Long-term Culture Experiments Research Articles

Biological culture module for plant research from seed-to-seed on the Chinese Space Station

The long-term cultivation of higher plants in space plays a substantial role in investigating the effects of microgravity on plant growth and development, acquiring valuable insights for developing a self-sustaining space life supporting system. The completion of the Chinese Space Station (CSS) provides us with a new permanent space experimental platform for long-term plant research in space. Biological Culture Module (GBCM), which was installed in the Wentian experimental Module of the CSS, was constructed with the objective of growing Arabidopsis thaliana and rice plants a full life cycle in space. The techniques of LED light control, gas regulation and water recovery have been developed for GBCM in which dry seeds of Arabidopsis and rice were set in root module of four culture chambers (CCs) and launched with Wentian module on July 24, 2022. These seeds were watered and germinated from July 28 and grew new seeds until November 26 within a duration of 120 days. To this end, both Arabidopsis and rice plants completed a full life cycle in microgravity on the CSS. As we know, this is the first space experiment achieving rice complete life cycle from seed-to-seed in space. This result demonstrates the possibility to cultivate the important food crop rice throughout its entire life cycle under the spaceflight environment and the technologies of GBCM have effectively supported the success of long-term plant culture experiments in space. These results can serve as invaluable references for constructing more expansive and intricate space plant cultivation systems in the future.

Involvement of a Rarely Used Splicing SD2b Site in the Regulation of HIV-1 vif mRNA Production as Revealed by a Growth-Adaptive Mutation.

We have previously reported an HIV-1 mutant designated NL-Y226tac that expresses Vif at an ultra-low level, being replication-defective in high-APOBEC3G cells, such as H9. It carries a synonymous mutation within the splicing SA1 site relative to its parental clone. In order to determine whether a certain mutant(s) emerges during multi-infection cycles, we maintained H9 cells infected with a relatively low or high input of NL-Y226tac for extended time periods. Unexpectedly, we reproducibly identified a g5061a mutation in the SD2b site in the two independent long-term culture experiments that partially increases Vif expression and replication ability. Importantly, the adaptive mutation g5061a was demonstrated to enhance vif mRNA production by activation of the SA1 site mediated through increasing usage of a rarely used SD2b site. In the long-term culture initiated by a high virus input, we additionally found a Y226Fttc mutation at the original Y226tac site in SA1 that fully restores Vif expression and replication ability. As expected, the adaptive mutation Y226Fttc enhances vif mRNA production through increasing the splicing site usage of SA1. Our results here revealed the importance of the SD2b nucleotide sequence in producing vif mRNA involved in the HIV-1 adaptation and of mutual antagonism between Vif and APOBEC3 proteins in HIV-1 adaptation/evolution and survival.

Evidence for the Growth Superiority and Delayed Ovarian Development in Tetraploid Loach Misgurnus anguillicaudatus

The diploids and tetraploids of cyprinid loach Misgurnus anguillicaudatus coexist in many natural habits. The tetraploids generally exhibit superior growth performance and delayed gonadal development compared with diploids. To investigate the regulation mechanism of growth superiority and ovarian development in tetraploids, we first conducted a long-term culture experiment and histological observation. The tetraploids exhibited a higher growth performance and delayed ovarian development. Meanwhile, at a genetic level, an average of 6891 differentially expressed genes (DEGs) between diploids and tetraploids were identified from the brain, pituitary, liver, gonad and muscle using the RNA-seq method. Among these DEGs, as expected, some candidate genes, including IGF family genes, somatostatin, leptin, cyp19a1b, gthα, lhβ and fshβ, were detected, which play critical roles in the regulation of growth and gonad development of fish. In particular, the genes related to GH/IGF axis and growth factors, signal transduction, gonadal hormone and appetite were significantly increased in tetraploids. The clustering analyses of the key candidate genes showed that most key genes were up-regulated in the pituitary and gonad of tetraploids instead of other tissues. The dynamics of these key genes provide valuable genetic evidence for clarifying the growth superiority and delayed gonadal development of tetraploids. Moreover, this study also provides some clues for unveiling the genetic superiority of polyploidy species in other phenotypes.

Hypomorphic mutation of PEX3 with peroxisomal mosaicism reveals the oscillating nature of peroxisome biogenesis coupled with differential metabolic activities.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.

Osmotrophy of dissolved organic carbon by coccolithophores in darkness.

Summary The evolutionary and ecological story of coccolithophores poses questions about their heterotrophy, surviving darkness after the end‐Cretaceous asteroid impact as well as survival in the deep ocean twilight zone. Uptake of dissolved organic carbon might be an alternative nutritional strategy for supply of energy and carbon molecules.Using long‐term batch culture experiments, we examined coccolithophore growth and maintenance on organic compounds in darkness. Radiolabelled experiments were performed to study the uptake kinetics. Pulse–chase experiments were used to examine the uptake into unassimilated, exchangeable pools vs assimilated, nonexchangeable pools.We found that coccolithophores were able to survive and maintain their metabolism for up to 30 d in darkness, accomplishing about one cell division. The concentration dependence for uptake was similar to the concentration dependence for growth in Cruciplacolithus neohelis, suggesting that it was taking up carbon compounds and immediately incorporating them into biomass. We recorded net incorporation of radioactivity into the particulate inorganic fraction.We conclude that osmotrophy provides nutritional flexibility and supports long‐term survival in light intensities well below threshold for photosynthesis. The incorporation of dissolved organic matter into particulate inorganic carbon, raises fundamental questions about the role of the alkalinity pump and the alkalinity balance in the sea.

Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9

To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34+ progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2mut) cells, whereas ASXL1mut as well as DNMT3Amut cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells.

Effect of root surface charge on the absorption and accumulation of Cu(II) by different japonica and indica rice varieties under acidic conditions

Excessive amounts of copper (Cu) in soils causes toxic effects on plants. In this study, 58 rice cultivars were classified into tolerant, moderately tolerant, and susceptible types for Cu(II) toxicity based on 50% germination (LC50). Nine japonica rice varieties (three each from the tolerant, moderately tolerant, and susceptible groups) and six indica rice varieties (three from the moderately tolerant and susceptible groups) were selected for the hydroponics experiments. In the short-term adsorption experiment, Cu(II) adsorbed on rice roots was differentiated into exchangeable, complexed, and precipitated forms. Similarly, it was done for long-term culture. Absorption of Cu(II) by rice roots and shoots was also measured. The results indicated that adsorbed Cu(II) mainly existed as complexed and exchangeable forms on rice roots in the short-term adsorption experiment, and the exchangeable and complexed Cu(II) levels were greater for indica rice than for japonica rice due to the larger negative charge on the indica rice roots. The adsorbed Cu(II) mainly existed as a complexed form in the long-term culture experiment, and the exchangeable Cu(II) level was much lower than that in the short-term adsorption experiment due to the absorption of Cu(II) by rice plants. The indica varieties absorbed more Cu(II) than the japonica varieties. Furthermore, the absorption and accumulation of Cu(II) by the susceptible varieties were greater than by the tolerant and moderately tolerant varieties for both the japonica and indica rice. The absorption and accumulation of Cu(II) in rice roots were much greater than in the shoots. Chlorophyll content, and the lengths and dry matter weights of the rice roots and shoots decreased with increasing Cu(II) concentration. The Cu(II) showed greater toxicity toward indica varieties than japonica varieties, and the greater negative charge on indica roots was one of reasons for the greater exchangeable Cu(II) on the roots, the increase in Cu(II) toxicity, and the higher uptake of Cu(II) by indica rice varieties compared to japonica rice varieties.

Long-term serial culture of Piscirickettsia salmonis leads to a genomic and transcriptomic reorganization affecting bacterial virulence

The propagation of intracellular bacteria in culture mediums implies physiological adaptation to an artificial environment. If these conditions are persistent enough time, a permanent adaptation to the new environment can be expected. This study aimed to explore the genomic and transcriptomic rearrangements that the intracellular bacterium Piscirickettsia salmonis undergo during a long-term serial culture experiment and the potential impact on virulence. Here, PacBio and Illumina sequencing were conducted after 200 passages (~2 years) of P. salmonis in a cell-free culture medium. The results evidenced genomic rearrangements of the P. salmonis genome during the continuous culture period, where a chromosome segment of 35 Kb was translocated from the P0 bacterial chromosome to a P200 plasmid. Notably, the genomic structure of this segment revealed the presence of the Dot/Icm secretion system, which has been previously associated with P. salmonis pathogenesis. On the other hand, a reduced transcriptomic response was evidenced in P. salmonis after 200 passages affecting the expression of different pathways including iron acquisition and metabolism. Furthermore, in vitro infections revealed that after 200 passages P. salmonis is less capable of generating cytopathic effects than the original P0 form. Overall, our results evidence that the continuous propagation of P. salmonis leads to genomic and transcriptomic rearrangement that impact on bacterial virulence. These results open new perspectives about the adaptation of intracellular bacteria to artificial culture conditions, providing useful information to develop live attenuated vaccines against P. salmonis in salmon aquaculture.

Decrease in bioavailability of soil heavy metals caused by the presence of microplastics varies across aggregate levels

Microplastics can alter the physicochemical and biogeochemical processes in soil, but whether these alterations have further the effects on the transformation of soil heavy metal speciation, and if so, whether these effects vary across soil aggregate levels remain unknown. Herein, long-term soil culture experiments and soil fractionation are combined to investigate the effects of microplastics on chemical speciation of Cu, Cr, and Ni with different particle-size soil aggregates. Results show that microplastics in soil decrease the exchangeable, carbonate-bound, and Fe-Mn oxide-bound fractions of metals but increase their organic-bound fractions via direct adsorption and indirect effects on the soil microenvironment conditions. The findings suggest that microplastics can promote the transformation of heavy metal speciation from bioavailable to organic bound. Such promotion exerts notable differences across soil aggregate levels. The transformation of soil heavy metal speciation is greater in larger aggregates than in smaller aggregates in the early incubation period with microplastics but shows the opposite trend in the later incubation period. Therefore, this process is more sensitive to long-term microplastic pollution in smaller aggregates than in larger aggregates, most likely owing to the lag in the influence of microplastics on metal speciation transformation in the smaller aggregates.

Complementary energy acquisition via aerobic anoxygenic photosynthesis and carbon monoxide oxidation by Planktomarina temperata of the Roseobacter group.

In marine pelagic ecosystems energy is often the limiting factor for growth of heterotrophic bacteria. Aerobic anoxygenic photosynthesis (AAP) and oxidation of carbon monoxide (CO) are modes to acquire complementary energy, but their significance in abundant and characteristic pelagic marine bacteria has not been well studied. In long-term batch culture experiments we found that Planktomarina temperata RCA23, representing the largest and most prominent subcluster of the Roseobacter group, maintains 2-3-fold higher cell numbers in the stationary and declining phase when grown in a light-dark cycle relative to dark conditions. Light enables P. temperata to continue to replicate its DNA during the stationary phase relative to a dark control such that when reinoculated into fresh medium growth resumed two days earlier than in control cultures. In cultures grown in the dark and supplemented with CO, cell numbers in the stationary phase remained significantly higher than in an unsupplemented control. Furthermore, repeated spiking with CO until day 372 resulted in significant CO consumption relative to an unsupplemented control. P. temperata represents a prominent marine pelagic bacterium for which AAP and CO consumption, to acquire complementary energy, have been documented.

Identification of alcohol stress tolerance genes of Synechocystis sp. PCC 6803 using adaptive laboratory evolution

BackgroundSynechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.ResultsIsobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.ConclusionsNovel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.

Sustained Inhibition of HIV-1 Replication by Conditional Expression of theE. coli-Derived Endoribonuclease MazF in CD4+T cells

Gene therapy using a Tat-dependent expression system of MazF, an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli, in a retroviral vector appears to be an alternative approach to the treatment of human immunodeficiency virus type 1 (HIV-1) infection. MazF can cleave HIV-1 RNA, since it has more than 240 ACA sequences. Significant inhibition of viral replication, irrespective of HIV-1 strains, was observed in CD4(+) T cells that had been transduced with the MazF-expressing retroviral vector (MazF-T cells). The growth and viability of MazF-T cells were not affected by HIV-1 infection. Interestingly, the infectivity of HIV-1 produced from MazF-T cells was found to be lower than that from control CD4(+) T cells. A long-term culture experiment with HIV-1-infected cells revealed that viral replication was always lower in MazF-T cells than in CD4(+) T cells transduced with or without a control vector for more than 200 days. MazF was expressed and mainly localized in the cytoplasm of the infected cells. Unlike in CD4(+) T cells, the expression level of Tat gradually decreased rather than increased in MazF-T cells after HIV-1 infection. As a consequence, the expression level of MazF appeared to be well regulated and sustained during HIV-1 infection in MazF-T cells. Furthermore, the levels of cellular mRNA were not affected by HIV-1 infection. Thus, the Tat-dependent MazF expression system has great potential for inhibition of HIV-1 replication in vivo without apparent toxicity and may be able to avoid the emergence of resistant strains.

Stabilization and modulation of the phycobilisome by calcium in the calciphilic freshwater red alga Bangia atropurpurea

The bangiophycean filamentous red alga Bangia atropurpurea is distributed in freshwater habitats such as littoral and splash zones of lakes or rapid currents distant from the sea. In these habitats, the distribution and growth of this alga appear to be related to hard water rich in calcium ions. To characterize the eco-physiological properties of this calciphilic red alga, we examined the effects of long-term and short-term Ca2+ depletion on photosynthetic growth of the thallus and on the phycobilisome. Long-term culture experiments suggested that higher Ca2+ concentrations (>50mgL−1) were required to sustain thallus growth and pigmentation of cells. In short-term Ca2+-depletion treatments, fluorescence derived from phycoerythrin (PE) fluctuated, although the absorption spectra of the thalli did not change. After 30min of Ca2+ depletion, the fluorescence lifetime of PE became markedly longer, indicating that the energy transfer from PE to phycocyanin (PC) was suppressed. The fluorescence lifetime of PE returned to its original value within a short time after 4h of Ca2+ depletion, however, energy transfer from PE to PC was still suppressed. This suggested that the excitation energy absorbed by PE was quenched during prolonged Ca2+ depletion. The efficient energy transfer from PC and allophycocyanin were unchanged during these treatments.

Combination of the Hedgehog Pathway Inhibitor LDE225 and Nilotinib Eliminates Chronic Myeloid Leukemia Stem and Progenitor Cells.

Abstract 1428Poster Board I-451Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder arising in a hematopoietic stem cell (HSC). CML is associated with expression of the Philadelphia chromosome (Ph) and its fusion gene product, BCR-ABL, a constitutively active tyrosine kinase. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, all show impressive rates of complete cytogenetic response in chronic phase (CP) CML. However, the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML stem cells not effectively targeted by TKIs. Recent studies have indicated that the Hedgehog (Hh) pathway, a developmental pathway with roles in primitive and adult hematopoiesis, is activated in CML stem cells via upregulation of Smoothened (SMO), a seven-transmembrane domain receptor protein (Dierks et al, Cancer Cell 2008;14:238). We found that Gli1, a downstream target of Hh signalling, is significantly upregulated (6-fold) at the mRNA level in CD34+ enriched CP CML cells compared to normal CD34+ hematopoietic cells by Taqman quantitative RT-PCR. Thus, inhibition of SMO may be an effective therapeutic strategy to reduce the quiescent CML stem cell pool. LDE225 (Novartis Pharma) is a small molecule SMO antagonist which has recently entered Phase 1 clinical evaluation in patients with solid tumors. We assessed the efficacy of LDE225, alone and in combination with nilotinib, in primary CD34+ CML cells in vitro using short- and long-term culture techniques, including colony forming cell (CFC) assays with replates, long-term culture-initiating cell (LTC-IC) assays, CFSE-based flow cytometry to track cell division, Annexin/Viaprobe to measure apoptosis and Ki-67/7AAD cell cycle analysis. In short-term culture, incremental concentrations of LDE225 (0.5 - 1000nM) had no effect on total viable cell counts up to 12 days and did not inhibit cell proliferation as assessed by BrDU incorporation and CFSE staining. LDE225 did not increase apoptosis or alter the cell cycle profile of CD34+ CML cells compared to untreated controls. The lack of response in short-term culture experiments is explained by the stem cell selective nature of LDE225 which affects self-renewal and not proliferation or apoptosis. For long-term culture experiments, CD34+ CML cells were cultured for either 3 or 7 days in serum-free media supplemented with physiological growth factors (IL-3, IL-6, G-CSF, FLT-3 and SCF) with and without LDE225; cells were then washed and put into CFC assays with no added LDE225. Increasing concentrations of LDE225 did not significantly reduce colony read-out in the CFC assays. However, when the CFCs were serially replated there was a highly significant reduction in secondary colony formation and replating efficiency with increasing concentrations of LDE225 up to a plateau at 50nM during the initial 72 hour culture. In the first re-plate, colony formation was reduced by 38% with 5nM (P=0.07) and 62% with 50nM LDE225 (P=0.011; n=3) compared to a “no drug” control. Further reductions in colony formation were seen in second and subsequent replates. In further CFC replating experiments, after 3 and 7 days initial exposure to the combination of LDE225 10nM + nilotinib 5μM, replating efficiency was reduced by 50% (P<0.03) and 74%,(P<0.005) respectively (n=3). Single agent nilotinib resulted in a non-significant increase in colony formation and replating efficiency. In LTC-IC assays, compared to the “no drug” control, CD34+ CML cells showed increased colony formation in the nilotinib arm, indicating that, by inhibiting proliferation, nilotinib exerts a protective effect on CML stem cells (as shown previously for imatinib and dasatinib; Copland et al, Blood 2008;111:2843). The arm containing LDE225 + nilotinib showed a reduction in colony formation compared to both the nilotinib arm (85% reduction) and the “no drug” control (50% reduction). These results confirm the stem cell selectivity of LDE225. In conclusion, LDE225 targets CML stem cells and, in combination with nilotinib, represents a novel therapeutic strategy for targeting both the primitive quiescent CML stem cell population and the bulk CML progenitor population. In vivo studies in murine models of CML are ongoing to help further evaluate the clinical potential of LDE225 in combination with nilotinib. DisclosuresHolyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dorsch: Novartis: Employment. Manley: Novartis: Employment. Bhatia: Novartis: Consultancy. Copland: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Involvement of Transcriptional Mediator Subunit TRAP220/MED1 in Action of Niche Cells to Support Hematopoietic Stem/Progenitor Cells

The mammalian TRAP/Mediator complex is a master transcriptional regulatory complex that integrates signals of diverse activators and recruits RNA polymerase II and other general factors to activate transcription. The TRAP220/MED1 subunit was originally identified as a ligand-dependent coactivator specific for nuclear receptors. We have previously shown through biochemical and mouse genetic studies that MED1 is essential for embryogenesis, cell growth/differentiation and homeostasis, and that it stimulates nuclear receptor-mediated myelomonopoiesis. MED1 also integrates other activators such as GATA-1 and C/EBPβ and appears to mediate erythropoiesis as well. The niche cells in the bone marrow plays a pivotal role in the maintenance of hematopoietic stem/progenitor cells (HSPCs). In this study, we employed mouse embryonic fibroblasts (MEFs) as a model to analyze the role of MED1 in the niche, since MEFs have a mesenchymal feature with the osteoblastic precursor lineage and are known to support HSPCs. To establish an experimental system, we crossed Med1 and p53 double knockouts to obtain Med1+/+/p53−/− and Med1−/−/p53−/− E10.0 embryos from a single female and prepared stable MEF lines. Then the Med1−/−/p53−/− MEFs were stably transfected with a MED1 expression vector (Rev-Med1−/− MEFs) or a control empty vector. When normal mouse bone marrow cells were cocultured with these MEFs treated with mitomycin C for a short period of 2 weeks, cell counts, live cells (MTT assay) and a DNA synthesis (BrdU incorporation) of marrow cells were measured. The number of live cells as well as DNA synthesis on Med1−/− MEFs was significantly decreased during this period, but those on Rev-Med1−/− MEFs recovered to the control levels. Thus the growth stress on MEFs appears to be attenuated on Med1−/− MEFs. When apoptosis of the marrow cells was measured, both the FITC-dUTP incorporation by TdT and annexin V/PI double positive cells were lower for Med1−/− MEFs, indicating that apoptosis was also attenuated. We next assessed the role of MED1 in MEFs to support long-term bone marrow culture. After bone marrow cells were cultured on mitomycin C-treated MEFs for 8 weeks in Myelocult M5300 (StemCell Technologies) or IMDM supplemented with BIT9500 (StemCell Technologies) and LDL, progenitor cells (adherent and nonadherent) were collected and cultured in complete methylcellulose (Methocult M3434; StemCell Technologies), and colonies were counted. The number of both myeloid and erythroid colonies were significantly attenuated (0 to 40% depending on experimental conditions) for cells on Med1−/− MEFs, but colonies for cells cultured on Rev-Med1−/− MEFs recovered to the control level. In order to exclude the possibility that lot differences among MEFs or p53 depletion might have affected the results, we next prepared primary Med1+/+ and Med1−/− MEFs by crossing Med1+/− mice and conducted the long-term culture experiments using these MEFs. The attenuated number of colonies for cells cocultured with Med1−/− MEFs (circa 10% of the control) was reproduced repeatedly, indicating that the observed role of MED1 in MEFs to support HSPCs is intrinsic. Since MED1 converges signals from a series of activators on specific promoters and activates transcription, one or some products of the downstream target genes in MEFs may be responsible for the observed activity to maintain HSPCs. In search for candidate MED1 target gene products among a series of known molecules that possess an activity on HSPCs, only the expression of osteopontin was found to be attenuated in Med1−/− MEFs and reverted in Rev-Med1−/− MEFs. Other factors including Angiopoietin-1 and Jagged-1 were comparable. This fact contrasts with the previous observation of osteopontin knockouts where the null niche cells that restricted the size of HSPC number overexpressed these factors. We next assessed the role of MED1 on the osteopontin promoter. We focused on vitamin D receptor (VDR) and Runx2 among the activators and tested MEFs by luciferase reporter assays. The basal level of transcription without any activators in Med1−/− MEFs was about half of the control. Moreover, both the activation by Runx2 and the liganddependent VDR function were significantly attenuated in Med1−/− MEFs. These results indicate that transcriptional coactivator MED1 in niche cells plays an important role in HSPCs support, and that osteopontin may be one of the downstream candidate target genes for MED1.

A cell culturing system that integrates the cell loading function on a single platform and evaluation of the pulsatile pumping effect on cells

In this paper, we present a novel microfluidic system with pulsatile cell storing, cell-delivering and cell culturing functions on a single PDMS platform. For this purpose, we have integrated two reservoirs, a pulsatile pumping system containing two soft check valves, which were fabricated by in situ photopolymerization, six switch valves, and three cell culture chambers all developed through a simple and rapid fabrication process. The sample volume delivered per stroke was 120 nl and the transported volume was linearly related to the pumping frequency. We have investigated the effect of the pulsatile pneumatic micropumping on the cells during transport. For this purpose, we pumped two types of cell suspensions, one containing human breast adenocarcinoma cells (MCF-7) and the other mesenchymal stem cells (hMSCs) derived from bone marrow. The effect of pulsatile pumping on both cell types was examined by short and long-term culture experiments. Our results showed that the characteristics of both cells were maintained; they were not damaged by the pumping system. Evaluations were carried out by morphological inspection, viability assay and immunophenotyping analysis. The delivered MCF-7 cells and hMSCs spread and proliferated onto the gelatin coated cell culture chamber. This total micro cell culture system can be applied to cell-based high throughput screening and for co-culture of different cells with different volume.

Fecal Water as a Non-Invasive Biomarker in Nutritional Intervention: Comparison of Preparation Methods and Refinement of Different Endpoints

: The assessment of cellular effects by the aqueous phase of human feces (fecal water, FW) is a useful biomarker approach to study cancer risks and protective activities of food. In order to refine and develop the biomarker, different protocols of preparing FW were compared. Fecal waters were prepared by 3 methods: (A) direct centrifugation; (B) extraction of feces in PBS before centrifugation; and (C) centrifugation of lyophilized and reconstituted feces. Genotoxicity was determined in colon cells using the Comet assay. Selected samples were investigated for additional parameters related to carcinogenesis. Two of 7 FWs obtained by methods A and B were similarly genotoxic. Method B, however, yielded higher volumes of FW, allowing sterile filtration for long-term culture experiments. Four of 7 samples were non-genotoxic when prepared according to all 3 methods. FW from lyophilized feces and from fresh samples were equally genotoxic. FWs modulated cytotoxicity, paracellular permeability, and invasion, independent of their genotoxicity. All 3 methods of FW preparation can be used to assess genotoxicity. The higher volumes of FW obtained by preparation method B greatly enhance the perspectives of measuring different types of biological parameters and using these to disclose activities related to cancer development.

Induction of centrosome and chromosome aberrations by imatinib in vitro

Imatinib (STI571, Gleevec/Glivec) is a potent selective tyrosine kinase inhibitor and is used successfully in the treatment of chronic myeloid leukemia (CML). While karyotype alterations, in addition to the Philadelphia chromosome, are a common phenomenon of progressing CML, the observation of BCR-ABL-negative leukemic clones with distinct aberrant karyotypes under an imatinib regimen is not yet understood. Here we test the hypothesis that such tumor clones may be induced de novo from normal cells by imatinib. In vitro experiments with varying drug concentrations (5-20 microM) were performed on normal human dermal fibroblasts (NHDF), Chinese hamster embryonal and Indian muntjak fibroblasts. After 3 weeks of treatment, analysis of cell cultures by centrosome immunostaining and conventional cytogenetics revealed that imatinib induced centrosome and chromosome aberrations in all cultures in a significant dose-dependent and species-independent manner. Moreover, the results of NHDF long-term culture experiments demonstrated that aberrant phenotypes, emerging under imatinib treatment for 12 weeks, were not reversible after prolonged propagation omitting the drug. These observations suggest a causative role of imatinib in the origin of centrosome and karyotype aberrations (genetic instability) and thus may explain the emergence of clonal chromosomal abnormalities in BCR-ABL-negative progenitor cells under imatinib therapy.

Long-term lens organ culture system with a method for monitoring lens optical quality

The Scan Tox System is a method for monitoring lens optical quality (focus or lack of focus) in culture conditions, which mimic conditions inside the eye. The ocular lens is an ideal organ for long-term culture experiments because it has no direct blood supply and no connection to the nervous system. The Scan Tox System makes it possible to keep lenses for long-term studies of up to a few weeks. The use of cultured lenses, mainly bovine, replaces the need for testing the effects of potentially damaging agents on live animals. This optical monitoring apparatus uses a computer-operated scanning laser beam, a video-camera system and a video frame analyzer to record the focal length and transmittance of the cultured lens. The scanner is designed to measure the focal length at points across the diameter of the lens. The lens container permits the lens to be exposed to a vertical laser beam from below. The laser source projects its light onto a plain mirror, which is mounted at 45 degrees C on a carriage assembly. The mirror reflects the laser beam directly up through the test lens. The mirror carriage is connected to a positioning motor, which moves the laser beam across the lens. The camera sees the cross section of the beams and, by examining the image at each position of the mirror, Scan Tox software is able to measure the quality of the lens by calculating the back vertex distance for each beam position. The cultured lenses continue to maintain their original refractive function. When foreign substances are introduced to a cultured lens, the Scan Tox System measures the resulting optical response. This provides a very sensitive means to follow early damage to the eye lens. Because the lens is maintained in an intact state in solutions that are similar to those inside the eye, the lens retains its normal recuperative powers. So in addition to measuring early damage, this system allows measurement of recovery from damage.

Comparative studies on phosphorus uptake and growth kinetics of the microalga Tetraselmis subcordiformis and the macroalga Ulva pertusa

Short-term uptake experiments and long-term semicontinuous culture experiments were performed under the condition of phosphorus (P) limitation to estimate and compare the P uptake and growth kinetics of the microalga Tetraselmis subcordiformis and the macroalga Ulva pertusa. Two new parameters, the maximum specific uptake rate (Vmsp) and the maximal growth efficiency (β), are introduced to achieve uniformity for the comparison of nutrient uptake and growth efficiency between microalgae and macroalgae. T. subcordiformis possesses 3 times lower half saturation uptake constant, 4 times higher maximal growth rate and 20 times higher maximum specific uptake rate than U. pertusa, while U. pertusa possesses 4 times higher maximal growth efficiency than T. subcordiformis.