No description available.

Each year the Organ Procurement and Transplantation Network (OPTN) reports an increase in patients requiring an organ transplant without an increase in available donor organs, leading to a transplant gap that continues to widen. Over 70% of donor hearts are deemed unsuitable for transplantation each year, and a large number of these organs (~50%) are discarded due to poor organ function, decreased ejection fraction, disease, or cardiac arrest (Scientific Registry of Transplant Recipients (SRTR) Annual Data Report 2011).We therefore set out to improve knowledge in the field of cardiac transplantation in terms of organogenesis, bioenergetics, and regeneration. The main goal through tissue bioengineering is to regenerate and salvage discarded hearts through organogenesis, or to lengthen the total organ preservation time such that organs would not be thrown away while a recipient was waiting to be found. Our first hypothesis was that an optimized acellular extracellular matrix scaffold would allow for cell adherence, growth and proliferation, and could potentially be grown into a clinically transplantable organ. To achieve these goals, an optimized protocol was developed for the total acellularization of a whole porcine heart, leaving behind a 3D bioscaffold. We showed that acellularized matrices could be successfully seeded using endothelial cells for acellular vasculature and stem cells for other acellular tissues, both as a 2D matrix and within a constantly perfused 3D Langendorff setup bioreactor. In order to best understand cell-cell and cell-matrix interactions, cellular bioenergetics were evaluated. We hypothesized that the bioenergetic demand of the type and anatomical origin of stem cells would affect the regeneration potential dependent on intrinsic metabolic demand. We therefore showed a differential of the bioenergetic profiles of human adipose-derived stem cells isolated from various adipose depots, concluding that the physiological microenvironment that supports stem cells in specific anatomic locations can regulate how stem cells participate in tissue regeneration, maintenance and repair, and also will vary based on donor-differences. During organ transplantation, organ preservation solutions are created for use at specific conditions, such as on ice or at room temperature. We hypothesized that hypothermia would slow down cellular metabolism, and that solutions containing a higher content of antioxidants and other protective substrates against ischemic reperfusion injury would create the best organ storage conditions. We tested three organ preservation solutions against control media and normal saline at 4 and 21 degrees C, for 4 to 8 hours, investigating the bioenergetics of organ preservation solution effects on cardiac cells. By simulating clinical conditions, we were able to determine that one of our solutions was ideal and had protective effects for cells for up to 8 hours at 4 degrees C. Finally, we believed that studying existing cardiac patches and optimizing cardiac matrix design would lead to improved cardiac physiological function and would aid in healing and repair during cardiac surgery. Following a clinical case report showing new cardiac tissue growth after implantation of an acellular porcine extracellular matrix, we devised a proof-of-concept study to show that clinical matrices could be easily cultured in vitro. We successfully seeded these clinical matrices using human amniotic stem cells, a commonly used cell type for regeneration and repair after surgery. Our preliminary studies suggest that preconditioned matrices can be potentially used clinically for greater efficacy and tissue regeneration.

Leaf discs of Saintpaulia ionantha x confusa hybrids (African Violets) were cultured and transferred between hormonal free medium (MS basal medium) and shoot-inducing medium (SIM) to determine whether there is a window of competence in shoot regeneration. The results showed that cultured cells were not responsive to shoot-inducing signals (i.e. not competent) until 3-5 days after explant isolation but the ability to regenerate shoots was not lost in surviving cells/tissues cultured on basal medium. Light microscopic observations found that first periclinal divisions of epidermal cells occurred at 3-5 days on SIM. Meristemoids were then formed from the derivatives of the original epidermal cells. It is proposed that cellular competence for shoot regeneration is acquired in culture. The pre-competent period consists of a resting phase and a "dedifferentiation" phase in which epidermal cells divide periclinally to form "dedifferentiated" cells which are the true target cells for shoot induction. Mutagenic treatments and propagation of a chimeral African Violet cultivar were carried out to study cell origin of adventitious shoots in African Violet leaf culture. The results suggest that adventitious shoots may originate from either multiple cells or single cells. The multiple cell origin is in contrast to the hypothesis of exclusively single cell origin for adventitious shoots proposed in several studies. Several factors were studied to identify the source of variations in shoot regeneration among individual explants and to define conditions favourable for shoot regeneration in African Violet in vitro culture. These factors include donor plant growth temperature, the presence of light and the quality of light in culture, the role of chlorophyll in cultured tissues, position of explants in leaves, age of explant materials, source and type of explants, wounding and leaf disc orientation on media. It was found that the presence of light in culture, age of leaf explants, source (in vitro cultured vs pot plant) and type (leaf vs petiole) of explants, wounding and leaf disc orientation on media all had a statistically significant effect on shoot production.

Sugar Maple (Acer saccharum Marsh.) is a very important tree species and is known not only for its sap in the production of maple syrup, but also for its superior hardwood quality and popular ornamental properties. In the effort to improve the diversity of the hardwood tree species in Manitoba, an effective propagation method for sugar maple is needed. The study tested several propagation techniques namely in vitro shoot organogenesis, induced embryo conversion and rooting of greenwood cuttings. Shoot multiplication was achieved using bud and embryo explants, however the rate of shoot production was very low implying that the culture conditions required some optimizations. Dormant isolated embryos were induced to germinate and convert into whole plants in vitro, eliminating the need for long stratification period. The study found the highest embryo conversion frequencies by the addition of cytokinin 6-benzylaminopurine (BAP) at 0.5-1.5 mg/L or thidiazuron (TDZ) at 0.01 mg/L into the culture medium. Greenwood cuttings of several hardy cultivars, including ‘Jefcan’, ‘Bailsta’ and ‘Green Mountain’ were compared for rooting capacity. In 2008, cutting type, rooting hormone and collection time were found to significantly influence rooting. In the following year, the study was expanded to compare different rooting conditions, using peat-perlite mix in fog system, sand beds under intermittent misting, and commercial peat plugs under automated misting system. Rooting was improved by selecting for medial-type cuttings and by promoting cutting survival through the the use of peat-based rooting medium and the maintenance of cool temperatures during the rooting period. The application of auxin did not increase rooting frequency of ‘Jefcan’ cuttings, but considerably improved the quality of roots produced, which may affect cutting survival upon transplantation.

Sugar Maple (Acer saccharum Marsh.) is a very important tree species and is known not only for its sap in the production of maple syrup, but also for its superior hardwood quality and popular ornamental properties. In the effort to improve the diversity of the hardwood tree species in Manitoba, an effective propagation method for sugar maple is needed. The study tested several propagation techniques namely in vitro shoot organogenesis, induced embryo conversion and rooting of greenwood cuttings. Shoot multiplication was achieved using bud and embryo explants, however the rate of shoot production was very low implying that the culture conditions required some optimizations. Dormant isolated embryos were induced to germinate and convert into whole plants in vitro, eliminating the need for long stratification period. The study found the highest embryo conversion frequencies by the addition of cytokinin 6-benzylaminopurine (BAP) at 0.5-1.5 mg/L or thidiazuron (TDZ) at 0.01 mg/L into the culture medium. Greenwood cuttings of several hardy cultivars, including ‘Jefcan’, ‘Bailsta’ and ‘Green Mountain’ were compared for rooting capacity. In 2008, cutting type, rooting hormone and collection time were found to significantly influence rooting. In the following year, the study was expanded to compare different rooting conditions, using peat-perlite mix in fog system, sand beds under intermittent misting, and commercial peat plugs under automated misting system. Rooting was improved by selecting for medial-type cuttings and by promoting cutting survival through the the use of peat-based rooting medium and the maintenance of cool temperatures during the rooting period. The application of auxin did not increase rooting frequency of ‘Jefcan’ cuttings, but considerably improved the quality of roots produced, which may affect cutting survival upon transplantation.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Abstract The mammalian metanephric kidney develops through ureteric bud branching morphogenesis and tubule formation and involves secreted inductive signals and possibly their antagonists to regulate the process. Sprouty (spry) genes encode antagonists of FGFs and the EGF signalling pathways. To get an insight to potential developmental roles of the spry genes, the expression of spry1, 2 and 4 was analyzed in developing kidney. Spry1 is expressed in the ureteric bud, and spry2 and 4 in the ureteric bud, the kidney mesenchyme and the nephrons deriving from it suggesting developmental roles for the sprys in kidney development. Spry function was addressed in vivo in the kidney by targeting hspry2 expression to the ureteric bud with a Pax2 promoter. Hspry2 expression led to development of small, ectopic and cystic kidneys. Ureter branching was reduced and there was less glomeruli in a smaller kidney compared to the wild type controls. Spry2 may antagonize signalling of FGF2 and lead to changes in FGFR1 and FGFR3 expression. In organ culture ectopic FGFs restored ureteric branching of the hSpry2 transgenic kidneys suggesting that hSpry2 may antagonize FGF signalling in embryonic kidney. In addition to changes in FGFs, hspry2 expression also lead to downregulation of GDNF and BMP4. We conclude that the Sprouty-FGFs-FGFR signaling is important for kidney development. Wnt2b is a recently identified member of the Wnt family of secreted growth factors, but its function in organogenesis is unknown. In the kidney Wnt2b is localized to the perinephric mesenchymal cells at the initiation of organogenesis. Wnt2b signalling supported ureteric bud growth and branching in vitro. Ureteric bud that was co-cultured with Wnt2b expressive cells or incubated with a known Wnt pathway regulator lithium, and then recombined with isolated kidney mesenchyme led to recovery of the expression of some ureteric epithelial marker genes and reconstitution of early kidney development. Hence, Wnt2b signalling is critical for induction of ureteric branching in vitro. Type XVIII collagen is a matrix molecule and may be involved in Wnt signalling. Roles of type XVIII collagen in kidney and lung organogenesis was analysed. Type XVIII collagen expression correlated with the differences in epithelial branching in both of these organs and its expression in the epithelial tissue was mutually exclusive. In recombinants of ureteric bud and lung mesenchyme, type XVIII collagen expression pattern shifted from kidney to lung type and was accompanied by a shift in epithelial Sonic Hedgehog (Shh) expression and by ectopic lung Surfactant Protein C in the ureteric bud. Blocking of type XVIII collagen function prevented ureteric development with lung mesenchyme and associated with reduction in the expression of Wnt2. Taken together, the findings suggest critical roles for Sprouty2, Wnt2b and type XVIII collagen in controlling pattern formation and the mode of ureteric bud branching in the embryonic kidney.

Abstract How cell and tissue interactions lead to complex structures and differentiated cell types during organogenesis is still one of the most fundermental questions in modern molecular biology. Laminin appears to have a role in branching morphogenesis during organ development. Laminin5 (α3β3γ2) is an epithelium-specific isoform of laminin and previous report has shown that two alternative transcripts for the γ2 chain, the longer γ2 and the shorter γ2*, result from alternative use of the last exon in the human LAMC2 gene. But the transcription of murine laminin γ2 and γ2* and their biological significance have remained unclear. Type XVIII collagen is a newly identified member of the collagen family. It may be involved in the Wnt signaling pathway, since its longest N-terminal variant contains a frizzled domain, which is part of the Wnt receptor and could antagonize Wnt signaling when secreted. Wnt2b is a new member of the Wnt family. Also its function in organogenesis is unknown. In this study, we have investigated the expressions of laminin γ2 and γ2*, type XVIII collagen and Wnt2b during mouse organogenesis. The function of type XVIII collagen in developing lung, kidney and a recombination of ureteric bud and lung mesenchyme tissue and the function of the Wnt-2b gene during kidney organogenesis were studied by using the combined methods of traditional experimental embryology and modern molecular biology. Two alternative laminin γ2 transcripts were demonstrated in mouse. In the developing kidney, the shorter γ2* form was localized in the mesenchyme, whereas the longer γ2 form was only present in the epithelium of the Wolffian duct and in the ureteric bud, indicating different functions for the γ2 variants. Type XVIII collagen was expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomed localized to the epithelial tips in the early-stage lung, while it was confined to the epithelial stalk region and was absent from the nearly formed ureteric tips in the kidney. In recombinants of ureteric bud and lung mesenchyme, the type XVIII collagen expression pattern in the ureteric bud shifted from the kidney to the lung type, accompanied by a shift in epithelial Sonic Hedgehog expression. The lung mesenchyme was also sufficient to induce ectopic lung Surfactant Protein C expression in the ureteric bud. A blocking antibody for the type XVIII collagen reduced the number of epithelial tips in the lung and completely blocked ureteric development with lung mesenchyme, which was associated with a notable reduction in the expression of Wnt2. The shift in type XVIII collagen expression in ureteric bud and lung mesenchyme tissue recombinant was also accompanied by the significant morphological changes in the branching pattern in ureteric bud development. Wnt2b was expressed in numerous developing organs in the mouse embryo, but it was typically localized in the perinephric mesenchymal cells in the region that partly overlaps the presumptive renal stroma at E11.5. Functional studies of the kidney demonstrated that 3T3 cells expressing Wnt2b were not capable of inducing tubule formation but rather stimulated ureteric development. Recombination of ureteric bud treated with cells expressing Wnt2b and isolated kidney mesenchyme resulted in recovery of the expression of epithelial marker genes and better reconstituted organogenesis. Lithium, a known activator of Wnt signaling, was also sufficient to promote ureteric branching in reconstituted kidney in a manner comparable to Wnt2b signaling. Our data suggest that different organ morphogenesis is regulated by an intraorgan patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen. In the mouse kidney, Wnt2b may act as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and the Wnt pathway may regulate ureteric branching directly.

No description available.

<p>As vertebrate embryos grow and develop into adults, their organs must acquire mass and mature tissue architecture to maintain proper homeostasis. While juvenile growth encompasses a significant portion of life, relatively little is known about how individual cells proliferate, with respect to one another, to orchestrate this final maturation. For its simplicity and ease of genetic manipulations, the teleost zebrafish (Danio rerio) was used to understand how the proliferative outputs of individual cells generate an organ from embryogenesis into adulthood. </p><p>To define the proliferative outputs of individual cells, a multicolor clonal labeling approach was taken that visualized a large number of cardiomyocyte clones within the zebrafish heart. This Brainbow technique utilizes Cre-loxP mediated recombination to assign cells upwards of ~90 unique genetic tags. These tags are comprised of the differential expression of 3 fluorescent proteins, which combine to give rise to spectrally distinct colors that represent these genetic tags. Tagging of individual cardiomyocytes was induced early in development, when the wall of the cardiac ventricle is a single myocyte thick. Single cell cardiomyocyte clones within this layer expanded laterally in a developmentally plastic manner into patches of variable shapes and sizes as animals grew into juveniles. As maturation continued into adulthood, a new lineage of cortical muscle appeared at the base of the ventricle and enveloped the ventricle in a wave of proliferation that fortified the wall to make it several myocytes thick. This outer cortical layer was formed from a small number (~8) of dominant cortical myocyte clones that originated from trabecular myocytes. These trabecular myocytes were found to gain access to the ventricular surface through rare breaches within the single cell thick ventricular wall, before proliferating over the surface of the ventricle.</p><p> </p><p>These results demonstrated an unappreciated dynamic juvenile remodeling event that generated the adult ventricular wall. During adult zebrafish heart regeneration, the primary source of regenerating cardiomyocytes stems from this outer wall of muscle. Regenerating cardiomyocytes within this outer layer of muscle are specifically marked by the cardiac transcription factor gene gata4, which they continue to express as they proliferate into the wound area.</p><p>Using heart regeneration to guide investigation of juvenile cortical layer formation, we found that both processes shared similar molecular and tissue specific responses including expression and requirement of gata4. Additional markers suggested that juvenile hearts were under stress and that this stress could play a role to initiate cortical morphogenesis. Indeed, experimental injury or a physiologic increase in stress to juvenile hearts caused the ectopic appearance of cortical muscle, demonstrating that injury could trigger premature morphogenesis.</p><p>These studies detail the cardiomyocyte proliferative events that shape the heart and identify molecular parallels that exist between regeneration and cortical layer formation. They show that adult zebrafish heart regeneration utilizes an injury/stress responsive program that was first used to remodel the heart during juvenile growth.</p> / Dissertation