genes following exposure to fractionated irradiation.

Ex vivo expansions of megakaryocytopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free culture supplemented with proteoglycans extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of whale.

As a possible approach to the treatment of thrombopocytopenia, the ex vivo expansion of megakaryocytic progenitor cells may be a useful tool to accelerate platelet recovery in vivo. Our objective was to assess the promoting effect of proteoglycans in a serum-free culture condition using human cord blood CD34(+) cells. Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of a whale were applied to the ex vivo expansion of megakaryocytopoiesis and thrombopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free cultures stimulated with a combination of thrombopoietin (TPO) and interleukin-3 (IL-3). Each PG (0.5 and 5 mug) was applied to the culture with three different concentrations of TPO (50, 5 and 0.5 ng/ml) and IL-3 (100, 10 and 1 ng/ml). Both of the PGs showed no promoting effects on the mononuclear cell proliferation rate in any of the cultures. However, the whale-PG promoted the generation of megakaryocytic progenitor cells and megakaryocytes in the culture with a lower dose of cytokines, respectively. In addition, whale-PG led to a significant increase in CD42a(+) particles which seemed to be platelets. While the salmon-PG failed to promote such production in almost all of the cultures. Although whale-PG is an attractive molecule for the ex vivo expansion of human megakaryocytopoiesis, its action may depend on the glycosaminoglycans sulfation pattern and the ability of the binding affinity and the kinetics to interact with the cytokines and hematopoietic stem/progenitor cells.