174 research outputs found

    Dedifferentiation: A new approach in stem cell research

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    Dedifferentiation is an important biological phenomenon whereby cells regress from a specialized function to a simpler state reminiscent of stem cells. Stem cells are self-renewing cells capable of giving rise to differentiated cells when supplied with the appropriate factors. Stem cells that are derived by dedifferentiation of one's own cells could be a new resource for regenerative medicine, one that poses no risk of genetic incompatibility or immune rejection and provokes fewer ethical debates than the use of stem cells derived from embryonic tissue. Until now, it has not been quite clear why some differentiated cell types can dedifferentiate and proliferate, whereas others cannot. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine and even tumor biology. If so, dedifferentiation will offer an ethically acceptable alternative route to obtain an abundant source of stem cells. Dedifferentiation is likely to become a new focus of stem cell research. Here we compile recent advances in this emerging but significant research, highlighting its central concepts, research findings, possible signaling pathways, and potential applications.published_or_final_versio

    Transcriptional Correlates of Proximal-Distal Identify and Regeneration Timing in Axolotl Limbs

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    Cells within salamander limbs retain memories that inform the correct replacement of amputated tissues at different positions along the length of the arm, with proximal and distal amputations completing regeneration at similar times. We investigated the possibility that positional memory is associated with variation in transcript abundances along the proximal-distal limb axis. Transcripts were deeply sampled from Ambystoma mexicanum limbs at the time they were administered fore arm vs upper arm amputations, and at 19 post-amputation time points. After amputation and prior to regenerative outgrowth, genes typically expressed by differentiated muscle cells declined more rapidly in upper arms while cell cycle transcripts were expressed more highly. These and other expression patterns suggest upper arms undergo more robust tissue remodeling and cell proliferation responses after amputation, and thus provide an explanation for why the overall time to complete regeneration is similar for proximal and distal amputations. Additionally, we identified candidate positional memory genes that were expressed differently between fore and upper arms that encode a surprising number of epithelial proteins and a variety of cell surface, cell adhesion, and extracellular matrix molecules. Also, genes were discovered that exhibited different, bivariate patterns of gene expression between fore and upper arms, implicating dynamic transcriptional regulation for the first time in limb regeneration. Finally, 43 genes expressed differently between fore and upper arm samples showed similar transcriptional patterns during retinoic acid-induced reprogramming of fore arm blastema cells into upper arm cells. Our study provides new insights about the basis of positional information in regenerating axolotl limbs

    Gene Expression and Discovery During Lens Regeneration in Mouse: Regulation of Epithelial to Mesenchymal Transition and Lens Differentiation

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    Purpose: It has been shown that after extracapsular lens removal by anterior capsulotomy in the mouse, the lens can be regenerated. However, as the capsular bag is filled with fibers, epithelial to mesenchymal transition (EMT), an event which is common after cataract surgery as well, takes place during early stages. This study, using a unique mouse model, was undertaken to identify novel regulators and networks in order to more clearly understand secondary cataracts at the molecular level

    Network based transcription factor analysis of regenerating axolotl limbs

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    <p>Abstract</p> <p>Background</p> <p>Studies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs.</p> <p>Results</p> <p>We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-β1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1.</p> <p>Conclusions</p> <p>Our study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem cell markers in regeneration.</p

    Cell cycle re-entry and the plasticity of myotubes in newt limb regeneration

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    Urodele amphibians regenerate various structures including the limb, tail, lens, and heart. Underlying this regenerative ability may be the plasticity of cells at the site of injury. Muscle is a key tissue to study these issues because myogenesis proceeds by fusion of mononucleate precursor cells into a multinucleate syncytium which is in a state of stable post-mitotic arrest. Following implantation into a regenerating limb, newt myotubes re-enter the cell cycle and give rise to proliferating mononucleate progeny. In culture, generation of mononucleate cells has never been observed, but the myotube nuclei re-enter the cell cycle and arrest in G2. This is in contrast to their mammalian counterparts, which are refractory to growth factor stimulation, and suggests that cell cycle re-entry may be one aspect of myotube plasticity. In initial experiments mouse and newt myoblasts were fused to create hybrid myotubes. In these hybrids, DNA synthesis was observed in both mouse and newt nuclei demonstrating that the post-mitotic arrest of mammalian nuclei can be destabilised. Other experiments addressed the hypothesis that mononucleate cells are generated from multinucleate myotubes by fragmentation of the syncytium, as the nuclei progress through mitosis and cytokinesis. I pursued several strategies to overcome the G2 arrest of cultured myotubes. Transfection with SV-40 large T antigen induced endoreplication of DNA in myotubes, but did not lead to mitosis. Treatment with caffeine resulted in the appearance of fragmented nuclei, which are indicative of aberrant mitosis. This response was dependent on traversal of S-phase by the myotube nuclei. These observations suggested that the block to mitosis is stable in cultured myotubes. A critical question is whether cell cycle re-entry is required for generation of mononucleate cells following implantation of myotubes into regenerating limbs. Cell-cycle re-entry was blocked in myotubes by X-irradiation or expression of the cdk4/6 inhibitor pl6. These myotubes were fluorescently labelled and implanted. Both arrested and control myotubes gave rise to mononucleate progeny, demonstrating that cell cycle re-entry is not required for generation of mononucleate cells

    Transcriptome profiling and digital gene expression by deep-sequencing in normal/regenerative tissues of planarian Dugesia japonica

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    AbstractPlanarians exhibit an extraordinary ability to regenerate lost body parts which is attributed to an abundance of pluripotent somatic stem cells called neoblasts. In this article, we report a transcriptome sequence of a Planaria subspecies Dugesia japonica derived by high-throughput sequencing. In addition, we researched transcriptome changes during different periods of regeneration by using a tag-based digital gene expression (DGE) system. Consequently, 11,913,548 transcriptome sequencing reads were obtained. Finally, these reads were eventually assembled into 37,218 unique unigenes. These assembled unigenes were annotated with various methods. Transcriptome changes during planarian regeneration were investigated by using a tag-based DGE system. We obtained a sequencing depth of more than 3.5million tags per sample and identified a large number of differentially expressed genes at various stages of regeneration. The results provide a fairly comprehensive molecular biology background to the research on planarian development, particularly with regard to its regeneration progress

    Mechanisms of Urodele Limb Regeneration

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    This review explores the historical and current state of our knowledge about urodele limb regeneration. Topics discussed are (1) blastema formation by the proteolytic histolysis of limb tissues to release resident stem cells and mononucleate cells that undergo dedifferentiation, cell cycle entry and accumulation under the apical epidermal cap. (2) The origin, phenotypic memory, and positional memory of blastema cells. (3) The role played by macrophages in the early events of regeneration. (4) The role of neural and AEC factors and interaction between blastema cells in mitosis and distalization. (5) Models of pattern formation based on the results of axial reversal experiments, experiments on the regeneration of half and double half limbs, and experiments using retinoic acid to alter positional identity of blastema cells. (6) Possible mechanisms of distalization during normal and intercalary regeneration. (7) Is pattern formation is a self-organizing property of the blastema or dictated by chemical signals from adjacent tissues? (8) What is the future for regenerating a human limb

    PHYSIOLOGICAL GENOMICS OF SPINAL CORD AND LIMB REGENERATION IN A SALAMANDER, THE MEXICAN AXOLOTL

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    Salamanders have a remarkable ability to regenerate complex body parts including the limb, tail, and central nervous system. Although salamander regeneration has been studied for several hundred years, molecular-level studies have been limited to a relatively few important transcription factors and signaling molecules that are highly conserved among animals. Physiological genomic approaches were used here to investigate spinal cord and limb regeneration. Chapter 2 reports that hundreds of gene expression changes were identified during spinal cord regeneration, showing that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. Chapter 3 presents results that identify the salamander ortholog of mammalian Nogo-A, a gene known to inhibit mammalian nerve axon regeneration. Nogo-A gene expression was characterized during salamander development and adulthood in order to address the roles of Nogo-A in the nervous system. Chapters 4 and 5 use physiological genomic approaches to examine limb regeneration and why this process is dependent upon an intact nerve supply. Results presented in Chapter 4 showed that many processes regulated during early limb regeneration do not depend upon nerve-derived factors, but striking differences arise between innervated and denervated limbs by 14 days after amputation. Chapter 5 identified genes associated with peripheral nerve axon regeneration and identified gene candidates that may be secreted by nerves to support limb regeneration. Lastly, chapter 6 characterizes the expression of a developmentally important family of genes, matrix metalloproteinases, during tail regeneration. These results suggest that matrix metalloproteinases play multiple roles throughout the regeneration process. Primarily, this dissertation presents data from the first genomic studies of salamander regeneration. The results suggest genes such as matrix metalloproteinases, and molecular pathways such as the Wnt and FGF signaling pathways that can be exploited to enhance regenerative ability in humans
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