Cell fate switch during in vitro plant organogenesis

Plant mature cells have the capability to reverse their state of differenUation and produce new organs under cultured conditions. Two phases, dedifferentiation and redifferentiation, are commonly characterized during in vitro organogenesis.In these processes, cells undergo fate switch several times regulated by both extrinsic and intrinsic factors, which are associated with reentry to the cell cycle, the balance between euchromatin and heterochromatin, reprogramming of gene expression, and so forth. This short article reviews the advances in the mechanism of organ regeneration from plant somatic cells in molecular, genomic and epigenetic aspects, aiming to provide important information on the mechanism underlying cell fate switch during in vitro plant organogenesis.     

When plant teratomas turn into cancers in the absence of pathogens

Habituated calli have long been classified as neoplasms together with tumors from different origins. The general opinion is that habituation is a reversible process with an epigenetic basis. This is probably true in most cases examined. However, we show here that there might be several degrees of habituation, which can be considered as steps of a neoplastic progression leading to cancerisation in the absence of an introduced oncogenic pathogen. Cell rejuvenation, loss of the capacity to organize meristematic centers, and loss of totipotency are proposed to define plant cancer through this neoplastic progression of a callus.
Habituated tissues share many morphological and biochemical similarities with so-called vitreous shoots from micropropagation. Vitrification and hyperhydric malformations of shoots raised in vitro may be considered as steps of another neoplastic progression, which leads to cancerisation also in the absence of introduced oncogenic pathogens. In this case death of the whole organism occurs either through direct apex necrosis or indirectly, from the loss of the capacity for the primary meristems to function normally, which gives rise to completely anarchic structures. As in the animal kingdom, carcinogenesis in plants is the final result of a multistep process involving the irreversible conversion of a stem cell to a terminal-differentiation-resistant cell.     

In vitro gametogenesis from embryonic stem cells

Many insights into mammalian germ cell development have been gained through genetic engineering and in vivo studies, but the lack of an in vitro system for deriving germ cells has hindered potential advances in germ cell biology. Recent studies have demonstrated embryonic stem cell differentiation into germ cells and more mature gametes, although significant unanswered questions remain about the functionality of these cells. The derivation of germ cells from embryonic stem cells in vitro provides an invaluable assay both for the genetic dissection of germ cell development and for epigenetic reprogramming, and may one day facilitate nuclear transfer technology and infertility treatments.     

Epigenetic cell reactions induced by ionizing radiation

The importance of radiation modification of epigenetic activity in the general mechanism of radiobiological reactions is proved. The inheritable epigenetic changes induced by irradiation are one of the basic reasons of formation of the remote radiation pathology. It is noted that epigenetic inheritable changes of cells have the determined character distinguishing them from mutation changes, being individual and not directed. It is underlined the ability of ionizing radiation to modify a level of spontaneous genetic instability inherited in a number of cell generations on the epigenetic mechanism.     

DNA methylation as a key process in regulation of organogenic totipotency and plant neoplastic progression?

Summary Progressive loss of organogenic totipotency appears to be a common event in long-term plant tissue culture. This loss of totipotency, which has been proposed to be a typical trait of plant neoplastic progression, is compared to some mechanisms that occur during the establishment of animal differentiation-resistant cancer lines in vitro. Evidence is presented that alteration in DNA methylation patterns and expression of genes occur during long-term callus culture. An effect of the auxin, 2,4-dichlorophenoxyacetic acid, in the progressive methylation, is moreover suggested. Methylation of genes relevant to cell differentiation and progressive elimination of cells capable of differentiation is proposed as being responsible for this progressive loss of organogenic potential. Finally, the epigenetic alteration (DNA methylation) that occurs during prolonged periods of culture may induce other irreversible genetic alterations that ultimately make the loss of totipotency irreversible.     

What keeps cells in tissues behaving normally in the face of myriad mutations?

The use of a reporter gene in transgenic mice indicates that there are many local mutations and large genomic rearrangements per somatic cell that accumulate with age at different rates per organ and without visible effects. Dissociation of the cells for monolayer culture brings out great heterogeneity of size and loss of function among cells that presumably reflect genetic and epigenetic differences among the cells, but are masked in organized tissue. The regulatory power of a mass of contiguous normal cells is expressed in its capacity to normalize the appearance and growth behavior of solitary homophilic neoplastic cells, and to redirect differentiation of solitary heterophilic stem-like cells. Intimate contact between the interacting cells is required to induce these changes. The normalization of the neoplastic phenotype does not require gap junctional communication between cells, though transdifferentiation might. These varied relationships are manifestations of the unifying biological principle of "order in the large over heterogeneity in the small".     

In vitro neoplastic transformation of plant callus tissue by γ-radiation

Tumors have been induced by γ-radiation in callus tissue derived from a monocotyledonous flowering plant, Haworthia mirabilis Haw. The transformed tissue exhibited compact texture, excessive cell proliferation and loss of capacity for organogenesis. Tumors were characterized by their ability to undergo continuous autonomous growth on minimal media in the subsequent 4 generations of subculture. In contrast, the nonirradiated control tissue grew with friable texture, required inositol or growth hormones and showed prolific differentiation of vegetative buds.     

Resistance of cultured human fibroblasts and other cells to purine and pyrimidine analogues in relation to mutagenesis detection

In vitro enumeration of diploid human cell variants that are resistant to purine analogues is a possible method of detecting mutagenesis. Their incidences can be increased by the known mutagens, X-rays and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Usefulness of this method depends on the kinds of hereditary changes that confer analogue-resistance on somatic cells. If resistance usually results from changes in genetic material, in vitro studies could be useful indicators of mutagenic effects on somatic cells and germ cells in vivo. If epigenetic changes are primarily responsible for analogue-resistant variants, their enumeration might not provide information relevant to germinal mutations but would still be a useful way to detect induction of general kinds of stable phenotypic changes that could cause cancer. This article outlines hypothetical epigenetic and genetic causes of somatic cell variation and a prospective genetic analysis of human cell variants that are resistant to 8-azaguanine (AG) or 2,6-diaminopurine ( (DAP).Recent evidences and arguments favoring epigenetic origins of resistance to base-analogues are inconclusive. The often cited high rate of changes causing impermeability to BUdR in hamster cells is based on one improperly executed determination. Comparisons of rates of variation conferring BUdR-resistance on cultured haploid and diploid frog cells included diploid variants that did not behave as mutants and ignored major sources of error in estimating mutation rates. AG-resistance could result from recessive mutations in X-chromosomal genes but comparisons of rates of mutation in hamster cells of different ploidies did not provide information about the numbers of X-chromosomes in the variants. Reports that normal rodent HGPRT reappeared in hybrids of enzyme-deficient rodent cells and HGPRT-containing cells of other species or in the rodent cells alone in response to the conditions of cell hybridization did not include adequate controls for reversions in mutant genes of the rodent cells. Questions about the epigenetic and genetic origins of analogue-resistance are mostly unanswered. It remains possible that some kinds of abnormal epigenetic changes cause somatic disease. Specific methods for detecting their occurrence and responsiveness to environmental factors should be devised by focusing efforts on traits that are normally subject to epigenetic regulation. Derepression of genes on the inactive X-chromosome and of liver phenylalanine hydroxylase production are presented as possible examples of abnormal epigenetic changes that could be quantitatively studied by direct selection in vitro.     

Epigenetics in plant tissue culture

Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at the molecular level, viz., changes in DNA methylation and alterations of histone methylation or acetylation. Various studies have been carried out with animals, and with plant cells or tissues that have grown in tissue culture but only little work has been done with shoots generated by axillary branching. We present various molecular methods that are being used to measure epigenetic variation. In micropropagated plants mostly differences in DNA methylation have been examined. Epigenetic changes are thought to underlie various well-known tissue-culture phenomena including rejuvenation, habituation, and morphological changes such as flower abnormalities, bushiness, and tumorous outgrowths in, among others, oil palm, gerbera, Zantedeschia and rhododendron.     

Keep-ING balance: Tumor suppression by epigenetic regulation

Cancer cells accumulate genetic and epigenetic changes that alter gene expression to drive tumorigenesis. Epigenetic silencing of tumor suppressor, cell cycle, differentiation and DNA repair genes contributes to neoplastic transformation.     

In vitro organogenesis using multipotent cells

The establishment of efficient methods for promoting stem cell differentiation into target cells is important not only in regenerative medicine, but also in drug discovery. In addition to embryonic stem (ES) cells and various somatic stem cells, such as mesenchymal stem cells derived from bone marrow, adipose tissue, and umbilical cord blood, a novel dedifferentiation technology that allows the generation of induced pluripotent stem (iPS) cells has been recently developed. Although an increasing number of stem cell populations are being described, there remains a lack of protocols for driving the differentiation of these cells. Regeneration of organs from stem cells in vitro requires precise blueprints for each differentiation step. To date, studies using various model organisms, such as zebrafish, Xenopus laevis , and gene-targeted mice, have uncovered several factors that are critical for the development of organs. We have been using X. laevis , the African clawed frog, which has developmental patterns similar to those seen in humans. Moreover, Xenopus embryos are excellent research tools for the development of differentiation protocols, since they are available in high numbers and are sufficiently large and robust for culturing after simple microsurgery. In addition, Xenopus eggs are fertilized externally, and all stages of the embryo are easily accessible, making it relatively easy to study the functions of individual gene products during organogenesis using microinjection into embryonic cells. In the present review, we provide examples of methods for in vitro organ formation that use undifferentiated Xenopus cells. We also describe the application of amphibian differentiation protocols to mammalian stem cells, so as to facilitate the development of efficient methodologies for in vitro differentiation.     

Red cell mechanics: what changes are needed to adversely affect in vivo circulation

The ability of red cells to deform is essential to allow their circulation. However the degree of rheological abnormality which can be tolerated before flow is impaired is not so clear. Red cell rheology has been characterised in a number of physiological, pathological and genetic conditions, and some inferences can be drawn. In vivo aging causes a small loss of cell deformability attributable to increased membrane and internal viscosity; volume and surface area are also lost. These changes cannot be sufficient to cause cellular removal, since the cells sampled had continued to circulate. In sickle cell disease, the oxygenated blood contains dense cells that are more severely abnormal than dense, aged cells from normal individuals. Melanesian ovalocytes have comparable rigidity to dense SS cells, but this condition has no marked circulatory pathology. Thus circulatory problems in SS disease probably stem from deoxygenation-induced sickling which causes extreme loss of deformability, rather than from the abnormal cells in oxygenated blood. In falciparum malaria, immature parasites cause appreciable loss of deformability but continue to circulate. Maturation of the parasites causes much greater rheological changes, including attachment to vascular endothelium, and the cells cease to circulate. In summary, quite marked changes in cell mechanics can occur without loss of ability to circulate. It thus seems that slight rheological alterations reported in some clinical studies are unlikely to cause appreciable flow disruption.     

Diversification and progression of malignant tumors

Tumor-cell diversification mechanisms insure that malignant neoplasms contain diversified tumor-cell subpopulations. Because of the instability of tumor cell phenotypes, some malignant cells will evolve with the most favorable properties for their progression to highly metastatic cells. The rates of cellular phenotypic diversification vary greatly among different tumors, and they are probably modulated, in part, by genetic and chromosome defects and by epigenetic events that may vary widely depending upon the nature of the tumor cells and their microenvironments. As tumor diversification and selection proceed, the most malignant cell subpopulations may eventually become dominant and gradually lose their microenvironmental responsiveness. Tumor-cell diversification mechanisms may be similar or identical to normal, developmentally regulated diversification mechanisms that are used during embryonic cell diversification and differentiation.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

Genetic and epigenetic stability of stem cells: Epigenetic modifiers modulate the fate of mesenchymal stem cells

Stem cell research has progressed widely and has been receiving a considerable attention for its advantages and drawbacks. Despite their extensive therapeutic potential in regenerative medicine, they are debatable for their genetic and epigenetic stability. In fact lineage specific differentiation is mediated via epigenetic changes in DNA methylation, acetylation, histone modifications etc. Thus epigenetics plays an important role in stem cell biology. For therapeutic interventions stem cells need to be genetically and epigenetically stable for their maximum paracrine secretions for bringing about expected tissue repair and regeneration. In this review we have focused on the current status of genetic and epigenetic stability in stem cells and their importance in regenerative medicine. We have also touched upon the possibility of considering tissue resident mesenchymal stem cells as epigenetic modifiers. This is likely to open a new era in stem cell therapeutic intervention by reversing disease inducing epigenetic changes.     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

Role of epigenetic reprogramming of host genes in bacterial pathogenesis

The genomes are regularly targeted by epigenetic regulatory mechanisms (DNA methylation, histone modifications, binding of regulatory proteins) in infected cells. In addition, proteins encoded by microbial genomes may disturb the action of a set of cellular promoters by interacting with the same epi-regulatory machinery. The outcome of this may result in epigenetic dysregulation and subsequent cellular dysfunctions that may manifest in or contribute to the development of pathological changes. How epigenetic methylation decorations on DNA and histones are started and established remains largely unknown. The inherited nature of these processes in regulation of genes suggests that they could play key roles in chronic diseases associated with microbial persistence; they might also explain so-called hit-and-run phenomena in infectious disease pathogenesis. Microbes infecting mammals may cause diseases by causing hyper-methylation of key cellular promoters at CpG di-nucleotides and may induce pathological changes by epigenetic reprogramming of host cells they are interacting with elucidation of the epigenetic consequences of microbe–host interactions may have important therapeutic implications because epigenetic processes can be reverted and elimination of microbes inducing patho-epigenetic changes may prevent disease development.     

Genic DNA methylation changes during in vitro organogenesis: organ specificity and conservation between parental lines of epialleles

During differentiation, in vitro organogenesis calls for the adjustment of the gene expression program toward a new fate. The role of epigenetic mechanisms including DNA methylation is suggested but little is known about the loci affected by DNA methylation changes, particularly in agronomic plants for witch in vitro technologies are useful such as sugar beet. Here, three pairs of organogenic and non-organogenic in vitro cell lines originating from different sugar beet (Beta vulgaris altissima) cultivars were used to assess the dynamics of DNA methylation at the global or genic levels during shoot or root regeneration. The restriction landmark genome scanning for methylation approach was applied to provide a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence that is particularly adapted for studies on crop plants without a fully sequenced genome. The cloned sequences had putative roles in cell proliferation, differentiation or unknown functions and displayed organ-specific DNA polymorphism for methylation and changes in expression during in vitro organogenesis. Among them, a potential ubiquitin extension protein 6 (UBI6) was shown, in different cultivars, to exhibit repeatable variations of DNA methylation and gene expression during shoot regeneration. In addition, abnormal development and callogenesis were observed in a T-DNA insertion mutant (ubi6) for a homologous sequence in Arabidopsis. Our data showed that DNA methylation is changed in an organ-specific way for genes exhibiting variations of expression and playing potential role during organogenesis. These epialleles could be conserved between parental lines opening perspectives for molecular markers.