Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

INTRODUCTION: Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. MATERIALS AND METHODS: NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. RESULTS: Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. CONCLUSIONS: 3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.

Osmolarity effects on bovine articular chondrocytes during three-dimensional culture in alginate beads.

OBJECTIVE: With the development of engineered cartilage, the determination of the appropriate culture conditions is vital in order to maximize extracellular matrix synthesis. As osmolarity could affect the fate of chondrocytes, the purpose of this study was to determine the effects of osmolarity on chondrocytes during relatively long-term culture. DESIGN: Bovine articular chondrocytes were cultured in alginate beads in a biocarbonate free system at 280, 380 and 550 mOsm at pH 7.4 for up to 12 days, respectively. Cell volume, intracellular pH (pH(i)), cell number, glucosaminoglycan (GAG) and collagen retention were measured at day 5 and 12. Cell viability and volume were monitored over the 12 days of culture. RESULTS: By day 5 and 12, compared to the cell volume at 380 mOsm, around 20% (P<0.01) swelling and 15% (P<0.05) shrinkage were observed when the cells were cultured at 280 and 550 mOsm. The pH(i) over the 12 days of culture varied with osmolarity of the culture medium. In comparison with fresh cells, pH(i) became slightly more acidic by 0.15 pH units at 280 mOsm at day 5. However, by day 12, an alkalization of pH(i), by 0.2 pH units, was noted. A higher proliferation rate was seen at 280 mOsm than at other osmolarities while less GAG was produced. CONCLUSIONS: Chronic exposure to anisotonic conditions results in cell swelling at 280 mOsm and shrinkage at 550 mOsm. The osmolarity of 280 mOsm appears to encourage proliferation of chondrocytes, but inhibits matrix production.

Application of multiple parallel perfused microbioreactors and three-dimensional stem cell culture for toxicity testing.

In this study, a multiple parallel perfused microbioreactor platform, TissueFlex, was developed which can be used to perform cell and tissue culture under almost uniform and precisely controlled environment in a mid-throughput and parallel manner. These microbioreactors were used to culture human bone marrow cells (hBMCs) in three-dimensional (3D) scaffolds and also in two-dimensional (2D) monolayer for comparison for upto 7 days. Several scaffolding materials were evaluated for this purpose in terms of easiness in handling, ability to support the hBMC growth, and feasibility for non-destructive optical assays. The feasibility and efficacy of using the developed 3D-hBMCs-based model tissue-constructs cultured in TissueFlex microbioreactors for drug evaluation and toxicity testing was then studied. As a demonstration case study, the cultured cells were challenged with two chemicals, trimethoprim and pyrimethamine, both known to be harmful to cellular activities, with different protocols. Cytotoxicity in terms of cell viability and growth was determined using the AlamarBlue assay. The 3D spatial variations in cell morphology and cell survival were also monitored using 3D optical imaging using non-linear multiphoton microscopy. The results show that (i) the data obtained from 3D hBMCs culture and from (2D) monolayer cultures on the effect of the tested chemicals on cell growth are significantly different, and that (ii) the perfused microbioreactor technology could provide a highly controlled and prolonged cell culture environment for testing of various drugs and chemicals. The outcome of this study demonstrated the feasibility and potentials of the using 3D stem cell based model tissues in TissueFlex microbioreactors for drug evaluation and toxicity testing of chemicals as an efficient and standardized alternative testing method.

Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death.

Two-photon excitation-based near-infrared (NIR) laser scanning microscopy is currently emerging as a new and versatile alternative to conventional confocal laser scanning microscopy, particularly for vital cell imaging in life sciences. Although this innovative microscopy has several advantages such as highly localized excitation, higher penetration depth, reduced photobleaching and photodamage, and improved signal to noise ratio, it has, however, recently been evidenced that high-power NIR laser irradiation can drastically inhibit cell division and induce cell death. In the present study we have investigated the cellular responses of unlabeled rat kangaroo kidney epithelium (PtK2) cells to NIR femtosecond laser irradiation. We demonstrate that NIR 170-fs laser pulses operating at 80-MHz pulse repetition frequency and at mean power of > or = 7 mW evoke generation of reactive oxygen species (ROS) such as H2O2 that can be visualized in situ by standard in vivo cytochemical analysis using Ni-3,3'-diaminobenzidine (Ni-DAB) as well as with a recently developed fluorescent probe Jenchrom px blue. The formation of the Ni-DAB reaction product as well as that of Jenchrom was relatively more pronounced when irradiated cells were incubated in alkaline solution (pH 8) than in those incubated in acidic solution (pH 6), suggesting peroxisomal localization of these reaction products. Two-photon time-lapse imaging of the internalization of the cell impermeate fluorescent dye propidium iodide revealed that the integrity of the plasma membrane of NIR irradiated cells is drastically compromised. Visualization of the nuclei with DNA-specific fluorescent probes such as 4',6-diamidino-2-phenylindole 24 h postirradiation further provided tangible evidence that the nuclei of these cells undergo several deformations and eventual fragmentation. That these NIR irradiated cells die by apoptosis has been established by in situ detection of DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Because the reactive oxygen species such as H2O2 and OH* can cause noxious effects such as cell membrane injury by peroxidation of polyunsaturated lipids and proteins and oxidative phosphorylation, and alterations of ATP-dependent Ca2+ pumps, these ROS are likely to contribute to drastic cytological alterations observed in this study following NIR irradiation. Taken together, we have established that NIR laser irradiations at mean power > or = 7 mW delivered at pulse duration time of 170 fs generally used in two- and multiphoton microscopes cause oxidative stress (1) evoking production of ROS, (2) resulting in membrane barrier dysfunction, (3) inducing structural deformations and fragmentation of the nuclei as well as DNA strand breaks, (4) leading to cell death by apoptosis.

Femtosecond near-infrared lasers as a novel tool for non-invasive real-time high-resolution time-lapse imaging of chloroplast division in living bundle sheath cells of Arabidopsis.

Non-linear excitation of fluorophores by contemporaneous absorption of two or more near-infrared (NIR) photons following diffraction-limited focusing with high-numerical-aperture objectives circumvents out-of-focus fluorescence (without a confocal pinhole) and spatially limits photobleaching and photodamage to the minute sub-femtoliter focal volume. This is in contrast to optical events in conventional confocal imaging systems using ultraviolet (UV) or visible laser sources wherein the entire sectors of the specimen above and below the plane of focus experience massive photostress and photodamage. In addition, NIR wavelengths penetrate deeper into the highly scattering environs of plant tissue than UV and visible wavelengths. We delineate a novel non-invasive technique using NIR femtosecond laser pulses at lambda = 740, 760, 780, and 800 nm for induction of chlorophyll fluorescence by the two-photon effect as well as for intra-tissue time-lapse vital three-dimensional imaging of fundamental events of chloroplast division in deeply seated bundle sheath cells of Arabidopsis thaliana (L.) Heynh. leaves. Our findings establish that (i) mature bundle sheath chloroplasts are indeed capable of division, (ii) the dividing chloroplasts assume a distinct constricted/dumbbell-shaped profile with an average lifespan of 20-25 min, (iii) the complete division of the pre-existing chloroplasts occurs within 50 min, and (iv) the two derivative daughter chloroplasts are invariably unequal in size. This novel NIR-laser-based technique has any number of potential applications, including (i) non-invasive intra-vital imaging of molecular and ion dynamics, (ii) non-destructive screening of mutants impaired in photosynthesis, (iii) diagnosis of physiological states of plants and (iv) bio-optical taxonomy.

Femtosecond near-infrared laser pulse induced strand breaks in mammalian cells.

Multiphoton laser scanning microscopy (MPLSM) is based on non-resonant simultaneous absorption of two or three near infrared (NIR) photons by a fluorophore in the subfemtoliter focal volume of a high numerical aperture (N.A. 1.3) objective. The higher penetration depth of NIR radiation enables optical sectioning across thick biological specimens and because of the absence of efficient single photon absorbers in the NIR spectral region of 700 to 1200 nm there is hardly any out-of-focus photodamage and photobleaching. Recent in vitro studies (14) have demonstrated that irradiation of supercoiled plasmid DNA with intense multiphoton NIR 810 nm of 140 fs pulse width, 76 MHz pulse repetition rate results in single strand breaks as a result of simultaneous absorption of three or more photons. Herein, we have investigated the influence of 800 nm NIR 170 fs laser pulses, 80 MHz pulse repetition frequency at mean powers of 2 to 20 mW on nuclear DNA of unlabelled PtK2 cells. In situ TdT-mediated dUTP-nick end labelling (TUNEL) revealed that cells exposed to the NIR irradiation above > or =5 mW mean laser power alone contained TUNEL-positive nuclei. The intensity of TUNEL fluorescence was relatively higher at increased mean NIR laser power. These results provide evidence that DNA strand breaks also occur in vivo when mammalian cells are exposed to high average power > or =5 mW NIR irradiation during MPLSM possibly due to multiphoton absorption process. Because intense DNA fragmentation is one of the hall marks of programmed cell death it is hypothesised that NIR induced cell death is by apoptosis.

Technical advance: near-infrared femtosecond laser pulses as a novel non-invasive means for dye-permeation and 3D imaging of localised dye-coupling in the Arabidopsis root meristem.

We have used near-infrared femtosecond Titanium: Sapphire laser pulses as novel non-invasive means for dye loading into various cell types of the Arabidopsis root meristem, and by 3D imaging have assessed the extent of dye coupling between the meristematic cells. The post-embryonic primary root of Arabidopsis thaliana has an invariant ontogeny and fixed cellular organisation which makes it an attractive model system to study developmental events involving cell fate determination, cellular differentiation and pattern formation. Local intercellular communication and local transmission of positional signals are likely to play a pivotal role in cell proliferation and regulation of differentiation. We have therefore examined the extent to which the constituent cells in the root meristem are symplastically coupled. Following laser-assisted loading of membrane impermeate fluorescent dye propidium iodide (PI) in single cells, we show by time-lapse and 3D imaging that in the root tip all undifferentiated cells are dye-coupled. When PI is permeated into the central cells, it rapidly moved into the adjacent initials of the columella, cortex, pericycle and stele. Interestingly, when only either of the initials were loaded with the dye, it never moved into any of the central cells. Amongst the epidermal cells, the differentiated hair cells are symplastically isolated. Our data provide evidence (1) for differential dye-coupling behaviour between quiescent centre cells and the neighbouring initials; (2) that cells in the root are coupled during stages at which the cell-lineage pattern is formed and that it becomes progressively secluded as they differentiate and the pattern is fixed. Taken together, our NIR-laser mediated approach is highly efficient and has numerous potential applications for non-invasive permeation of dyes in different cell types.

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis.

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.

Evidence for UV-B-induced DNA degradation in Euglena gracilis mediated by activation of metal-dependent nucleases.

It is demonstrated that in vivo irradiation with artificial UV-B for several hours significantly reduces the amount of large DNA extractable from immobilized Euglena in comparison with non-irradiated controls. This UV-B effect can be eliminated by a drastic reduction of the divalent ion concentration in the extracellular medium, i.e. the substitution of the culture medium by Tris-buffered agarose. Moreover, in vitro degradation of large DNA is demonstrated for crude protein extracts isolated from non-irradiated or UV-B-irradiated Euglena. The nuclease activity is shown for both crude protein extracts and purified nucleases; in both cases, two protein bands possessing nuclease activity are obtained with apparent molecular masses of 26 and 40 kDa and their activity is inhibited by specific nuclease inhibitors, i.e. aurintricarboxylic acid and ATP, applied at a concentration as low as 10(-8) M. Moreover, in vitro, nuclease activity clearly depends on the pH, with an optimum around pH 4.5, and on the ion composition of the extracellular medium. A strong stimulating effect is shown for Ca2+ with an optimum around 10(-4) M; this effect is potentiated by Zn2+ and Mn2+, but strongly counteracted by Mg2+ and the calmodulin inhibitors trifluoperazine and N- (6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W5). These results favour the concept which explains the lethal UV-B effect on Euglena as arising from a change in the general metabolic state of the cell and an activation of a DNA-degrading system, i.e. activation of metal-dependent nucleases (U.K. Tirlapur, D.-P. Häder and R. Scheuerlein, UV-B mediated damage in the photosynthetic flagellate, Euglena gracilis, studied by image analysis, Beitr. Biol. Pflanzen, 67 (1992) 305-317).

Confocal imaging and immunogold electron microscopy of changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L.

Using anti-myosin antibodies, standard immunocytochemical techniques in conjunction with confocal scanning laser microscopy and colloidal gold immunoelectron microscopy we compare changes in the distribution patterns of myosin during the early stages of pollen hydration, germination, tube growth, and myosin associated with isolated vegetative nucleus and the generative cell in Nicotiana tabacum L. Furthermore, on the Western blots of pollen tube proteins, the antimyosin antibodies crossreact only with one polypeptide of approximately 174 kDa. Confocal immunofluorescence microscopy reveals that in hydrated pollen, myosin is discretely associated with the cytoplasmic organelles and numerous punctate structures present in the center of the pollen. Within 30 min following transfer of pollen into the germination medium, that is, with the onset of germination, the centrally located punctate structures are displaced, and we find accumulation of myosin-associated organelles towards one of the germinal apertures from which the pollen tube would emerge. Subsequently, after 45 min of germination with the emergence of germination structure, few punctate structures are detected in the vegetative cytoplasm while intense immunostain is detected just below the plasma membrane of the emerging pollen tube tip. In the older parts of both short and long pollen tubes after 90 to 120 min of pollen germination, few fluorescent structures were found in the pollen tubes, however, numerous punctate fluorescent spots were concentrated in the tip region over a distance of 2 to 3 microns below the plasma membrane of the tube tip. This is further substantiated by colloidal gold immunoelectron microscopy wherein clusters of gold particles are associated with vesicle-like structures in the tip region of the pollen tubes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of isolated egg cells, in vitro fusion products and zygotes of Zea mays L. using the technique of image analysis and confocal laser scanning microscopy.

Changes in membrane Ca2+, calcium receptor protein calmodulin, endoplasmic reticulum (ER), mitochondria and cellulose in unfixed, living, isolated egg cells and fusion products of pairs of one egg and one sperm cell of Zea mays L. have been investigated using chlorotetracycline, fluphenazine, immunocytochemical techniques, 3,3'-dihexyloxa-carbocyanine iodide (DiOC6(3)) and calcofluor white in conjunction with computer-controlled video image analysis. In addition, confocal laser scanning microscopy has been used in conjunction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetracycline (CTC) fluorescence reveal that egg cells contain high levels of membrane Ca2+ in organelles present around the nucleus while the cytosolic signal is relatively low. Intense CTC fluorescence is invariably present just below the plasma membrane of egg cells and a certain degree of regionalised distribution of Ca2+ in cytoplasm is also discernible. Similarly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that localised immunocytochemically using monoclonal anti-CaM antibodies reveal high levels of CaM in the vicinity of the nucleus in egg cells. Only a few ER profiles and mitochondria could be visualised in the egg cell and no calcofluor fluorescence could be detected. Following in vitro fertilisation of single isolated eggs substantial changes in the Ca2+ levels occur which include an increase in the membrane Ca2+ of the fusion product, particularly in the cytosol and around the nucleus. Unlike in the eggs the fine CTC fluorescence signal below the plasma membrane is not detectable in the fusion products.(ABSTRACT TRUNCATED AT 250 WORDS)

Polar distribution of annexin-like proteins during phytochrome-mediated initiation and growth of rhizoids in the ferns Dryopteris and Anemia.

Although the calcium requirement of phytochrome-mediated fern spore germination and early rhizoid growth is well established, the calcium-binding proteins that serve as transducers for these responses are not known. Here we report the presence of annexin-like proteins in germinating spores of Dryopteris filix-mas (L.) Schott and Anemia phyllitidis (L.) Sw. and evidence that they may be important participants in early photomorphogenic changes in gametophytes. Immunolocalization and immunoblot assays of these proteins were carried out using polyclonal antibodies raised either against a 35-kDa annexin-like protein from pea or against anchorin CII from chicken. Western-blot analysis showed that crude protein extracts obtained from both species after red-light treatment contained two cross-reactive protein bands with molecular weights around 70 kDa. These proteins were annexin-like in that they bound to a phosphatidylserine affinity column in a calcium-dependent fashion. Using this column, two protein bands around 70 kDa, i.e. 67 and 73 kDa, were partially purified together with proteins at 36 kDa and a doublet at 54 kDa. Proteins of these latter molecular weights are suggested to be members of the annexin family, but no cross-reactivity could be found between these and the two antibodies used in our investigations. Immunodetectable levels of these proteins were observed only after light-mediated induction of spore germination. Imaging of the immuno-localization patterns observed with both antibodies showed that the annexin-like proteins are concentrated at the extreme tips of the rhizoids in D. filix-mas and A. phyllitidis during rhizoid initiation and all stages of elongation. We suggest that these proteins may play a major role in the tip-oriented exocytosis events that are critical for the initiation and growth of fern rhizoids.

Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L.

Using monoclonal anti-calmodulin antibodies in conjunction with confocal scanning laser microscopy we have analysed the spatial variations in the distribution pattern of calmodulin (CaM) during the sequential events of pollen hydration, germination and tube growth in Nicotiana tabacum. These immunocytochemical observations have been complemented by immunochemical studies wherein the anti-calmodulin antibody raised against pea CaM recognises a polypeptide of c. 18 kDa in the pollen extracts. Digitisation of confocally acquired optical sections of immunofluorescence images reveals that in hydrated pollen a high level of CaM is consistently present in the region of the germinal apertures. Subsequently, with the onset of germination a high CaM concentration was found associated with the plasma membrane of the germination bubble and in the cytoplasm in its vicinity, while in the vegetative cytoplasm a weak diffuse and intense punctate signal was registered. CaM immunostain was also detected in association with the plasma membrane of the tube tips in both short and long pollen tubes. Furthermore, the cytosol of the tubes invariably manifested an apically focused CaM gradient. We were, however, unable to detect any vacuolar association of CaM in the older regions of the pollen tubes. Although punctate immunostain was obvious across the pollen tube numerous punctate structures were invariably present in the extreme tip. The possible implications of these findings in development of cell polarity, polarised growth, maintenance of calcium homeostasis and CaM interactions with other mechanochemical motor proteins in effecting propulsion of organelles during pollen hydration, germination and pollen tube growth are discussed.

Spatial variation of sequestered calcium in the multicellular stage of Dictyostelium discoideum as assayed by chlortetracycline fluorescence.

We have used chlortetracycline (CTC) as a fluorescent probe to detect the distribution of sequestered calcium in multicellular stages of Dictyostelium discoideum. Tips of late aggregates, slugs and early culminating masses fluoresce very strongly. Most of the fluorescence is intracellular in origin and emanates from a small number of intense punctate sources. The sources correspond in part to autophagic vacuoles vis. neutral-red staining, acidic digestive vesicles, and may also include intracellular organelles; cytoplasmic fluorescence is much weaker in comparison. The level of fluorescence drops in the middle portion of slugs and rises again in the posteriormost region, though not to as high a level as in the tip. This holds good irrespective of whether CTC is applied only in the neighbourhood of the aggregate centre, only in the aggregate periphery, or to the whole aggregate. We infer that there must be a good deal of mixing in the stages leading from aggregation to slug formation; thus the serial order in which cells enter an aggregate does not bear any relation to their ultimate fates. The other implication of our study is that calcium sequestration is much more extensive in prestalk and anterior-like cells than in prespore cells. These findings are discussed with regard to possible implications for pattern formation.