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.     

The mouse karyotype in somatic cells cultured in vitro

Summary An idiogram of the karyotype of the mouse somatic cells in culture was constructed by arranging the chromosomes according to decreasing order of length. The length of the individual chromosomes was then measured in 25 colchicin-blocked, hypotonic-treated metaphases from male C3H mice.It was shown that the 40 chromosomes of the diploid chromosome set of the mouse, although they appeared to be very similar to one another, can be subdivided into five distinct groups, each including chromosomes of similar length. The X chromosome belongs to the second group, the Y chromosome to the fifth group. It is thus possible to identify the sex also in somatic cells.In order to test the reliability of such a classification, a statistical analysis of the chromosome measurements in 25 mitosis of male C3H mice was carried out. The analysis has shown that the length differences between the chromosomes belonging to different groups are highly significant. A high variability among mitoses is however present.     

Chromosomal aberrations after induced pluripotent stem cells reprogramming

Induced pluripotent stem cells (iPSCs) are generated from adult cells that have been reprogrammed to pluripotency. However, in vitro cultivation and genetic reprogramming increase genetic instability, which could result in chromosomal abnormalities. Maintenance of genetic stability after reprogramming is required for possible experimental and clinical applications. The aim of this study was to analyze chromosomal alterations by using the G-banding karyotyping method applied to 97 samples from 38 iPSC cell lines generated from peripheral blood or Wharton’s jelly. Samples from patients with long QT syndrome, Jervell and Lange-Nielsen syndrome and amyotrophic lateral sclerosis and from normal individuals revealed the following chromosomal alterations: acentric fragments, chromosomal fusions, premature centromere divisions, double minutes, radial figures, ring chromosomes, polyploidies, inversions and trisomies. An analysis of two samples generated from Wharton’s jelly before and after reprogramming showed that abnormal clones can emerge or be selected and generate an altered lineage. IPSC lines may show clonal and nonclonal chromosomal aberrations in several passages (from P6 to P34), but these aberrations are more common in later passages. Many important chromosomal aberrations were detected, showing that G-banding is very useful for evaluating genetic instability with important repercussions for the application of iPSC lines.     

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.     

Growth properties and biochemical characterization of mouse Schwann cells cultured in vitro

Purified secondary cultures of mouse Schwann cells (less than 5% fibroblast contamination) have been obtained by taking advantage of the differential adhesion of Schwann cells and fibroblasts during trypsinization. The growth properties of the purified subcultures changed with time in culture. Cells passaged after 5 days in vitro (DIV) divided rapidly (doubling time 22 h), whereas cells that had been in vitro for longer periods progressively decreased their growth rate, becoming quiescent after 20 or more days. Schwann cells lacked the Thy 1.2 surface antigen, but were positively stained with antigalactocerebroside antibodies after prefixation. Biochemical analyses showed Schwann cells to be enriched in the activities of enzymes characteristic of the myelin-forming cells: 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNP), cerebroside sulfotransferase (CST) and UDP-galactose: ceramide galactosyltransferase (CGalT).     

The status of nucleolus organizing regions in hybrids of pluripotent and somatic mouse cells cultured under different conditions

In the present work we examined the status of nucleolus organizing regions of mitotic chromosomes (NOR) in hybrid cells obtained by fusion of the mouse teratocarcinoma cells PCC4aza1 and adult mouse spleenocytes upon cultivation of hybrid cells under different conditions. We have shown that extended cultivation of hybrid cells in medium supplemented with HAT (hypoxanthine, aminopterin, thymidine) promotes the maintenance of NO-chromosomes, whereas under nonselective conditions elimination of NO-chromosome occurs. In nonselective medium the number of active, i. e. Ag-positive, NORs has been augmented comparatively to that observed under selective conditions. This observation directly indicates that reprogramming of the parental cell genomes in hybrid cells includes changes in the status of chromosomal NORs. The number of active NORs depends on conditions of hybrid cells culturing and may be changed by either of the two major ways--by elimination of NO-chromosomes (under nonselective conditions) or by inactivation of some NORs, when the general number of NO-chromosomes remains unaltered (under selective conditions).     

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.     

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated on day 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.     

In vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells

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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.     

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.     

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.     

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.     

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     

Extended pluripotent stem cells facilitate mouse model generation

Mouse embryonic stem(mES)cells,established in 1981(Evans and Kaufman,1981;Martin,1981),were derived from the inner cell mass(ICM)of blastocysts and can be expanded in vitro for many passages,maintaining normal karyotype and differentiation potential.Upon introduction into blastocysts,mES cells can differentiate into all three germ layers,contributing to all the somatic lineages and germline.     

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.