Biosynthesis of methionine in mouse cells cultured in vitro

In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.     

Chromosomal aberration arises during somatic reprogramming to pluripotent stem cells

Recurrence of Glioblastoma multiforme (GBM) seems to be the rule despite combination therapies. Cell invasion and cell proliferation are major reasons for recurrence of GBM. And insulin-like growth factor binding protein 5 (IGFBP5) is the most conserved of the IGFBPs and is frequently dysregulated in cancers and metastatic tissues. By studying the human glioma tissues, we find that IGFBP5 expression associate to the histopathological classification and highly expressed in GBM. Using IGFBP5 mutants we demonstrate that knockdown of IGFBP5 inhibited cell invasion, whereas promoting cell proliferation in GBM cells. Mechanistically, we observed that promoting GBM cell proliferation by inhibiting IGFBP5 was associated with stimulating Akt (Protein kinase B) phosphorylation. However, IGFBP5 promote GBM cell invasion was related to the epithelial-to-mesenchymal transition (EMT). Furthermore, the Chinese Glioma Genome Altas (CGGA) database show that IGFBP5 is significantly increased in recurrent glioma and it predicted worse survival. The obtained results indicate that IGFBP5 has two sides in GBM—inhibiting cell proliferation but promoting cell invasion.     

Chromosomal changes in two strains of human fibroblasts cultured in vitro

The chromosomal changes that occurred in two freshly initiated human cell strains, during their in vitro life were followed. Both strains are characterized by slow growth, and both show a period of delayed growth. In one of the strains the chromosomal composition showed changes just before the period of slow growth. After this period both cell strains showed several kinds of aneuploid chromosome complements, mainly in the hypotetraploid region. Aberrant chromosomes were present at the end of the in vitro life of both strains.The work in this paper was supported by the Foundation for Basic Medical Research (FUNGO), and the Association between Euratom and the University of Leiden, contract No. 052-64-1-BIAN.     

Chromosomal recombination and breakage associated with instability in mouse centrometric satellite DNA

A mouse L cell line containing the centromeric insertion of herpes thymidine kinase genes (tk) was previously shown to undergo a high frequency of DNA rearrangement at the site of tk insertion. Analysis of TK- revertants had demonstrated that DNA rearrangements were usually associated with DNA deletion and were always mediated by intrachromosomal recombinations. In this study, we further analyzed several TK+ subclones to examine the mode of DNA rearrangements in the absence of negative selection pressure. In two clones, LC2-3F and LC2-3E17, rearrangements were accompanied by DNA amplification and were mediated by intrachromosomal recombination. In subclone LC2-3E17-19, we further detected perturbations in the pattern of centromeric heterochromatization. This was associated with chromosome instability, as evidenced by chromosome breakage at the centromere. The analysis of three other sibling clones, LC2-3, LC2-6 and LC2-15, further suggests that reciprocal recombination events may play a role in such centromeric rearrangements. These results suggest that DNA rearrangements in the centromere may be mediated by a number of different mechanisms, and generally do not affect chromosome stability except when accompanied by changes in the pattern of heterochromatization.     

In vitro organogenesis from pluripotent stem cells

Pluripotent stem cells (PSCs) have the ability to spontaneously generate structured tissues in vitro reminiscent of embryonic tissue development. Recently, complex organoids such as cortical tissues, cerebral brain organoids, optical cups, intestinal tissues, and liver buds have been generated from PSCs derived from healthy individuals and patients with genetic diseases, providing powerful tools to understand morphogenesis and disease pathology. This article highlights recent advances in the state-of-art generation of organoids from PSCs, possible signaling pathways and mechanisms involved in organogenesis, and the understanding of extracellular microenvironment. Challenges involved in the organoid generation such as increasing organoid size, enhancing the tissue complexity, and improving functional maturation are also discussed.     

In vitro organogenesis from pluripotent stem cells

Pluripotent stem cells (PSCs) have the ability to spontaneously generate structured tissues in vitro reminiscent of embryonic tissue development. Recently, complex organoids such as cortical tissues, cerebral brain organoids, optical cups, intestinal tissues, and liver buds have been generated from PSCs derived from healthy individuals and patients with genetic diseases, providing powerful tools to understand morphogenesis and disease pathology. This article highlights recent advances in the state-of-art generation of organoids from PSCs, possible signaling pathways and mechanisms involved in organogenesis, and the understanding of extracellular microenvironment. Challenges involved in the organoid generation such as increasing organoid size, enhancing the tissue complexity, and improving functional maturation are also discussed.     

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O2/1% CO2, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16-0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 X CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.     

Attachment of Mycoplasma pulmonis to rat and mouse synovial cells cultured in vitro

The attachment of Mycoplasma pulmonis m53 organisms to mouse and rat synovial cells was examined by using the organisms and the synovial cells treated in various ways. M. pulmonis treated with trypsin attached to the synovial cells, but the organisms treated with pronase, formaldehyde, glutaraldehyde, or heat did not. These findings suggest that the sites for binding M. pulmonis to the mouse and rat synovial cells are of polypeptide nature. Treatment of M. pulmonis with sialic acid and treatment of the synovial cell sheets with neuraminidase did not affect the attachment. The synovial cell surface for receptors M. pulmonis organisms would be different from those on respiratory cells or erythrocytes for M. pneumoniae or M. gallisepticum. Even nonviable organisms and M. pulmonis membranes attached to the mouse or rat synovial cells. The nature of the receptor of mouse synovial cells would be different from that of rat cells, since rat cells were affected by treatment with formaldehyde or glutaraldehyde, but mouse cells were not.     

Germ cells from pluripotent stem cells: mouse versus human

<正>Currently embryonic stem cells(ESCs)derived from fertilized embryos or cloned embryos by somatic cell nuclear transfer and induced pluripotent stem cells(i PSCs)from somatic cells represent two major types of pluripotent stem cells(PSCs).The nave PSCs functionally can produce all ESC/i PSC mice by tetraploid embryo complementation,and     

X-ray-induced telomeric instability in Atm-deficient mouse cells

The gene responsible for ataxia telangiectasia (AT) encodes ATM protein, which plays a major role in the network of a signal transduction initiated by double strand DNA breaks. To determine how radiation-induced genomic instability is modulated by the dysfunction of ATM protein, we examined radiation-induced delayed chromosomal instability in individual cell lines established from wild-type Atm(+/+), heterozygote Atm(+/-), and knock-out Atm(-/-) mouse embryos. The results indicate that Atm(-/-) mouse cells are highly susceptible to the delayed induction of telomeric instability and end-to-end chromosome fusions by radiation in addition to the elevated spontaneous telomeric instability detected by telomere fluorescence in situ hybridization (FISH). The telomeric instability was characterized by abnormal telomere FISH signals, including loss of the signals and the extra-chromosomal signals that were associated and/or not associated with chromosome ends, suggesting that Atm deficiency makes telomeres vulnerable to breakage. Thus, the present study shows that Atm protein plays an essential role in maintaining telomere integrity and prevents chromosomes from end-to-end fusions, indicating that telomeres are a target for the induction of genomic instability by radiation.     

Generation of pluripotent stem cells from neonatal mouse testis

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.     

X chromosome inactivation in human and mouse pluripotent stem cells

Since the groundbreaking hypothesis of X chromosome inactivation (XCI) proposed by Mary Lyon over 50 years ago, a great amount of knowledge has been gained regarding this essential dosage compensation mechanism in female cells. For the mammalian system, most of the mechanistic studies of XCI have so far been investigated in the mouse model system, but recently, a number of interesting XCI studies have been extended to human pluripotent stem cells, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Emerging data indicate that XCI in hESCs and hiPSCs is much more complicated than that of their mouse counterparts. XCI in human pluripotent stem cells is not as stable and is subject to environmental influences and epigenetic regulation in vitro. This mini-review highlights the key differences in XCI between mouse and human stem cells with a greater emphasis placed on the understanding of the epigenetic regulation of XCI in human stem cells.     

Zajdela hepatoma cells cultured in vitro

The goal of this work was to study cellular mechanisms of tumor progression and metastasizing. As a result of explantation of cells of rat Zajdela ascitic hepatoma, we obtained two transplantable cell cultures—monolayer (ZH-ad) and suspension (ZH-fl)—that differ in levels of cell differentiation and tumorigenicity. By using tumor-specific immune serum, we revealed tumor-associated antigens, synthesis of which is reduced or inhibited in ZH-ad cells, in outer membranes of the ZH-fl cells. Intraperitoneal injection into rat of 0.5–12 × 10⁶ ZH-fl cells leads to development of an ascitic tumor and death of 100% of animals, whereas, in the case of administration of ZH-ad cells, to achieve a tumorigenic effect, the minimal dose needs to be elevated to 20 × 10⁶ cells. Clonogenic analysis of the ZH-fl cells revealed three types of the formed clones—nonadhesive sphere colonies and two types of monolayer clones differing in proliferative potential, shape of colonies, and cell composition. Upon reaching a critical size, the spheres disintegrated, with separation of single cells and islands of different sizes, some of them being attached with monolayer formation. Three clonal cell lines were obtained: 1C as a result of expansion of a spherical clone and 4G and 10E from monolayer clones. We established that there is tumorigenicity of the 1C cell line, which, at a dose of 107 cells, led to the development of ascites and to the death of 50% of animals. The presented results indicate the existence in the ZH-fl cell population of tumor-initiating cells generating spherical clones—floating multicellular islets that, in culturing in the complete growth medium, are partly differentiated and are attached with monolayer formation.