Growth and differentiation of murine cartilage cells in vitro followng a short-term exposure to triamcinolone acetonide

Summary Measurements of ³H-thymidine incorporation, quantitative autoradiography and morphometry were used to evaluate cell behavior during the recovery of mandibular condylar cartilage cultures following short-term exposure to a corticosteroid hormone in vitro. Apical segments of mandibular condyles of newborn mice were initially incubated in the presence of the hormone triamcinolone acetonide (10^-6 M) for 24 h and were thereafter cultured for additional 6 days in hormone-free medium. The present results indicated that the treatment led to a decrease in the rate of incorporation of ³H-thymidine, a feature that lasted for 48 h following the removal of the hormone. Quantitative ³H-thymidine autoradiography of explants that were labeled in the presence of the hormone further substantiated the initial suppressive effect of the hormone on cellular proliferation, a feature that was followed by a recovery. Differences were noted in the pattern of distribution of labeled cells: in control explants, labeled cells progressively moved from the chondroprogenitor compartment into the differentiated portion of the cartilage; in hormone-treated explants, ³H-thymidine labeled cells were confined to the progenitor layer up to 5 days after the treatment and only then appeared in the chondrocytic compartment. The hormone's adverse effect upon differentiation was manifested by both morphology, and by causing a significant increase in the size of the progenitor layer (up to 50.5% on 4th post-treatment day) along with a 70.5% reduction in the size of chondroblastic layer. We conclude that a short-term exposure to a glucocorticoid hormone in vitro interferes with proliferation of chondroprogenitor cells and their subsequent differentiative pathway. While the proliferative activity was restored within 48 h, the hormone's effect on differentiation lasted for a considerably longer period of time.     

Comparison of in vitro and in vivo cell cycle parameters of lymphoid cells in Pig spleens

Summary Parameters of the cell cycle of lymphoid cells were estimated by analyzing percent labeled mitoses curves after a ³H-thymidine flash. Either anaesthetized pigs were labeled and multiple biopsies taken from the spleen in vivo or isolated perfused pig spleens were labeled in vitro. The data from in vivo and in vitro experiments were very similar.The mean values for cell cycle parameters were: 20.2 to 20.5 hours for the generation time, about 0.5 to 1 hour for G₂, about 1.2 to 1.3 hours for M; about 17 to 16.5 hours for S and about 1.5 to 1.7 hours for G₁. The mean grain count halving time of labeled mitoses was in accordance with the measured generation time. The isolated perfused spleen seems to give results equal to in vivo data and could, therefore, be employed as a model for studying cell cycle parameters not only in animal but also in human lymphoid tissue.The expert technical assistance of Mrs. A. Fischer is gratefully acknowledged. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 112.     

Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to human lung epithelial cells

Particulate hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen. It is currently a major public health concern, there is widespread exposure to it in occupational settings and to the general public. However, despite the potential widespread exposure and the fact that the lung is its target organ, few studies have considered the toxic effects of particulate Cr(VI) in human lung cells. Accordingly, we used lead chromate as a model particulate Cr(VI) compound and determined its cytotoxicity and genotoxicity in cultured human bronchial epithelial cells, using BEP2D cells as a model cell line. We found that lead chromate induced concentration-dependent cytotoxicity in BEP2D cells after a 24 h exposure. Specifically, the relative survival was 78, 59, 53, 46 and 0% after exposure to 0.5, 1, 5, 10 and 50 μg/cm² lead chromate, respectively. Similarly, the amount of chromosome damage increased with concentration after 24 h exposure to lead chromate. Specifically, 0.5, 1, 5 and 10 μg/cm² damaged 10, 13, 20 and 28% of metaphase cells with the total amount of damage reaching 11, 15, 24 and 36 aberrations per 100 metaphases, respectively. Lead chromate (50 μg/cm² lead chromate) induced profound cell cycle delay and no metaphases were found. In addition we investigated the effects of soluble hexavalent chromium, sodium chromate, in this cell line. We found that 1, 2.5, 5 and 10 μM sodium chromate induced 66, 35, 0 and 0% relative survival, respectively. The amount of chromosome damage increased with concentration after 24 h exposure to sodium chromate. Specifically, 1, 2.5 and 5 μM damaged 25, 34 and 41% of metaphase cells with the total amount of damage reaching 33, 59 and 70 aberrations per 100 metaphases, respectively. Ten micromolar sodium chromate induced profound cell cycle delay and no metaphases were found. Overall the data clearly indicate that hexavalent Cr(VI) is cytotoxic and genotoxic to human lung epithelial cells.     

Prematurely condensed chromosomes of dividing and non-dividing cells in aging human cell cultures.

Chromosomes of dividing and non-dividing aging cells were examined by fusing senescent WI38 cells with mitotic HeLa cells to induce premature chromosome condensation (PCC). Exposure of the WI38 cells to ³H-thymidine 48 h prior to fusion allowed autoradiographic identification of cells that did not synthesize DNA (non-dividing cells). Ninety-six percent of the non-dividing cells, diploid or tetraploid, induced into PCC had single chromatids and were therefore blocked in the G1 phase of the cell cycle. Anomalous centromeric pairing of chromatids was noted in the remaining 4% of the non-dividing cells. Typical G2 configurations (double chromatids) were observed only among labeled (dividing) cells. The efficiency of PCC induction was independent of culture age. In addition, the efficiency of PCC induction was independent of phase in the cell cycle, as shown by comparison of observed frequencies with expected frequencies.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

Cell cycle and DNA content of mitotic cells in brain ganglia ofdrosophila larvae

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae ofDrosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and ofDrosophila nasuta were examined by autoradiography of [³H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [³H]thymidine for 24 hin vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae ofDrosophila melanogaster andDrosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase.     

Differentiation of spermatogonial stem cell-like cells from murine testicular tissue into haploid male germ cells in vitro

In vitro differentiation of spermatogonial stem cells (SSCs) promotes the understanding of the mechanism of spermatogenesis. The purpose of this study was to isolate spermatogonial stem cell-like cells from murine testicular tissue, which then were induced into haploid germ cells by retinoic acid (RA). The spermatogonial stem cell-like cells were purified and enriched by a two-step plating method based on different adherence velocities of SSCs and somatic cells. Cell colonies were present after culture in M1-medium for 3 days. Through alkaline phosphatase, RT-PCR and indirect immunofluorescence cell analysis, cell colonies were shown to be SSCs. Subsequently, cell colonies of SSCs were cultured in M2-medium containing RA for 2 days. Then the cell colonies of SSCs were again cultured in M1-medium for 6–8 days, RT-PCR and indirect immunofluorescence cell analysis were chosen to detect haploid male germ cells. It could be demonstrated that 10⁻⁷ mol l⁻¹ of RA effectively induced the SSCs into haploid male germ cells in vitro.     

Incorporation of Radioactive Pyrimidine Nucleosides into DNA and RNA of Eimeria tenella (Coccidia) Cultured In Vitro*

Autoradiographic methods were used to study the incorporation of tritiated cytidine, thymidine, and uridine into asexual stages of Eimeria tenella cultured in embryonic chick kidney cells. Developing parasites did not incorporate ³H-thymidine either when host cells were labeled prior to infection or when the cultures were labeled for 30 min, 48–72 hr after infection. Continuous exposure of infected cultures to ³H-thymidine for up to 18 hr resulted in light labeling of cell cytoplasm and schizonts. ³H-cytidine and ³H-uridine were incorporated into parasites developing in cultures that were labeled before infection. When the cultures were labeled for 30 min, 48–72 hr postinfection and fixed immediately, schizonts were labeled lightly with ³H-cytidine but contained dense accumulations of ³H-uridine.     

The Role of Cytokinin in the Regulation of Growth, DNA Synthesis and Cell Proliferation in Cultured Soybean Tissues (Glycine max var. Biloxi)

Changes in protein content and cell proliferative activity were followed after a cytokinin-requiring strain of cultured Glycine max tissue was transferred to freshly prepared media which either contained or lacked cytokinin. Cell numbers doubled within the first two days after transfer, both in the presence and absence of cytokinin. However, after the second day no further increase in cell number was observed in the absence of cytokinin, while cell numbers continued to increase logarithmically in the presence of cytokinin. The size of the cell population attained after the first six days of growth was a function of the cytokinin concentration of the culture medium. However, the amount of ³H-thymidine incorporated into nuclear DNA bore no relation to the rate of cell proliferation. Tissues cultured on medium lacking cytokinin incorporated the greatest amount of ³H-thymidine per microgram of DNA, while the actively dividing tissues incorporated somewhat less. Using autoradiography and isopycnic CsCl gradient centrifugation, it was shown that the radioactivity derived from ³H-thymidine was associated with nuclear DNA in the cytokinin-deprived cells. Biochemical measurements demonstrated that cells cultured for six days without cytokinin had approximately twice the DNA content of the actively proliferating cells cultured on cytokinin-containing medium. Furthermore, in autoradiographs labeled cells were found to average nearly three times as many silver grains per nucleus in tissues cultured without cytokinin as the cytokinin-grown tissues. This suggests that the ³H-thymidine incorporation in the non-proliferating soybean cells results from nuclear DNA synthesis and that some of the cells became polypoid in the absence of cytokinin. These findings would be consistent with the idea that cytokinin acts as a specific trigger for cytokinesis.     

DNA synthesis in Hela cells at low temperature

It has been proved that ³H-thymidine is incorporated into DNA of HeLa cells cultured at 4 °C and its labelling distribution in DNA is homogeneous. This incorporation of ³H-thymidine increased with the duration of incubation and only 30% of the cell population was labelled after 12 h. When synchronous cell populations were used, the rate and extent of DNA synthesis at 4 °C was proportional to the relative number of cells in S phase at that temperature. Thus, cellular labelling at 4 °C does not result from a non-specific absorption phenomenon, but indicates a DNA synthesis process.     

Increase in DNA replication sites in cells held at the beginning of S phase

CHO cells were pulse labeled with ³H-thymidine after synchronization and blockage at the beginning of S phase for various intervals. The distribution of initiation sites for DNA replication and rates of chain growth were measured in autoradiographs prepared from these cells. Origins used for replication are widely distributed at or near the beginning of S phase, but usable origins increase continuously for many hours when FdU is used to block the synthesis of thymidylate. Potential origins are located about four microns apart, but in normal replication in these fibroblasts only one in 15 to 20 potential origins are used for initiation. On the other hand, when cells are held at the beginning of S phase for 12–14 h, about one-half of the potential origins are activated in part of the DNA and utilized when the cell is released from the block by supplying ³H-thymidine (10^–6M). Chain growth during a short pulse decreases with time of the blockage at what appears to be a linear rate. However, cells can replicate long continuous stretches of their DNA with only 2×10^–8M thymidine available in the medium for several hours when synthesis is blocked by FdU. The total amount of DNA replicated is, however, much less than when a concentration of 10^–6 M thymidine is supplied for the same period. The origins which are finally used under any experimental condition appear to be a random sample of the total potential origins which are distributed in a regular repeating sequence along the DNA at about 12 kilobase intervals.     

Length of cell cycle in vitro and sister-chromatid exchange (SCE) frequency in bone marrow cells from severely malnourished rats

Cell proliferation and SCE frequency were evaluated through differential staining of sister chromatids in cultured bone marrow cells from rats malnourished during the lactation period. Cell proliferation was studied in vitro in sequential analysis every 5 h in cultures from 20 to 40 h of incubation. Results show a longer generation cycle in malnourished rat cells, revealing a delay in cell proliferation. Cells of this group of animals showed a higher percentage of first-cycle metaphases and lacked third-cycle metaphases even after 40 h of culture. This shows that the damage caused to cells of undernourished organisms used in this experiment persists even when they are placed in a nutrient-rich medium. The SCE frequency did not show differences between malnourished rats and their controls.     

Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder‐free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round‐shaped cells resembling immature oocytes. Mvh⁺ cells formed clumps by co‐aggregation with differentiation‐supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency. Mol. Reprod. Dev. 77: 802–811, 2010. © 2010 Wiley‐Liss, Inc.     

Families of replicating units in cultured hamster fibroblasts

An examination of the patterns of DNA replication in pseudodiploid Don C and diploid Don cell lines in culture has been made. Pulse-chase labelling experiments with ³H-thymidine in both synchronized and log-phase cells indicate that the newly replicated DNA can be divided into two and three large temporally distinct fractions in Don C and Don cells, respectively. This is shown radiochemically by fluctuations in the incorporation of ³H-thymidine into the DNA of synchronized cells and autoradiographically by fluctuations in counts of labelled metaphases and grain over mitotic figures. Pulse-chase experiments and fluorometric determinations indicate that the periodic incorporation of ³H-thymidine can be accounted for by discontinuous synthesis and turnover of DNA during the cell cycle.A survey of the literature reveals that fluctuations in DNA synthetic activity during the S phase are to be found in a large number of published graphs of cell population kinetics. The phenomenon is observable in both diploid and non-diploid cells. A change in the timing of DNA replicon synthesis during the S phase according to the developmental stage and age of the cell is proposed.     

Changes in proteins synthesized by rabbit endometrial epithelial cells following primary culture

Summary Morphological and biochemical changes occurring in rabbit endometrial epithelial cells when placed in culture were investigated. Cells were examined by scanning- and transmission electron microscopy and freeze-fracture. Morphologically, cultured cells are shorter and broader than the columnar epithelial cells in vivo, but retain their polarity as indicated by the presence of apical microvilli and a well-developed junctional belt. To study changes in biochemical function, proteins synthesized by cells in primary culture were analyzed by two-dimensional gel electrophoresis. Proteins were labeled during a 24-h incubation with ³⁵S-methionine and gels examined by fluorography. The pattern of proteins changed after cells had been in culture for 48 h. On day 3 new proteins were synthesized and several protein species labeled during days 1 or 2 of culture, including uteroglobin, no longer appeared. On days 3–8 of culture the protein patterns were similar. Addition of progesterone, estradiol, prolactin, or combinations of these hormones to the culture medium for 24–144 h failed to elicit consistent changes in the pattern of labeled proteins established after 3 days of culture. Minor differences in protein patterns among unrelated cultures appear to have been derived from the original cells of the culture. These results indicate that after 48 h in primary culture, cells grown in vitro resemble endometrial epithelial cells morphologically, but no longer reflect functionally the character of epithelial cells in the uterus.     

Neuropeptide Y-Y1 receptor agonist worsens while antagonist improves survival of cultured Y1-expressing neuronal cells following oxygen and glucose deprivation

In this in vitro study, we investigated the influence of neuropeptide Y (NPY) Y1 receptor activation or inhibition on the viability of cultured neuronal or glial cells following oxygen glucose deprivation (OGD). Viability of cultured cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. When compared to the vehicle-treated control group, treatment with NPY or [Leu³¹,Pro³⁴]-NPY (Y1 agonist) reduced viability of cultured SK-N-MC (Y1-expressing) human neuronal cells at 24 h after 1 h of OGD, while BIBP3226 (Y1 antagonist) improved viability. Except at the highest concentration of NPY used in the study, treatment with NPY or NPY3-36 (Y2 agonist) did not influence viability of cultured SH-SY5Y (Y2-expressing) human neuronal cells at 24 h after 1 h of OGD. In addition, treatment with NPY, [Leu³¹,Pro³⁴]-NPY, NPY3-36, or BIBP3226 did not affect viability of cultured primary astrocytes at 24 h after 4 h of OGD. The present results agree with those of a recent in vivo study. Activation of NPY-Y1 receptors may mediate ischemic pathophysiological processes, and inhibiting the Y1 receptors may be protective. The combination of OGD and cultured neuronal cells may be useful in future studies on the neuroprotective and harmful mechanisms of NPY-Y1 receptor inhibition and activation during ischemia, respectively.     

Effect of dibutyryl cyclic AMP on the restoration of contact inhibition in tumor cells and its relationship to cell density and the cell cycle

KB cell cultures exposed to 10⁻⁴ M dibutyryl cyclic AMP were significantly inhibited and exhibited contact inhibition of growth at cell densities of 8 × 10⁴/cm² irrespective of the initial plating density. Control cultures reached densities of 2.5 × 10⁵/cm². Inhibition of growth did not occur in KB cells when the density was below 1 × 10⁴ cells/cm². When dibutyryl cyclic AMP was removed from KB cells in the contact-inhibited state, growth resumed with DNA synthesis beginning in about 6 h. Labeled metaphases increased rapidly after 22 h without the appearance of an early rise in unlabeled metaphases. This suggests that the inhibitory effect of dibutyryl cyclic AMP is on the G1 phase of the cell cycle.     

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.     

Evidence for a four micron replication unit in CHO cells

CHO cells in culture were synchronized by mitotic selection, allowed to reattach to plastic flasks, and reach S phase in the presence of fluorodeoxyuridine at concentrations known to completely block the synthesis of thymidylate. The cells were released from the block with ³H-thymidine for pulses of 4, 8, 12, 24 and 40 min and DNA fiber autoradiographs prepared. An analysis of the spacing between origins of replication indicates that sites are available at intervals of about 4 m along most of the DNA. Chain growth proceeds at about 1,000 nucleotides per minute and some of the closely situated sites become continuous, labeled segments after 8–12 min. However, unlabeled segments are still present between the replicated segments after 40 min. The data may be interpreted as evidence for regularly spaced initiation sites which are available in CHO cells, even though only one in 10–15 of these may be utilized for initiation each cycle under normal growth conditions in these cultures.     

Phenotypic Plasticity of Mouse Spermatogonial Stem Cells

Background

Spermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro.

Methodology and Principal Findings

In this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit⁻ and Kit⁺ cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo.

Conclusions/Significance

These results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.