Lineage potential, plasticity and environmental reprogramming of epithelial stem/progenitor cells.

Recent evidence supports and reinforces the concept that environmental cues may reprogramme somatic cells and change their natural fate. In the present review, we concentrate on environmental reprogramming and fate potency of different epithelial cells. These include stratified epithelia, such as the epidermis, hair follicle, cornea and oesophagus, as well as the thymic epithelium, which stands alone among simple and stratified epithelia, and has been shown recently to contain stem cells. In addition, we briefly discuss the pancreas as an example of plasticity of intrinsic progenitors and even differentiated cells. Of relevance, examples of plasticity and fate change characterize pathologies such as oesophageal metaplasia, whose possible cell origin is still debated, but has important implications as a pre-neoplastic event. Although much work remains to be done in order to unravel the full potential and plasticity of epithelial cells, exploitation of this phenomenon has already entered the clinical arena, and might provide new avenues for future cell therapy of these tissues.

Structure-function characterization and optimization of a plant-derived antibacterial peptide.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Role of major histocompatibility complex class II expression by non-hematopoietic cells in autoimmune and inflammatory disorders: facts and fiction.

It is well established that interactions between CD4(+) T cells and major histocompatibility complex class II (MHCII) positive antigen-presenting cells (APCs) of hematopoietic origin play key roles in both the maintenance of tolerance and the initiation and development of autoimmune and inflammatory disorders. In sharp contrast, despite nearly three decades of intensive research, the functional relevance of MHCII expression by non-hematopoietic tissue-resident cells has remained obscure. The widespread assumption that MHCII expression by non-hematopoietic APCs has an impact on autoimmune and inflammatory diseases has in most instances neither been confirmed nor excluded by indisputable in vivo data. Here we review and put into perspective conflicting in vitro and in vivo results on the putative impact of MHCII expression by non-hematopoietic APCs-in both target organs and secondary lymphoid tissues-on the initiation and development of representative autoimmune and inflammatory disorders. Emphasis will be placed on the lacunar status of our knowledge in this field. We also discuss new mouse models-developed on the basis of our understanding of the molecular mechanisms that regulate MHCII expression-that constitute valuable tools for filling the severe gaps in our knowledge on the functions of non-hematopoietic APCs in inflammatory conditions.

Organisation and role of actin microfilaments during cellular growth and morphogenesis in the moss Physcomitrella patens

ABSTRACT The network of actin cytoskeleton is composed of actin filaments (F-actin) that are made by polymerisation of actin monomers and actin binding proteins. It is required for growth and morphogenesis of eukaryotic cells. The labelling of F-actin with constitutively expressed GFP-Talin (Kost et al., 1998) reveals the organisation of cellular actin networks in plants. Due to the lack of information on actin cytoskeleton through gametophytic development of the model moss plant Physcornitrella patens, stable transgenic lines overexpressing GFP-Talin were generated to detect F-actin structures. It is shown that the 35S promoter driven expression is not suitable for F-actin labelling in all cells. When it is replaced by the inducible heat-shock promoter Gmhsp17.3 from soybean, one hour mild heat stress at 37°C followed by recovery at 25°C is enough to induce efficient and transient labelling in all tissues without altering cellular morphology. The optimal observations of F-actin structures at different stages of moss development can be done between 12-18 hours after the induction. By using confocal microscopy, we demonstrate that stellated actin arrays were densely accumulated at the growing tip in regenerating protoplasts, apical protonemal cells and rhizoids and connected with a fine dispersed F-actin mesh. Following three-dimensional growth, the cortical star-like structures are widespread in the meristematic cells of developing bud and young gametophores. On the contrary, undulating networks of actin cables are found at the final stage of cell differentiation. During redifferentiation of mature leaf cells into protonemal filaments the rather stagnant web of actin cables is replaced by diffuse actin meshwork. In eukaryotes, nucleation of the actin monomers prior to their polymerization is driven by the seven-subunit ARP2/3 complex and formins. We cloned the gene encoding the ARP3 subunit of P. patens and generated arp3 mutants of the moss through gene disruption. The knockout of ARP3 affects the elongation of chloronemal cells and blocks further differentiation of caulonemal cells and rhizoids, and the gametophores are slightly stunted compared to wild-type. The arp mutants were created in the heat-shock inducible GFP-Talin strains allowing us to visualise a disorganised actin network and a lack of star-like actin cytoskeleton arrays. We conclude that ARP2/3 dependent nucleation of actin filaments is critical for the growth of filamentous cells, which in turn influences moss colonization. In complementation assays, the overexpression of Physcomitrella and Arab idopsis ARP3 genes in the moss arp3 mutant results in full recovery of wild type phenotype. In contrast the ARP3 subunit of fission yeast is not able to complement the moss arp3 mutant of moss indicating that regulation of the ARP2/3 dependent actin nucleation diverged in different kingdoms. RESUME Le réseau d'actine est composé de filaments de F-actine et d'un ensemble de protéines s'y attachant (Actin binding proteins). Le réseau d'actine est nécessaire à la croissance et à la morphogenèse de toutes les cellules eucaryotes. Chez les plantes, le marquage ainsi que l'étude de l'organisation du réseau d'actine ont été réalisés en utilisant une fusion GFP-Talin (Kost et al., 1998) exprimée sous le control d'un promoteur constitutif. Afin d'étudier les structures F-actine dans les cellules de Physcomitrella Patens et pour combler le manque d'information sur le développement des gamétophores, des lignées transgéniques stables surexprimant GFP-Talin ont été crées. Nous avons démontré que l'utilisation du promoteur 35S est inadéquate pour le marquage complet et homogène des filaments d'actine dans toutes les cellules de P. patens. Par contre, l'utilisation du promoteur inductible Gmhsp17.3 nous a permis de réaliser un marquage transitoire et général dans tous les tissus de la mousse. Une heure de choc thermique à 37°C suivis d'un temps de récupération de 12-18h à 25°C sont les conditions optimales (sans dommages cellulaires) pour l'observation des structures F-actine à différentes étapes de développement de la mousse. En utilisant la microscopie confocale, nous avons observé l'existence de structures F-actine accumulées en forme d'étoiles. Ces structures, qui sont liées au réseau de microfilaments d'actine, ont été observées dans les protoplastes en régénération, les cellules des protonema apicales ainsi que dans les rhizoïdes. En suivant la croissance tridimensionnelle, ces structures en étoiles ont été observées dans les cellules meristématiques des bourgeons et des jeunes gamétophores. Par contre, dans les cellules différentiées ces structures laissent place à des réseaux de câbles épais. Nous avons également remarqué que durant la redifferentiation des cellules foliaires le réseau de câbles de F-actine est remplacé par un réseau de F-actine diffus. Dans les cellules eucaryotes, la nucléation des filaments d'actirie précédant leur polymérisation est contrôlé par sept sous unités du complexe ARP2/3 et par des formines. Nous avons isolé le gène codant pour la sous unité ARP3 de P. patens et nous avons crée des mutants arp3 par intégration ciblée (Knockout). L'élongation des cellules chloronema est clairement affectée dans les mutants arp3. La différentiation des caulonemata et des rhizoïdes est bloquée et les gametophores sont légèrement plus courts comparé au type sauvage. A fin d'étudier l'organisation des filaments d'actines dans les mutants arp3, nous avons aussi réalisé un arp3-knockout dans la lignée Hsp-GFP-Talin. La nouvelle lignée générée nous a permis de visualiser une désorganisation du réseau d'actine et une absence complète de structures de F-actine accumulée en forme d'étoiles. Les résultats obtenus nous amènent à conclure que la nucléation (ARP2/3 dépendante) des filaments d'actine est indispensable à la croissance des cellules filamenteuses. Par conséquent, les filaments d'actine semblent avoir un rôle dans la colonisation des milieux par les mousses. Nous avons également procédé à des essais de complémentation du mutant arp3. La surexpression des gènes ARP3 de Physcomitrella et d'Arabidopsis dans les cellules du mutant arp3 rétabli complètement le phénotype WT. Par contre, le gène ARP3 des levures n'est pas suffisant pour complémenter la même mutation dans les cellules de mousses. Ce résultat démontre que les mécanismes de régulation de la nucléation des filaments d'actine (ARP2/3 dépendante) sont différents entre les différents groupes d'eucaryotes.

Phenotypic heterogeneity of human cardiac stem cell clones and cardiosphere-forming cells

Although cardiac stem cells have been isolated based on stem cell surface markers, no single marker is stem cell-specific. Clonogenicity is a defining functional property of stemness. We therefore analyzed cardiac cell clones derived from human hearts.Methods: Clonogenic cells were derived from adult human atrial samples. Cells were either cultured in the absence of an initial marker selection or, in separate experiments, they were initially selected for c-kit (CD117), CD31 or CD164 by magnetic immunobeads, or for high aldehyde dehydrogenase activity (ALDH) by FACS. High ALDH activity has been linked to stem/progenitor cells in several tissues. Surface marker analysis was performed by flow cytometry. Cultured cells were also exposed to different factors that modulate cell differentiation, including Dikkopf-1, Noggin, and Wnt-5.Results: Clonogenic cells mainly showed fibroblast-like morphology, ability to grow for more than 30 passages in vitro, and a heterogeneous marker profile even in clones derived from the same cardiac sample. The predominant phenotype was positive for CD13, CD29, CD31, CD44, CD54, CD105 and CD146, but negative for CD10, CD11b, CD14, CD15, CD34, CD38, CD45, CD56, CD106, CD117, CD123, CD133, CD135 and CD271, primarily consistent with endothelial/vascular progenitor cells. However, a minority of clones showed a different profile characterized by expression of CD90, CD106 and CD318, but not CD31 and CD146, consistent with mesenchymal stem/progenitor cells. When initial cell selection was performed, both phenotypes were observed, similarly to unselected cells, irrespective of the selection marker used. Of note, CD117+ sorted cell clones were CD117-negative in culture. Regardless of the immunophenotype, several clones were able to form spheric cell aggregates (cardiospheres), a distinct stem cell property. Dikkopf-1 induced marked CD15 and CD106 upregulation, consistent with stromal differentiation; this effect was prevented by Noggin.Conclusions: The adult human heart contains clonogenic stem/progenitor cells that can be expanded for many passages and form cardiospheres. The surface marker profile of these cells is heterogeneous, consistent with a majority of clones being comprised of endothelial or vascular progenitor cells and a minority of clones consisting of mesenchymal stem/progenitor cells. Dikkopf-1 and Noggin showed opposing effects on stromal differentiation of human cardiac cell clones.

Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Background Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging. Results The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension. Conclusions This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

Association of T-zone reticular networks and conduits with ectopic lymphoid tissues in mice and humans.

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node-like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin(+) T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell-rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter-CXCL13-transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell-rich zone.

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

Wound-healing gene family expression differences between fetal and foreskin cells used for bioengineered skin substitutes.

For tissue engineering, several cell types and tissues have been proposed as starting material. Allogenic skin products available for therapeutic usage are mostly developed with cell culture and with foreskin tissue of young individuals. Fetal skin cells offer a valuable solution for effective and safe tissue engineering for wounds due to their rapid growth and simple cell culture. By selecting families of genes that have been reported to be implicated in wound repair and particularly for scarless fetal wound healing including transforming growth factor-beta (TGF-beta) superfamily, extracellular matrix, and nerve/angiogenesis growth factors, we have analyzed differences in their expression between fetal skin and foreskin cells, and the same passages. Of the five TGF-beta superfamily genes analyzed by real-time reverse transcription-polymerase chain reaction, three were found to be significantly different with sixfold up-regulated for TGF-beta2, and 3.8-fold for BMP-6 in fetal cells, whereas GDF-10 was 11.8-fold down-regulated. For nerve growth factors, midkine was 36-fold down-regulated in fetal cells, and pleiotrophin was 4.76-fold up-regulated. We propose that fetal cells present technical and therapeutic advantages compared to foreskin cells for effective cell-based therapy for wound management, and overall differences in gene expression could contribute to the degree of efficiency seen in clinical use with these cells.

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate.

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.

Membrane lipid composition affects plant heat sensing and modulates Ca ( 2+) -dependent heat shock response.

Understanding how plants sense and respond to heat stress is central to improve crop tolerance and productivity. Recent findings in Physcomitrella patensdemonstrated that the controlled passage of calcium ions across the plasma membrane regulates the heat shock response (HSR). To investigate the effect of membrane lipid composition on the plant HSR, we acclimated P. patens to a slightly elevated yet physiological growth temperature and analysed the signature of calcium influx under a mild heat shock. Compared to tissues grown at 22°C, tissues grown at 32°C had significantly higher overall membrane lipid saturation level and, when submitted to a short heat shock at 35°C, displayed a noticeably reduced calcium influx and a consequent reduced heat shock gene expression. These results show that temperature differences, rather than the absolute temperature, determine the extent of the plant HSR and indicate that membrane lipid composition regulates the calcium-dependent heat-signaling pathway.

TLR5 signaling stimulates the innate production of IL-17 and IL-22 by CD3(neg)CD127+ immune cells in spleen and mucosa.

In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3(neg)CD127(+) immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3(neg)CD127(+) cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues.

Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres) of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1) and cancer stem cell markers (ABCG2, CD44 and ALDH1) genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7). Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells

Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.