Dedifferentiation is the process by which specialised quiescent cells give rise to heterotrophic, dividing cells. This process may be initiated in vivo as a response to wounding, or in vitro during culture initiation. This thesis is concerned with evaluating whether the process of dedifferentiation and maintenance of the fast-dividing, dedifferentiated state by culture, is dependant upon major changes in gene expression. In particular, the role of transcription, as mirrored by changes in steady state mRNA levels, in these putative changes in gene expression has been investigated. Mechanically isolated Asparagus officinalis mesophyll cells were used to study dedifferentiating cells, and suspension cultures of Petunia hybrida to investigate the established dedifferentiated state. This thesis shows that dedifferentiation in Asparagus officinalis is accompanied by major changes in the steady state mRNA profiles of the cells. A group of novel transcripts appearing in dedifferentiating asparagus cells were termed DDl, and targeted for further study. Two cDNA clones coding for DDl transcripts were isolated and characterised, and antibodies to DDl raised for serological work. Only minor differences were found between the steady state mRNA populations of Petunia hybrida cultured cells and seedlings, and these were mainly caused by transcripts disappearing in culture; no transcripts specific to the suspension culture system were detected. The results presented in this thesis are used to foward the hypothesis that changes in gene expression involving de novo transcription may only occur in response to major changes in environmental conditions. It is suggested that the basal transcription pattern for cells in established state is probably common to all cell types with regards to primary cell functions such as growth, division and catabolism. In such established states, the control of metabolism probably resides within the biochemical pathways utilised by the cell at any moment in time.

To obtain a cell source for cartilage tissue engineering primary cells are passaged on polystyrene dishes to increase cell number however, this stiff environment results in dedifferentiation. This study evaluates the role of microenvironment stiffness on regulation of passaged chondrocyte phenotype. Results show passaged cells on soft polyacrylamide gels (0.5kPa) become round, less proliferative, less contractile, have higher levels of globular actin (g-actin) compared to filamentous actin (f-actin), MRTF localization in the cytoplasm and down-regulation of MRTF associated genes such as type I collagen, alpha-smooth muscle actin, transgelin, tenascin C and vinculin. This suggests that the chondrogenic phenotype during passaging is regulated by actin polymerization and activation of MRTF signaling that induces expression of non-chondrogenic genes, and has functional effects as the cells become proliferative and contractile. Modulating substrate stiffness maybe a way to influence aspects of the chondrogenic phenotype in order to obtain sufficient cells suitable for cartilage tissue engineering.

Bronchopulmonary dysplasia (BPD), the chronic lung disease that develops in premature babies following mechanical ventilation and oxygen exposure, is the most common complication of extreme prematurity. Currently, there is no cure for BPD. Increasing evidence indicates early-onset emphysema and pulmonary vascular disease in survivors with BPD (Aukland et al., 2006; Wong et al., 2008), suggesting an irreversible arrest in lung growth and/or premature lung aging resulting in life-long health problems (J. Sucre et al., 2021). Transient in vivo cellular reprogramming through the activation of the Yamanaka reprogramming factors Oct4, Sox2, Klf4, c-Myc (OSKM), ameliorate cellular and physiological hallmarks of aging and to promote tissue regeneration and improve organ function after injury. (Chen et al., 2021a; Hishida et al., 2022b; Lu et al., 2020) This thesis focuses on determining if transient in vivo cellular reprogramming can regenerate an established lung injury in a BPD mouse model. Two strategies, (a) Adeno-Associated virus (AAV) mediated transient overexpression of the OSK factors and (b) using a transgenic reprogrammable mouse line to overexpress the OSKM factors were employed to test the efficiency of in vivo cellular reprogramming in regenerating the lungs. Both the strategies, under the conditions tested, did not regenerate established lung injury in a BPD mouse model but the feasibility of both these strategies was established here laying a foundation for the next phase of the study.

Extrinsic repair techniques for the treatment of acute chondral injuries continue to yield suboptimal repair. The inability of these techniques to produce hyaline cartilage underscores the limitations in our understanding of basic chondrocyte biology. Conversely, intrinsic repair tissue has not been extensively studied despite the fact that it can yield hyaline-like cartilage and is commonly observed in osteoarthritis. Attempts at extrinsic repair could therefore benefit from a better understanding of the successes and failures inherent in the intrinsic repair process. Chondrocyte culture has typically been conducted under non-physiologic conditions whereby chondrocytes readily dedifferentiate. Consequently, much of the knowledge gained about chondrocytes has been misleading thus hindering advancements in chondrocyte biology and attempts at extrinsic articular cartilage (AC) repair. Hypoxic culture conditions, which are beneficial towards the preservation of the chondrocyte phenotype, remain insufficient due to elevated collagen I gene expression. As such, an appropriate model system does not yet exist in which to study physiologically-relevant chondrocyte biology. The presence and prevalence of collagen I in both degenerate and de novo osteoartritic tissue was examined immunohistochemically. Collagen I deposition during osteoarthritic progression was compared against IHC staining for collagen II and aggrecan. A novel model system was also evaluated for chondrocytic phenotype retention. To this end, hypoxic, high-density-monolayer-chondrocyte (HDMC) cultures were compared to freshly isolated chondrocytes for their ability to maintain a chondrocytic extracellular matrix (ECM) gene expression profile. HDMC culture conditions prevented the severe loss of the phenotype typically associated with conventional monolayer culture. Moreover, prolonged HDMC culture resulted in the formation of a complex ECM and a marked suppression of collagen I expression. This study also demonstrated that collagen I deposition occurs in osteoarthritic AC at the onset of structural damage and increases in response to increasing structural damage. Collagen I deposition was also found in different types of de novo cartilage associated with osteoarthritic joints and suggests that it plays an important role in intrinsic cartilage repair. Taken together, this work demonstrates that collagen I is a common feature in the ECM of structurally immature and structurally damaged AC and hence may play a role in tissue stabilization. / Experimental Surgery

No description available.

The adoption and subsequent retention of distinct cellular fates upon cell division is a critical phenomenon in the development of multicellular organisms. A well-studied example of this process is stem cell divisions; stem cells must possess the capacity to self-renew in order to maintain a stem cell population, as well as to generate differentiated daughters for tissue growth and repair. Drosophila neuroblasts are the neural stem cells of the central nervous system and have emerged as an important model for stem cell divisions and the genetic control of daughter cell identities. Neuroblasts divide asymmetrically to generate daughters with distinct fates; one retains a neuroblast identity and the other, a ganglion mother cell, divides only once more to generate differentiated neurons and glia. Perturbing the asymmetry of neuroblast divisions can result in the failure to self-renew and the loss of the neural stem cell population; alternatively, ectopic self-renewal can occur, resulting in excessive neuroblast proliferation and tumorigenesis. Several genetic lesions have been characterized which cause extensive ectopic self-renewal, resulting in brains composed of neuroblasts at the expense of differentiated cells. This contrasts with wild type brains, which are composed mostly of differentiated cells and only a small pool of neuroblasts. We made use of these mutants by performing a series of microarray experiments comparing mutant brains (consisting mostly of neuroblasts) to wild type brains (which are mostly neurons). Using this approach, we generated lists of over 1000 putatively neuroblast-expressed genes and over 1000 neuronal genes; in addition, we were able to compare the transcriptional output of different mutants to infer the neuroblast subtype specificity of some of the transcripts. Finally, we verified the self-renewal function of a subset of the neuroblast genes using an RNAi-based screen, resulting in the identification of 84 putative self-renewal regulators. We went on to show that one of these genes, midlife crisis (mammals: RNF113a), is a well-conserved RNA splicing regulator which is required in postmitotic neurons for the maintenance of their differentiated state. Our data suggest that the mammalian ortholog performs the same function, implicating RNF113a as an important regulator of neuronal differentiation in humans.

Tissue regeneration in mammals does not occur via the process of dedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state. McGann et al. (2001) showed that mouse C2C12 myotubes treated with newt extract derived from regenerating limbs can re-enter the cell cycle, fragment and proliferate ( a characteristic of muscle dedifferentiation). However, the validity of these studies has been called into question since others have been unable to repeat them. My research attempts to replicate the results of McGann et al, and to carry them further. I examined several strategies for tracking the extract-treated cells and I also repeated the studies in a primary muscle culture system. Furthermore, I examined the effect of the extract on myoblast differentiation. The most effective dedifferentiation assay that I developed involved the microinjection of myotubes with extract and with a GFP plasmid that allowed tracking of the injected cells. Cells were then examined for cell cycle re-entry using BrdU incorporation or Ki-67 immunostaining. In addition, immunocytochemistry and RT-PCR analysis were used to examine the expression or down-regulation of muscle-specific markers. Finally, a preliminary GeneChip analysis was conducted to examine which genes were up or down regulated following extract treatment. The results show that newt extract is able to block the differentiation of confluent myoblasts, resulting in fewer multinucleated, myosin heavy chain expressing myotubes. However, when myoblasts were differentiated into myotubes and subsequently treated with newt extract, the results suggest that cell cycle re-entry and down-regulation of differentiation markers can occur in C2C12 myotubes, but not in primary myotubes. Fragmentation though, was seen in both C2C12 and primary myotubes following treatment or injection with newt extract. Moreover, the fragmented cells appeared to be viable. Transcriptional profiling indicated that newt extract affects genes implicated in cell cycle, transcription, stress, chromatin modification, growth, cell adhesion, extracellular matrix, wound healing and microtubule binding. These findings confirm that mammalian myotubes can be induced to dedifferentiate following treatment with newt extract; however, a differential response was observed between C2C12 and primary muscle cells.

Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts, or can be made by dedifferentiation of adult cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient's own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create in vitro cell lines with which to study regenerative medicine. First, we used siRNA targeted against myogenin to induce the dedifferentiation of murine C2C12 myotubes into myoblasts. Timelapse photography, immunofluorescence, and western blot analysis support successful dedifferentiation into myoblasts. However, the inability to separate the myotubes and myoblasts prior to siRNA treatment confounded the results. This system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing, but a better method for separating out the myoblasts needs to be developed before this will be achievable. Second, we used a doxycycline-inducible lentiviral vector encoding the transcription factors Oct4, Sox2, cMyc, and Klf4 to create a line of naive-like porcine induced pluripotent stem cells (iPSCs). This reprogramming vector was verified first in murine cells, the system in which it was developed. Successful production of both murine and porcine iPSC lines was achieved. Both showed alkaline phosphatase activity, immunofluorescence for pluripotency marker (Oct4, Sox2, and Nanog) expression, PCR for upregulation of endogenous pluripotency factors (Oct4, Sox2, cMyc, Klf4, and Nanog), and the ability to form embryoid bodies that expressed markers of all three germ layers. Additionally, we were able to create secondary porcine iPSC lines by exposing cellular outgrowths from embryoid bodies to doxycycline to initiate more efficient production of porcine iPSCs. The secondary porcine iPSCs were similar to the primary porcine iPSCs in their morphology, behavior, alkaline phosphatase expression, and Nanog expression with immunofluorescence. The porcine iPSCs were dependent on doxycycline to maintain pluripotency, indicating that they are not fully reprogrammed. Despite this dependence on doxycyline, this system can be used in the future to study the process of reprogramming, to develop directed differentiation protocols, and to model diseases. / Master of Science

Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is known of the underlying mechanisms. Here, the proteome map of Arabidopsis cotyledons has been established and investigated the dynamic change of the cotyledon proteome in the time course of cell dedifferentiation. Among the 353 distinct genes, corresponding to 500 2-DE gel protein spots identified with high confidence, 12% have over twofold differential regulations within the first 48 h of induction of cell dedifferentiation. The distributions of these genes among different Gene Ontology categories and gene differential regulations within each of the categories have been examined. In addition, the cotyledon phosphoproteome has been investigated using Pro-Q Diamond Phosphoprotein in Gel Stain followed by mass spectrometry analyses. Among the 53 identified putative phosphoproteins, nine are differentially regulated during cell dedifferentiation. Arabidopsis cotyledon proteome at four different time points after the induction of cell dedifferentiation with MudPIT approach has been investigated and analyzed the protein quantity change using two labelree methods, the Spectral Count (SC) and SEQUEST Cross Correlation Coefficient (ÓXcorr) methods. Among the 662 MudPIT identified proteins, one hundred forty eight displayed differential regulation. The up-regulated proteins include transcription factors, calmodulins, translational regulators, and stress response proteins. The Spectral Count and the cross correlation coefficient quantification results are highly consistent in over 81% of the differentially regulated proteins. These studies have provided significant new insight into cell dedifferentiation process in Arabidopsis thaliana and also enhanced the Arabidopsis cotyledon proteome database established using gel based and non gel based methods. The results show that cell dedifferentiation involves extensive protein quantitative and qualitative changes in almost every cellular compartment and cellular process. Proteins like 14-3-3 proteins, Translational controlled tumor protein (TCTP) and its possible interaction protein-Translational elongation factor eEF1 alpha chain, GTP binding nuclear protein RAN2, GTP binding protein SAR1B and several other hypothetical and expressed proteins and nine other phosphoproteins showed significant differential expression during early dedifferentiation. Deciphering the molecular mechanisms regulating the cellular dedifferentiation certainly enhances the understandings and mechanisms of reprogramming all types of differentiated cells including animal cells.

Cell dedifferentiation is a cell fate regression process in which the cell fate memory of a differentiated cell is erased, leading to regain stem cell characteristics. Auxin regulates both cell dedifferentiation and differentiation in plants. It is unknown how auxin controls the two opposite processes. Here the minimal auxin requirements for cell dedifferentiation were found, molecular markers associated with the cell dedifferentiation event were identified. When cellular auxin concentration exceeds the level of meristem cell, most differentiated cells undergo dedifferentiation. In differentiated cells, the polar auxin efflux system prevents cell dedifferentiation by reducing auxin accumulation, particularly in the presence of exogenous auxin. Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, it was found that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced. These results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. In this study, as a part of interaction between auxin and cytokinin it was identified that the induction of ARR5 and ARR6 expression by cytokinin is subjected to the regulation of auxin. The expression of ARR5 and ARR6 follows a mutual exclusive pattern in response to the induction of exogenous auxin in Arabidopsis seedlings and calli. The results suggest that auxin interacts with the cytokinin via a gene and tissue specific induction of the negative regulators in the cytokinin signaling pathway.