Thesis (doctoral)--Ludwig-Maximilians-Universität München, 1993.

A sucrose gradient technique was developed for the preparation of plasma membrane material from SV40 3T3, Py3T3 and HeLa cells. A modification of this method yielded purified nuclei. The plasma membrane material, which was enriched in both 5'-nucleotidase and (Na+K)-ATP ase activities, was used to establish some of the properties of the latter in 3T3 and SV40 3T3 cells. Many of these properties were similar in both cell lines. The effect of conditioning of the growth medium, on the transport of 86Rb into 3T3 and SV40 3T3 cells was investigated. It was shown that conditioning reduced the uptake from growth media and that when the cells were then transferred to Krebs solution, the uptake was apparently increased. The effect of cell population density on several of the properties of 3T3, SV40 3T3, Py3T3 and HeLa cells was examined. When transformed 3T3 cells were grown in medium containing 10% calf serum, the activities of the cell surface enzymes, (Na+K)-ATPase and 5'-nucleotidase, decreased differently with increasing density. The (Na+K)-ATPase activity decreased sharply as the density exceeded 5 x 10⁴ cells/cm2 while the 5'-nucleotidase activity decreased gradually as the density increased. The activity of the latter plateaued so that the overall decrease in the activities of both enzymes was similar. When 3T3 cells were grown under the same conditions, the activities of the cell surface enzymes increased with cell population density. The cell surface enzymes of HeLa cells were shown to decrease with density in a manner similar to those of the transformed 3T3 cells but the decrease in the (Na+K)-ATPase did not exactly follow that of the specific ouabain binding. The effect of culture density on the intracellular enzyme was shown to depend on the cell type as well as the particular enzyme. In all of the 3T3 cell lines, the acid phosphatase activity was observed to increase with increasing density whereas in HeLa cells this enzyme did not alter. In the transformed 3T3 cell lines, the monoamine oxidase activity did not alter with density and the rotenone insensitive NADH-ferricyanide reductase activity increased but in 3T3 cells, the former increased and the latter decreased. In foetal calf serum, the cell surface enzymes were observed to alter differently with respect to cell population density. The cell protein tended to decrease as did the cell volume but these measurements were not directly related. The specific Concanavalin A binding also decreased with cell population density but the decreases were not related to the volume changes. It was concluded that the activity of many of the enzymes of cultured cells is dependent both on the cell population density and the medium in which the cells are grown.

Remyelination is the restoration of myelin sheaths to denuded axons following demyelinating events, which occurs spontaneously in adult mammals, including humans. The principal cells which participate in remyelination are the Oligodendrocyte Progenitor Cells (OPCs). Similar to other regenerative processes, remyelination efficiency declines with ageing. It is still unknown how much of this decline can be attributed to intrinsic changes in the OPCs themselves rather than environmental changes arising from changes in the cellular niche. Thus, we currently have a fundamental gap in our knowledge regarding the basic biology of adult OPCs, and therefore the changes that occur to them with ageing. In order to address these questions, I have developed a method to reliably isolate all cell types of the oligodendrocyte (OL) lineage from adult rats. This allowed me to identify the specific transcriptome state unique to adult OPCs, which is different to the transcriptome of neonatal OPCs, upon which previous studies have focused. This included genes which support the notion that following the initial phases of developmental myelination, adult OPCs enter a quiescent mode, in a manner similar to other tissue resident stem cells. Moreover, using a recently established isolation method, I was able to isolate aged OPCs, and develop a transcriptional database that can allow researchers to explore the changes in aged OPCs and identify new targets for enhancing their function. Lastly, I present in this thesis novel ideas regarding the influence of microglia cell surface molecules on OPC differentiation. I show that changes in the cell surface of aged microglia are inhibitory for OPC differentiation into OLs, and that these changes in microglia are a result of the increase in TGFb levels with ageing. In summary, this dissertation introduces new tools and methods that will allow x further in-depth study of adult OPCs, and specifically will help to shed light on the role of adult OPCs in the CNS in homeostasis. Furthermore, I explore the changes that occur within OPCs as they age, and show how such changes reduce aged OPCs ability to efficiently facilitate the process of remyelination.

Doctor of Philosophy / Department of Animal Sciences and Industry / Duane L. Davis / Monocytes are leukocytes in peripheral blood that differentiate into macrophages in the context of the inflammatory response. Leukocytes are easy to isolate from a blood sample by inexpensive standardized methods, such as the Ficoll-based density gradient. We have found that monocytes isolated from peripheral blood of pigs and grown using simple procedures produce large numbers of mesenchymal cells that exhibit differentiation into mesodermal lineages in vitro. Peripheral blood samples were obtained from 2, 4, and 6 months old male pigs. The cells were isolated by a Ficoll-based density gradient and cultured in 20% FBS in DMEM media, on uncoated tissue culture vessels. All isolates exhibited mesenchymal morphology and continued to expand at least to passage 7. The expansionary potential was greatest for the cells obtained from the 2 mo. old pigs. We isolated similar cells from porcine fetal livers (gestation day 60), at which time hematopoiesis is occurring in the liver. Therefore, these cells are present from at least mid-gestation through 6 months, the approximate age of puberty in pigs. In regards to immune-phenotype, the cells are strongly positive for the leukocyte maker CD 14 and SLA-DR-II. Approximately 50% of the cells are positive for CD 45, and they are negative for CD 105, CD 31, and CD 90. The monocyte-derived cells express mRNAs for TLR-3,4,5, 7, and 9. They also express the pluripotency-associated gene Nanog but only weakly express Sox-2 and Oct-4. In vitro the cells are capable of differentiation into adipogenic, osteogenic, and chondrogenic lineages. They also exhibit phagocytosis as measured by in vitro assay. We tested their ability to support the porcine reproductive and respiratory virus in vitro but they were not supportive using standard techniques. Initial attempts have also failed to support myogenic differentiation. The cells isolated in this study represent a novel subset of monocytes with characteristics overlapping those of mesenchymal stem cells. Swine are physiologically similar to humans and further work is needed to characterize these cells for regenerative medicine applications.

Understanding cell behavior at the material-host tissue interface is a fundamental prerequisite for designing the next generation of biomaterials capable of directing cellular events towards a desired biological outcome (e.g. faster tissue integration). In addition, unraveling the relationship between cell activity and nanoscale surface features will further the present knowledge of the fundamental cellular mechanisms that control how cells sense and respond to natural (e.g. extracellular matrix) and synthetic (e.g. biomaterials) surfaces. It is now well-known that the nanoscale physicochemical features of surfaces dictate cell fate by affecting phenomena such as proliferation, differentiation, genetic transcription and protein translation. In particular, nanotopographical features play a pivotal role during cell-surface interactions by exerting a direct mechanotransductive effect on cells, which, in turn, dictate biochemical signaling. In this context, several studies have addressed different aspects of the relationships between nanofeatures and specific cellular functions, including morphological changes, the establishment of focal adhesions (FAs, clusters of adhesion molecules that regulate cell structure and activity, determining how cells sense and respond to natural and synthetic substrates) and differentiation. However, the precise interplay between the morphological characteristics of nontopographical features not only on the surface but also along a third dimension (height) and cellular response still needs to be fully elucidated. Once revealed, such knowledge will shed new light on how cells sense and respond to 2- and 3-dimensional nanoscale patterns. In this context, anodization, a simple yet effective electrochemical treatment, allows to engender on titanium, the gold standard in medicine, arrays of nanotubes with tailor-made diameters. Notably, although nanotubular surfaces on anodized titanium have been extensively studied in relation to their effect on cell response, none of the previous studies has precisely assessed the effects of the morphological features and geometrical arrangement of the nanotubes. This is an important aspect, since the morphological characteristics and the spatial placement of nanofeatures has been shown to control cell response. In addition, by employing the same technique (i.e. anodization), a 3-dimensional hierarchical surface that mimics the frustule (i.e. silicified cell wall) of diatoms (a type of microalgae) can be created. Aside from enabling, for the first time, cellular studies on such bioinspired surface, this hierarchical nanoscale substrate will also allow to probe the effects of a 2-tier nanotopographical gradient along the depth of the nanotubular layer.

Background: Vascular endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. As such, the study of endothelial cells has sought to identify causal pathways and novel therapeutic approaches to promote vascular repair. Induced pluripotent stem (iPS) cell technology may be a particularly useful tool, and could be used to derive endothelial cells and their progenitors from individuals with endothelial dysfunction to explore these pathways and develop novel strategies for vascular regeneration. Whilst iPS cells are conventionally obtained from the reprogramming of dermal fibroblasts, it was hypothesised that endothelial cells could also be reprogrammed, and that these pluripotent cells would have enhanced capacity for endothelial differentiation and vascular regeneration. Objectives: To generate iPS cells from human fibroblasts and endothelial cells and to assess their potential for endothelial differentiation and vascular regeneration. Methods and Results: A) Reprogramming: Dermal fibroblasts and endothelial outgrowth cells from blood were obtained from healthy donors (n=5) and transfected with episomal vectors containing six reprogramming factors: Sox2, Klf4, Oct3/4, L-Myc, Lin28 and Shp53. Successfully reprogrammed fibroblast-derived iPS (fiPS) and endothelial cell-derived iPS (eiPS) arose as colonies, and were isolated and expanded. Reprogrammed cells expressed pluripotency markers SSEA3, SSEA4, TRA 1 60, Oct3/4 and NANOG, and developed into all three germ layers following embryoid body formation. B) Endothelial differentiation: iPS and ES cell lines were aggregated into embryoid bodies in stem cell growth media containing mesoderminducing cytokines. Embryoid bodies were then disaggregated and cultured in endothelial medium supplemented with VEGF. After seven days, a population of CD31+ cells was isolated and further cultured. Mature endothelial cell antigen expression was confirmed by flow cytometry. CD31+ cells were similar to mature endothelial cells in functional assays of proliferation, migration, nitric oxide production and angiogenesis. C) Comparison of fiPS versus eiPS: eiPS differentiated into endothelial cells with greater efficiency than fiPS (21±3% versus 3±2%, P < 0.05). fiPS-derived endothelial cells and eiPS-derived endothelial cells expressed similar levels of endothelial markers CD146, CD31, VEFGR2 and CD34 compared to control endothelial cells. When grown on Matrigel, they formed tubule-like structures with a similar number of vessel connections. In vivo, endothelial cells derived from fiPS and eiPS increased neovasculogenesis in a nude mouse model: vessel density was increased after implantation of endothelial cells from fiPS and eiPS by 3.50 vessel counts (P≤0.001) and 3.47 vessel counts (P≤0.001) respectively, when compared to controls. By comparison control endothelial cells did not increase vessel density compared to control (P > 0.05). Conclusions: Endothelial cells can be isolated from blood and reprogrammed to form pluripotent stem cells with enhanced capacity to differentiate into endothelial cells than those derived from dermal fibroblasts. Endothelial cells derived from both sources promote angiogenesis in vivo, and have major potential for therapeutic applications in vascular regeneration.

No description available.

The main aim of the work in this thesis was to incorporate two large fields of research in the development of new medical therapies: light therapy and stem cells.

Previous studies in mice have suggested that some if not all hemopoietic stem cells with long-term in vivo repopulating ability are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays. Since human hemopoietic stem cells cannot be assessed by expression of their in vivo repopulating potential, characterization of these cells requires an alternative endpoint. This thesis explores the use of clonogenic cell production in vitro in the presence of a competent stromal cell feeder layer for this purpose, based on the observation that this can continue for many weeks when unseparated human marrow cells are cultured under conditions that allow a stromal cell layer to form. Accordingly, a population of human clonogenic cell precursors referred to as long-term culture-initiating cells (LTC-IC) were postulated to exist as a biologically distinct compartment whose members could be quantitated by measuring the number of myeloid, erythroid and multi-lineage clonogenic progenitors present after 5 weeks of their culture on stromal feeder layers. LTC-IC in normal marrow assayed in this way were found to have a significantly lower forward light scatter, lower expression of HLA-DR, lower expression of CD 71 (transferrin receptor), and a higher expression of CD 34 as compared to clonogenic cells. Separation of marrow cells on the basis of these differences allowed a cell population enriched ~800 fold in LTC-IC to be obtained. This population contained only 0.06% of the marrow cells and 2% of the total clonogenic cells, but retained 50 - 60% of the LTC-IC present in the original marrow. Absolute numbers of LTC-IC and the proliferative and differentiative capability of individual LTC-IC were then determined by limiting dilution analysis following the demonstration that clonogenic cell output (at 5 weeks) is linearly related to input cell number over a wide range of cell concentrations. The frequency of LTC-IC in normal human marrow was determined to be ~1 per 2 x 10⁴ cells. Following purification this was increased to 1-2%. The proliferative capacity exhibited by individual LTC-IC as measured by the number of clonogenic cells per LTC-IC in 5 week-old cultures was found to range from 1 to 30 (the average being ~4). These studies also showed that a least some LTC-IC are multipotent as evident by their production of both erythroid and myeloid progeny. To study the effect of specific growth factors on LTC-IC maintenance and differentiation, highly purified LTC-IC were seeded onto irradiated murine marrow-derived stromal cells (from the M2-10B4 line) previously engineered to produce one of the human hemopoietic growth factors G-CSF, GM-CSF or IL-3. In the absence of any feeders, both the LTC-IC and their progeny in these purified suspensions decreased to very low levels within 5 weeks. However, in the presence of control M2-10B4 cells, LTC-IC maintenance and differentiation was supported as effectively as when standard human marrow feeders were present. The combined presence of G-CSF and IL-3-producing M2-10B4 cells further enhanced the maintenance and early differentiation of LTC-IC above levels obtained with control feeders, but only in the absence of GM-CSF producing feeders. In contrast, in the presence of GM-CSF-producing feeders the output of mature granulocytes and macrophages was maximal, and LTC-IC were inhibited. These studies describe and validate the use of the LTC-IC assay to selectively identify and quantitate a previously inaccessible population of very primitive human hemopoietic cells. Exploration of in vitro conditions and human growth factors able to influence their developmental behaviour points to G-CSF plus II-3 as the best candidates for future studies of LTC-IC activation and/or expansion in vitro. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

archives@tulane.edu / Off-the-shelf implantable biomaterials for tissue engineering and regenerative medicine (TERM) applications can treat numerous damaged organs/tissues. Such biomaterials have been investigated for their natural and synthetic biomimetic material properties to induce tissue regeneration in situ. Engineering biomaterials based off their mechanical properties like that of tensile strengths, porosity, nanofiber widths, or moduli can aid in tissue repair. However, tuning biomechanical properties alone is not always sufficient for biomaterials to induce effective tissue regeneration of large tissue/organ defects. The inclusion of biological properties to certain biomaterials such as stem/progenitor cells, growth factors, and/or cytokines can attenuate inflammation and stimulate proper tissue regeneration. In order to develop better off-the-shelf biomaterial implants for tissue repair, both the biomechanical and biological properties of the engineered biomaterial implant need to be assessed. In this dissertation, a decellularized polycaprolactone/chitosan hollow tube grown with MSC2s (De-PCL/CS) is investigated as a novel off-the-shelf tissue engineered graft (OTS-TEG) to repair hollow tubular organs like that of the esophagus. It is demonstrated that De-PCL/CS can retain extracellular matrix (ECM) factors from anti-inflammatory MSC2s post freeze-thaw decellularization. De-PCL/CS exhibits similar esophageal compressive biomechanics and is shown in an in vivo murine omentum model to be anti- inflammatory while robustly recruiting gastrointestinal (GI) cells. This gives great insight into De-PCL/CS as an immune modulating biomaterial to attenuate inflammation while promoting tissue repair for GI applications. Severe esophageal malignancies like esophageal cancer (EC), advanced Barrett’s Esophagus (BE), and pediatric atresia require GI reconstructive surgery (i.e. gastric pull up). De-PCL/CS has the translational capability as an implantable OTS-TEG to regenerate damaged esophageal tissue avoiding a gastric pull up. This not only increases the quality of life of the patient receiving this treatment but has an immediate potential impact of over $2.2 billion saved annually on the U.S. healthcare system. Nevertheless, the unique bioengineering patent-pending methods in developing De-PCL/CS allow it to be a platform OTS-TEG for treating a plethora of damaged organs/tissues. Translationally, De-PCL/CS can have a great therapeutic impact in the TERM global market that is expected to accrue over $100 billion by the mid 2020s. / 1 / Derek Cyrus Dashti