Neonatal hydrocephalus is a common neurological disorder leading to expansion of the cerebral ventricles. This disease is associated with significant morbidity and mortality and is often fatal if left untreated. Hydrocephalus was first described over 2500 years ago by Hippocrates, the father of medicine, and remains poorly understood today. Current therapies still rely on invasive procedures developed over 60 years ago that are associated with high failure and complication rates. Thus, the identification of molecular mechanisms and the development of non-invasive medical treatments for neonatal hydrocephalus are high priorities for the medical and scientific communities. The prevailing doctrine in the field is that hydrocephalus is strictly a "plumbing problem" caused by impaired cerebrospinal fluid (CSF) flow. Recently, animal models with impaired cilia have provided insight into the mechanisms involved in communicating (non-obstructive) hydrocephalus. However, as a result of a poor understanding of hydrocephalus, no animal studies to date have identified an effective non-invasive treatment. The goal of this thesis project is to investigate the molecular mechanisms underlying this disease and to identify a non-invasive, highly effective treatment strategy. In Chapter 2, we utilize a novel animal model with idiopathic hydrocephalus, mimicking the human ciliopathy Bardet-Biedl Syndrome (BBS), to examine the role of cilia in hydrocephalus. We find that these mice develop communicating hydrocephalus prior to the development of ependymal "motile" cilia, suggesting that this phenotype develops as a result of dysfunctional "primary" cilia. Primary cilia are non-motile and play a role in cellular signaling. These results challenge the current dogma that dysfunctional motile cilia underlies neonatal hydrocephalus and implicate a novel role for primary cilia and cellular signaling in this disease. Chapter 3 focuses on identifying the link between primary cilia and neonatal hydrocephalus. In this chapter, we report that disrupting the molecular machinery within primary cilia leads to faulty PDGFRα signaling and the loss of a particular class of neural progenitor cells called oligodendrocyte precursor cells (OPCs). We find that the loss of OPCs leads to neonatal hydrocephalus. Importantly, we identify the molecular mechanism underlying both the loss of OPCs and the pathogenesis of neonatal hydrocephalus. Chapter 4 explores the therapeutic potential of targeting the defective cellular signaling pathways to treat neonatal hydrocephalus. By targeting the faulty signaling, we restore normal development of oligodendrocyte precursor cells, and curtail the development of hydrocephalus. This work challenges the predominant view of hydrocephalus being strictly a "plumbing problem" treatable solely by surgical diversion of CSF. Here, we propose that hydrocephalus is a neurodevelopmental disorder that can be ameliorated by non-invasive means. Importantly, we introduce novel molecular targets and a non-invasive treatment strategy for this devastating disorder. To our knowledge, we are the first to successfully treat neonatal hydrocephalus in any model organism by targeting neural progenitor cells.

Bone marrow (BM) mesenchymal stem cells (MSC) support proliferation and differentiation of hematopoietic progenitor cells (HPC) in vitro. Since they represent a rare subset of BM cells, MSC preparations for clinical purposes involves a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on MSC supportive activity. MSC were expanded for up to 10 passages. MSC and CD34+ cells were seeded in cytokinefree co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed. Early passage MSC supported HPC expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage MSC did not support HPC and myeloid cell outgrowth but maintained B cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for one week in contact with MSC was effective until the fourth MSC passage and declined afterwards. CD34+ cells achieved higher levels of engraftment in NOD/SCID mice when co-injected with early passage MSC; however MSC expanded beyond 9 passages were ineffective in promoting CD34+ cell engraftment. Non-contact cultures indicated that MSC supportive activity involved diffusible factors. Among these, interleukin (IL)-6 and IL-8 contributed to the supportive activity of early passage MSC but not of late passage MSC. MSC phenotype as well as fat, bone and cartilage differentiation capacity did not change during MSC culture. Extended MSC culture alters their supportive ability toward HPC without concomitant changes in phenotype and differentiation capacity.

Neural stem and progenitor cells (NSPC) can differentiate to neurons and glial cells. NSPC are easily propagated in vitro and are therefore an attractive tool for tissue regeneration. Traumatic brain injury (TBI) is a common cause for death and disabilities. A fundamental problem following TBI is tissue loss. Animal studies aiming at cell replacement have encountered difficulties in achieving sufficient graft survival and differentiation. To improve outcome of grafted cells after experimental TBI (controlled cortical impact, CCI) in mice, we compared two transplantation settings. NSPC were transplanted either directly upon CCI to the injured parenchyma, or one week after injury to the contralateral ventricle. Enhanced survival of transplanted cells and differentiation were seen when cells were deposited in the ventricle. To further enhance cell survival, efforts were made to reduce the inflammatory response to TBI by administration of ibuprofen to mice that had been subjected to CCI. Inflammation was reduced, as monitored by a decrease in inflammatory markers. Cell survival as well as differentiation to early neuroblasts seemed to be improved. To device a 3D system for future transplantation studies, NSPC from different ages were cultured in a hydrogel consisting of hyaluronan and collagen. Cells survived and proliferated in this culturing condition and the greatest neuronal differentiating ability was seen in cells from the newborn mouse brain. NSPC were also used in a model of peripheral nervous system injury, and xeno-transplanted to rats where the dorsal root ganglion had been removed. Cells survived and differentiated to neurons and glia, furthermore demonstrating their usefulness as a tool for tissue regeneration.

Diabetes or diabetes mellitus which is the correct medical term is a medical condition were the affected person lack the ability to regulate his or her blood glucose levels. This inability is directly due to the fact that the insulin producing cells, residing in the pancreas, can’t meet the body’s demand for insulin. It is estimated that close to 200 million people are suffering from diabetes today and this number is predicted to double within 20 years. Of the approximately 200 million people suffering from diabetes today approximately 20 million are in dependent on daily injections of insulin. Being dependent on exogenous insulin is not only an inconvenience it also increase the risk for several medical complications such as stroke, heart disorders, kidney failure, retinopathy, atherosclerosis and impaired wound healing. The major risk factor for all these complications is long periods of high blood sugar levels that is damaging to thin blood vessels and nerves.  Even in the best of situations the blood sugar levels of a diabetic with need for daily insulin injections can never be as well controlled as in a healthy individual. Increased understanding in the developmental processes behind the formation of the pancreas, and more specifically the insulin producing β-cells could result in new treatments for diabetics. By imitating the in vivo conditions generating pancreatic development scientist are now able to induce embryonic stem cells to differentiate into pancreatic progenitors as well as insulin producing β-cells in vitro. These in vitro generated pancreatic cells might in the future serve as a donor source for transplantations, thereby restoring the insulin producing capability of diabetic patients. An alternative approach to restore insulin production in diabetics is to influence cells in the pancreas to generate more insulin producing cells. To successfully achieve this, what cell types have the capacity to generate β-cells needs to be appreciated. In this thesis papers concerning in vitro differentiating of embryonic stem cells towards a pancreatic fate as well as in vivo studies in basic pancreas development are presented and discussed.

<p>Synthetic small diameter vascular grafts fail clinically due to thrombosis and intimal hyperplasia. The attachment of endothelial cells (ECs) onto the inner lumen of synthetic small diameter vascular grafts can improve graft patency; however, significant challenges remain that prevent wide clinical adoption. These issues include difficulties in the autologous sourcing of ECs, the lack of attachment, growth and retention of the layer of ECs to the graft lumen, and the maintenance of an anti-thrombotic and anti-inflammatory profile by the layer of ECs. </p><p>This dissertation describes the isolation, characterization, and use of endothelial progenitor cells (EPCs) to improve the performance of small diameter vascular grafts. First, EPC isolation efficiency and expression of critical EC markers was compared between young healthy volunteers and patients with documented coronary artery disease (CAD). EPCs were isolated and expanded from patients with CAD and had a similar phenotype to EPCs isolated from healthy donors, and a control population of human aortic ECs. Second, we assessed the ability to enhance the anti-thrombotic activity of patient derived EPCs through the over expression of thrombomodulin (TM). In vitro testing showed TM-transfected EPCs had significantly increased production of key anti-thrombotic molecules, reduced platelet adhesion, and extended clotting times over untransfected EPCs. Finally, native and TM-transfected EPCs were seeded onto small diameter vascular grafts and tested for their ability to improve graft performance. EPCs sodded onto the lumen of small diameter ePTFE vascular grafts had strong adhesion and remained adherent during graft clamping and exposure to flow. TM-transfected EPCs improved graft anti-thrombotic performance significantly over bare grafts and grafts seeded with native EPCs. Based on these promising in vitro results, grafts were implanted bilaterally into the femoral arteries of athymic rats. Bare grafts and grafts with air removed clotted and had only 25% patency at 7 days. In contrast, graft sodded with native EPCs or TM-transfected EPCs had 87% and 89% respective patency rates. High patency rates continued with 28 day implant testing with EPC sodded grafts (88% Native; 75% TM). There were no significant differences in patency rates at 7 or 28 days between native and TM-transfected grafts. These in vivo data suggest patient blood-derived EPCs can be used to improve the performance of small diameter vascular grafts.</p> / Dissertation

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, in vitro approaches for functional assessments have been relatively limited. Herein, we describe the guidance of primary endothelial cells (ECs) in an agarose hydrogel scaffold that is chemically patterned with an immobilized concentration gradient of vascular endothelial growth factor 165 (VEGF165) using multiphoton laser patterning of VEGF165. This is the first demonstration of this patterning technology to immobilize proteins; and the first demonstration of immobilized VEGF165 to guide endothelial cell growth and differentiation in 3D environments. It is particularly compelling that this 3D hydrogels provide an excellent biomimetic environment for stem cell niche, thereby offering a new approach to study stem cell biology. In this thesis, we focused on the retinal stem cell niche, investigating cellular interactions between retinal stem and progenitor cells (RSPCs) and endothelial cells (ECs). By using this 3D in vitro model, we demonstrated the synergistic interactions between RSPCs and ECs wherein RSPCs migrated into 3D gels only in the presence of ECs and RSPCs stabilized EC tubular-like formations. Moreover, we characterized the contact-mediated effects of ECs on RSPC fate in terms of proliferation and differentiation.

Large-scale expansion of lineage-committed stem cells can provide an excellent ex vivo model for studying complex molecular pathways governing cell fate choices. Also, such cells could be useful for implementing cell therapeutic approaches for treatment of specific disorders involving extensive cellular damage within that lineage. Using growth factors, pluri- and multipotent stem cells have been successfully isolated and cultured from pre- and peri-implantation stage embryos, including trophectoderm, primitive ectoderm, epiblast and primitive endoderm. However, ex vivo expansion of lineage restricted cells from later embryonic lineages and adult tissues have been a challenge. N-myc is a well-characterized member of myc gene family that is known to be essential for the proliferation of numerous progenitor cell types during normal embryonic development of diverse organs including lungs, liver, heart, kidneys and brain. Considering this important role of N-myc, we hypothesized that its regulated activation in these progenitors might allow their expansion in culture. To test this hypothesis, we generated a novel doxycycline-inducible transgenic mouse line that expresses N-myc uniformly across all tissues. Using cortical precursors derived from mid-gestation embryos of these mice, we show that upon doxycycline induced N-myc expression, we can achieve at least a million-fold expansion of multipotent neural precursors within a short span of time in culture. When doxycycline is withdrawn, N-myc expression is turned off and the cells differentiate into neurons and glia. An extensive characterization of the expanded cells revealed that the cells retained their differentiation potential, genomic stability and commitment specific to their origin. The tetracycline-inducible N-myc expressing mouse line might also serve as a source for establishing other than neural lineage committed progenitor cell lines where N-myc has a known role in regulating cell proliferation and differentiation decisions.

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, in vitro approaches for functional assessments have been relatively limited. Herein, we describe the guidance of primary endothelial cells (ECs) in an agarose hydrogel scaffold that is chemically patterned with an immobilized concentration gradient of vascular endothelial growth factor 165 (VEGF165) using multiphoton laser patterning of VEGF165. This is the first demonstration of this patterning technology to immobilize proteins; and the first demonstration of immobilized VEGF165 to guide endothelial cell growth and differentiation in 3D environments. It is particularly compelling that this 3D hydrogels provide an excellent biomimetic environment for stem cell niche, thereby offering a new approach to study stem cell biology. In this thesis, we focused on the retinal stem cell niche, investigating cellular interactions between retinal stem and progenitor cells (RSPCs) and endothelial cells (ECs). By using this 3D in vitro model, we demonstrated the synergistic interactions between RSPCs and ECs wherein RSPCs migrated into 3D gels only in the presence of ECs and RSPCs stabilized EC tubular-like formations. Moreover, we characterized the contact-mediated effects of ECs on RSPC fate in terms of proliferation and differentiation.