How cells dedifferentiate: a lesson from plants

The remarkable regenerative capacity displayed by plants and various vertebrates, such as amphibians, is largely based on the capability of somatic cells to undergo dedifferentiation. In this process, mature cells reverse their state of differentiation and acquire pluripotentiality--a process preceding not only reentry into the cell cycle but also a commitment for cell death or trans- or redifferentiation. Recent studies provide a new perspective on cellular dedifferentiation, establishing chromatin reorganization as its fundamental theme.     

Maintenance of differentiated phenotype of articular chondrocytes by protein kinase C and extracellular signal-regulated protein kinase.

The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC alpha was essentially the same as that of type II collagen during de- and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC alpha, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC alpha in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC alpha did not affect type II collagen levels, suggesting that PKC alpha expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by down-regulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of N-cadherin, alpha-catenin, and beta-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.     

Two phases of chromatin decondensation during dedifferentiation of plant cells: distinction between competence for cell fate switch and a commitment for S phase

Cellular dedifferentiation is the major process underlying totipotency, regeneration, and formation of new stem cell lineages in multicellular organisms. In animals it is often associated with carcinogenesis. Here, we used tobacco protoplasts (plant cells devoid of cell wall) to study changes in chromatin structure in the course of dedifferentiation of mesophyll cells. Using flow cytometry and micrococcal nuclease analyses, we identified two phases of chromatin decondensation prior to entry of cells into S phase. The first phase takes place in the course of protoplast isolation, following treatment with cell wall degrading enzymes, whereas the second occurs only after protoplasts are induced with phytohormones to re-enter the cell cycle. In the absence of hormonal application, protoplasts undergo cycles of chromatin condensation/decondensation and die. The ubiquitin proteolytic system was found indispensable for protoplast progression into S phase, being required for the second but not the first phase of chromatin decondensation. The emerging model suggests that cellular dedifferentiation proceeds by two functionally distinct phases of chromatin decondensation: the first is a transitory phase that confers competence for cell fate switch, which is followed, under appropriate conditions, by a second proteasome-dependent phase representing a commitment for the mitotic cycle. These findings might have implications for a wide range of dedifferentiation-driven cellular processes in higher eukaryotes.     

3D chromatin architecture and epigenetic regulation in cancer stem cells

Dedifferentiation of cell identity to a progenitor-like or stem cell-like state with increased cellular plasticity is frequently observed in cancer formation.During this process,a subpopulation of cells in tumours acquires a stem cell-like state partially resembling to naturally occurring pluripotent stem cells that are temporarily present during early embryogenesis.Such characteris-tics allow these cancer stem cells (CSCs) to give rise to the whole tumour with its entire cellular heterogeneity and thereby support metastases formation while being resistant to current cancer therapeutics.Cancer devel-opment and progression are demarcated by transcrip-tional dysregulation.In this article,we explore the epigenetic mechanisms shaping gene expression dur-ing tumorigenesis and cancer stem cell formation,with an emphasis on 3D chromatin architecture.Comparing the pluripotant stem cell state and epigenetic repro-gramming to dedifferentiation in cellular transformation provides intriguing insight to chromatin dynamics.We suggest that the 3D chromatin architecture could be used as a target for re-sensitizing cancer stem cells to therapeutics.     

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.     

Dose-, time-, and tissue-dependent effects of 5-bromo-2′ -deoxyuridine on the in-vitro organogenesis of Arabidopsis thaliana

Vigorous organogenesis can be induced from hypocotyl and root explants of Arabidopsis thaliana using a two-step culture procedure consisting of preculture on callus-inducing medium (CIM) and subsequent culture on shoot-inducing medium (SIM) or root-inducing medium (RIM). With this culture system, we examined the influence of 5-bromo-2′-deoxyuridine (BrdU), a thymidine (dT) analogue, on plant organogenesis in vitro. Treatment with BrdU during SIM or RIM culture had negative effects on shoot and root redifferentiation over a broad range of concentrations. When explants were exposed to low concentrations of BrdU during preculture and then transferred onto BrdU-free SIM, shoot redifferentiation was accelerated significantly. At higher doses, BrdU treatment during the pre-culture inhibited shoot redifferentiation strongly in hypocotyl explants, but not in root explants. This suggests that a target of the BrdU action lies within the process of acquisition of cell proliferation competence specifically involved in hypocotyl dedifferentiation. These effects of BrdU were counteracted by the simultaneous addition of excess dT. BrdU-pretreated and untreated explants did not differ significantly in the phytohormone dependency of shoot redifferentiation. Our results provide a basis for future studies on plant organogenesis combining pharmacological analysis with BrdU as a probe and molecular genetics with Arabidopsis mutants.     

Plasticity of rat bone marrow-derived 5-hydroxytryptamine-sensitive neurons: dedifferentiation and redifferentiation

Inducing cellular dedifferentiation has been proposed as a potential method for enhancing endogenous regeneration in mammals. Here we demonstrate that phenotypic and functional neurons derived from adult rat bone marrow stromal stem cells (MSCs) can be induced to undergo dedifferentiation, then proliferation and redifferentiation. In addition to morphological changes and expression of neuronal markers, neuron-specific enolase and neurofilament H, functional differentiation was monitored by intracellular Ca2+ mobilization in response to a ubiquitous neurotransmitter, 5-hydroxytryptamine (5-HT) at different stages. The neurons derived from rMSCs were found to have increased 5-HT response. This 5-HT sensitivity could be reversed to basal level similar to that found in rMSCs when neurons, up to 3 days after neuronal induction, were induced to undergo dedifferentiation. Increase in 5-HT-induced Ca2+ mobilization was again observed when rMSCs derived from dedifferentiated neurons were induced to redifferentiate into neurons again. Variation in 5-HT1A receptor immunoreactivity was observed in stem cells, differentiated neurons, dedifferentiated neurons and redifferentiation neurons, consistent with their respective 5-HT sensitivity. These results suggest that adult bone marrow-derived 5-HT sensitive neurons are capable of dedifferentiation, then proliferation and redifferentiation, indicating their plasticity and potential use in treatment of neural degenerative diseases.     

Changes in cytoskeletal actin patterns in the Malpighian tubules of the fleshfly,Sarcophaga bullata (Parker) (Diptera : Calliphoridae), during metamorphosis

Using the rhodamine-labelled phalloidin staining method in combination with detergent extraction, metamorphic changes in actin filament patterns were investigated in the Malpighian tubules of the fleshfly, Sarcophaga bullata (Parker) (Diptera : Calliphoridae). Metamorphosis in this organ implies a process of dedifferentiation, followed by a process of redifferentiation. During dedifferentiation, the large basal actin bundles of the primary cells disappear and the microvillar membrane surface of these cells decreases. Concomitantly, several vesicles are pinched off from infoldings of the brush border. In older pupae, the Malpighian tubules redifferentiate to give rise to adult tubules with actin patterns similar to those of larvae. During redifferentiation of the tubules, the secondary cells display a marked increase in the number of actin filaments in their protrusions. The primary cells in the distal part of the anterior Malpighian tubules of late pupae display a well-developed basal pattern of thick parallel actin bundles. In most cases, major changes in actin filament patterns are found simultaneously with major changes in cell shape, indicating a close relationship between these actin filaments and the process of cellular remodelling.     

Redifferentiation of dedifferentiated chondrocytes on chitosan membranes and involvement of PKCalpha and P38 MAP kinase

To investigate the effects of chitosan on the redifferentiation of dedifferentiated chondrocytes, we used chondrocytes obtained from a micromass culture system. Micromass cultures of chick wing bud mesenchymal cells yielded differentiated chondrocytes, but these dedifferentiated during serial monolayer subculture. When the dedifferentiated chondrocytes were cultured on chitosan membranes they regained the phenotype of differentiated chondrocytes. Expression of protein kinase C (PKC) increased during chondrogenesis, decreased during dedifferentiation, and increased again during redifferentiation. Treatment of the cultures with phorbol 12-myristate 13-acetate (PMA) inhibited redifferentiation and down-regulated PKC. In addition, the expression of p38 mitogen-activated protein (MAP) kinase increased during redifferentiation, and its inhibition suppressed redifferentiation. These findings establish a culture system for producing chondrocytes, point to a new role of chitosan in the redifferentiation of dedifferentiated chondrocytes, and show that PKC and p38 MAP kinase activities are required for chondrocyte redifferentiation in this model system.     

Glucosaminidase and Dedifferentiation of Newt Iris Epithelium

To try to understand the mechanism of the dedifferentiation process which occurs during metaplastic transformation of iris epithelial cells into lens cells in newt lens regeneration, the activity of N -acetylglucosaminidase in iris and iris epithelium was studied as a function of time after lentectomy. The activity was found to increase during the dedifferentiation phase of the iris epithelium. The dorsal iris, where definite dedifferentiation occurs side by side with incomplete dedifferentiation, shows significantly greater enhancement of the activity than the ventral iris, where only incomplete dedifferentiation takes place. When the cells complete dedifferentiation and engage in redifferentiation into lens cells, the level of activity drops, approaching that of the normal lens. Evidence is also presented for release of the enzyme into the ocular fluid during dedifferentiation. The possibility that the enzyme is involved in surface alterations of iris epithelial ceils engaged in dedifferentiation is discussed.     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity     

Disruption of normal forelimb regeneration in adult Notophthalmus viridescens by sublethal concentrations of trypan blue.

The effects of the vital dye trypan blue (TpB) on the regeneration of amputated newt forelimbs were examined. Administration of the dye (10 mug/g body weight) via IP injection during the early wound healing and dedifferentiation phases of regeneration inhibited the normal regenerative response. The accumulation phases of regeneration are similarly halted but only by greater concentrations of TpB (50 mug/g body weight) while redifferentiation and morphogenesis are only affected by still greater concentrations of the dye (100 mug/g body weight). In addition to abolishing the regenerative response, low levels of TpB were also capable of inducing skeletal abnormalities in the regenerates as might be expected from previous reports on the teratogenicity of the dye. The in vitro action of newt hyaluronidase (as well as purified testicular hyaluronidase) on hyaluronate was diminished by TpB, with virtually complete inhibition observed at initial reaction mixture concentrations of 100 mug/ml. The results of this study suggest that TpB acts to disrupt the normal regenerative response by preventing dedifferentiation and remodeling, perhaps by inhibition of various necessary lytic enzyme functions or by interference with normal intercellular communications.     

'Open minded' cells: how cells can change fate

It has long intrigued researchers why some but not all organisms can regenerate missing body parts. Plants are remarkable in that they can regenerate the entire organism from a small piece of tissue, or even a single cell. Epigenetic mechanisms that control chromatin organization are now known to regulate the cellular plasticity and reprogramming necessary for regeneration. Interestingly, although animals and plants have evolved different strategies and mechanisms to control developmental processes, they have maintained many similarities in the way they regulate chromatin organization. Given that plants can rapidly switch fate, we propose that an understanding of the mechanisms regulating this process in plant cells could provide a new perspective on cellular dedifferentiation in animals.     

Differential protein profiles in interspecific hybrids between Elaeis oleifera and E. guineensis with contrasting responses to somatic embryogenesis competence acquisition

Understanding the molecular events that initiate somatic embryogenesis (SE) may help optimize clonal propagation protocols in oil palm. The objective of this work was to identify differentially abundant proteins during the induction of SE in two interspecific F1 hybrids of Elaeis oleifera?×?Elaeis guineensis with contrasting responses (responsive and non-responsive) to the SE process. Leaf explants were obtained and submitted to SE induction medium for up to 180 days. Explants were collected at 0, 14, 90 and 150 days of induction (doi). Proteins were extracted and analyzed by two-dimensional electrophoresis. Analyses were focused at 14 and 150 doi, representing the initial and late stages of embryogenic competence acquisition, respectively. The results indicate that at 14 doi a high amount of stress is present which results in cellular dedifferentiation. At 90 doi, cells (of the responsive genotype) seem adapted and have kept stress under control, allowing the use of energy for cellular proliferation that occurs at 150 doi. Therefore, the control of stress and oxidation seem to be crucial for callus development. We highlight proteins associated to oxidative stress, protein processing, energy metabolism and development as potentially involved in embryogenic competence acquisition.

     

Population doublings and percentage of S100‐positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57–4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90‐negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90‐positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100‐positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell‐based cartilage repair treatments. J. Cell. Physiol. 222: 411–420, 2010. © 2009 Wiley‐Liss, Inc.