The adrenal cortex is classically divided into three morphologically and biochemically distinct zones, covered by a thin, cellular capsule. The adult adrenal cortex is a dynamic tissue in which distinct regions of cell proliferation, movement and death have been identified. Several models for stem cell maintenance of the adult adrenal cortex have been proposed, but adrenocortical stem cells have not yet been identified. Adrenal cortices of 21OH/LacZ transgenic mice show similar mosaic patterns of β-galactosidase staining to X- inactivation mosaics and LacZ ↔ wildtype chimeras. 21OH/LacZ mice provide a tool for lineage analysis, which may help to i) identify clones of cells produced by stem cells in the adult, ii) determine when stem cells begin to function and iii) evaluate different models of how stem cells maintain the adrenal cortex. Analysis of 21OH/LacZ transgenic adrenal cortices showed that the randomly orientated clusters of fetal patches change progressively during the perinatal period to adult radial stripes. Correlation of changes in mosaic patterns and the locations of cell proliferation suggests that the stripes arise by edge-biased growth during the perinatal growth period. Although stem cells may not be involved in the initial formation of stripes, it seems likely that stem cells later maintain the stripes by producing clones of cells that move centripetally to displace the earlier fetal patterns and later replace aging cells. Various combinations of BrdU labelling and chase periods demonstrated that most cell division occurred in the outer 40% of the adrenal cortex, confirmed that cells moved towards the medulla and identified a population of label-retaining cells near the capsule, which could include stem cells. (Stem cells have been recognised as BrdU label-retaining cells in other tissues because they divide less frequently than their daughter cells so dilute the incorporated BrdU more slowly.) Stripe patterns in adult 21OH/LacZ transgenic adrenal cortices were examined to try to distinguish between various models proposed for stem cell maintenance of the adrenal cortex. The observed continuous radial stripe pattern favours the general hypothesis that a single population of stem cells in the periphery maintains the entire adrenal cortex, although other explanations are possible. Quantitative analysis of adult stripe patterns did not show the reduction in stripe number that might be predicted if an age-related decline in adrenocortical stem cell function occurs, as may happen in some other tissues.

No description available.

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo. They exhibit unlimited proliferation in culture and have the ability to differentiate into all three germ layers of the developing organism, a property defined as pluripotency. Previously it was reported that growth factor-bound protein 2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that harbours the pluripotent founder cells of the foetus. GRB2 is an adapter protein involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in response to extracellular signals. It has also been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in response to fibroblast growth factor (FGF) signaling. The work presented in this thesis examines the role of Grb2 in ES cells and describes previously unreported contributions of this adaptor protein in regulating ES cell growth and differentiation. It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow relatively normally in ES growth medium containing serum. However, in serum free conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA mini gene. Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog expression was uniformly upregulated, in contrast to the heterogeneous pattern reported in serum-based medium. Colony expansion on the substratum appears to be compromised, although there is no apparent defect in the initial attachment of Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in serum free medium could be due to lengthening of the G1 phase of the ES cell cycle. In an attempt to identify the signalling deficiency responsible for the growth defect of Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated component of the MAPK pathway that acts downstream of Grb2 in the MAPK pathway. Although both approaches increased MAPK signalling they were unable to rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive. It is hypothesised that under these conditions cells undergo hyper-self-renewal at the cost of growth. Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6, MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically and immunohistochemically identifiable neuronal cells confirmed differentiation of ectodermal cell types, indicating Grb2 is not required for neuronal differentiation. However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation pathway. This may be due to a defect in GATA factor expression since these factors are essential for cardiogenesis. In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells. Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to prevent this differentiation. However, biochemical analysis of the cells indicates that this occurs when ERK activation is very low, indicating differentiation was not MAPK-independent. Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is dispensable for differentiation in to some lineages. However as differentiation of Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.

Neural stem cell (NSC) transplantation is a promising therapy for the treatment of brain damage. Although the “proof of principle” for NSC transplantation therapy has been demonstrated in a variety of animal models of brain injury (stroke, traumatic brain injury, ageing) and in a clinical setting (Parkinson’s disease), the mechanisms by which grafted stem cells survive, migrate and differentiate in host brain are yet to be elucidated. Initial studies have demonstrated that, after transplantation of the MHP36 neural stem cell line in a focal ischaemia model, the lipid transport protein apolipoprotein E (apoE) is upregulated and co-localised to differentiated cells in parallel with functional recovery. ApoE has been shown to have a critical role in the response to brain injury and repair processes. Furthermore, in humans, three different forms of apoE exist (E2, E3, E4 encoded by the alleles e2, e3, e4) and each of these has a different ability to promote repair, with the E4 form associated with an impaired capacity. This thesis tests the hypothesis that apoE is critical in stem cell integration and investigates whether this effect is APOE genotype dependent, in a mouse model of global cerebral ischaemia. This model was chosen as it produces diffuse selective neuronal damage in the striatum and hippocampus, which also occurs in other conditions such as ageing and Alzheimer’s disease. The studies described in this thesis were designed to test the hypothesis and are outlined as follows: I. Characterisation of neural stem cell grafts in a mouse model of global ischaemia In order to investigate the potential influence of apoE on stem cell grafts, it was first essential to characterise stem cells grafts in mouse brain. Thus, the initial aim of the thesis was to characterise MHP36 grafts in a mouse model of ischaemic neuronal injury. The effect of cyclosporin A (CsA) immunosuppression was also investigated. C57Bl/6J mice underwent an episode of transient global ischaemia induced by bilateral common carotid artery occlusion. Three days following ischaemia, mice received a unilateral striatal graft of fluorescently labelled MHP36 neural stem cells or vehicle; the mice also received CsA or saline. The mice were terminated at either XVII 1 or 4 weeks post-transplantation. This study determined that MHP36 grafts survived and migrated robustly in host ischaemic brain at both 1 week and 4 weeks post-transplantation. Grafted MHP36 cells differentiated into neurons and were able to reduce the extent of ischaemic neuronal damage. An acute host inflammatory response was evoked following MHP36 grafting, but this decreased dramatically by 4 weeks post-transplantation. CsA immunosuppression did not affect MHP36 survival and migration or reduce the host inflammatory response. The successful transplantation and characterisation of MHP36 grafts in mouse brain allowed for future investigation into the genetic factors underlying stem cell graft integration via the use of apoE transgenic mice. II. Influence of apoE on neural stem cell grafts in a mouse model of global ischaemia The aim of this study was to investigate whether endogenous apoE influenced MHP36 survival, migration and differentiation and then to determine potential signalling pathways that may be involved. ApoE deficient mice on a C57Bl/6J background (APOE-KO) and control wildtype C57Bl/6J (WT) mice were subjected to an episode of transient global ischaemia, as in Experiment 1. Two weeks following ischaemia, all mice received unilateral striatal and hippocampal grafts of MHP36 cells. All mice received CsA immunosuppression. Mice were terminated 4 weeks post-transplantation. MHP36 survival and migration was significantly increased in WT as compared to APOE-KO mice. In addition, neuronal differentiation was significantly increased in WT as compared to APOE-KO mice. Increased astrocytic differentiation was observed in the hippocampus, but not striatum of WT as compared to APOE-KO mice. Measurement of the levels of signalling proteins associated with cell survival, extracellular signal-regulated kinase (ERKs) and c-Jun amino-terminal kinase (JNKs) and their phosphorylated forms (pERK and pJNK), indicated selective alterations in JNK with no change in ERK in APOE-KO as compared to WT mice, suggesting that JNK may underlie the apoE effects in stem cell integration. This study demonstrated that apoE strongly influences the survival, migration and differentiation of grafted MHP36 cells and provides initial evidence for the signalling pathways involved. XVIII III. Influence of APOE genotype on neural stem cell grafts in a mouse model of global ischaemia Following the demonstration that endogenous mouse apoE has a critical role in MHP36 graft survival, migration and differentiation, this study sought to investigate whether these effects are influenced by human APOE genotype. Transgenic mice expressing human APOE-e3 or e4, (on an APOE-KO background) and a control group of APOE-KO mice underwent transient global ischaemia and two weeks later MHP36 cells were transplanted unilaterally into the striatum and hippocampus. 1 week after grafting the mice were started on a series of tests for motor balance and coordination using the rotarod, and taken for histology 4 weeks post-transplantation. MHP36 graft survival was significantly improved in APOE-e3 mice compared to APOE-KO and APOE-e4 mice. However, the migration and differentiation of MHP36 cells and motor performance of grafted mice were similar in all three APOE groups, indicating a comparable fate and functional activity within a 4 week survival time. Thus the data indicate that APOE genotype may influence cell survival with minimal effect on stem cell migration and differentiation. The data presented in this thesis demonstrate that endogenous apoE strongly influences MHP36 graft survival, migration and differentiation. Although there was minimal evidence that human APOE genotype influences cell migration and differentiation, stem cell survival was markedly improved in a human APOE-e3 allelic environment, which may affect the effectiveness of stem cells in APOE-e4 individuals.

In allogeneic stem cell mobilization, peripheral blood stem cell mobilization with filgrastim can be considered standard of care. Poor mobilizers may be at risk for inadequate stem cell collection during apheresis. He we present a successful case of salvage plerixafor use with filgrastim in a haploidentical identical transplant patient.

Lau Fung Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 107-130). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Adult stem cells are vital for tissue maintenance. Stem cell over proliferation results in tumour formation, whilst loss of stem cells causes tissue degeneration and a variety of diseases. Stem cell maintenance and proliferation is regulated through somatic structures called niches. The germline stem cell niche in Drosophila ovary has been well defined and it is useful to better understand the interactions between niche and stem cells. Notch signalling is needed for germline stem cell niche creation and maintenance. The aim of this thesis is to better understand both the regulation of Notch signalling during development and its requirement in the adult niche. The first paper, "Reversible regulation of stem cell niche size through dietary control of Notch signalling", revolves around the dynamicity of the niche. The niche is found to respond to diet stimuli and has the ability to be restored. Notch was previously found to be involved in the maintenance of the niche. We found that Notch signalling is altered by diet, and we dissect its different maintenance and recovery roles in the ovary. In the second paper, "ZO-1 controls stem cell niche assembly by acting as an upstream regulator of Deltex-dependent Notch signalling", we show how Notch signalling is finely regulated during niche formation through interplay with the proteins Polychaetoid and Deltex. This paper leads to a better understanding of how the niche is assembled and how Notch signalling is regulated in a context-dependent way. The obtained results from both papers will help understand the dynamics of the model germline stem cell niche, and how Notch signalling is found at the convergence between internal and external stimuli regulating the ovary's response to a changing environment.

Embryonic stem (ES) cell maintenance and differentiation are dynamic processes controlled by various intrinsic and extrinsic factors. Identifying these factors will enhance the understanding about developmental process and improve the application of stem cells in clinic. Previous studies highlight a shift between non-oxidative and oxidative energy metabolism to play roles during differentiation. Oxidative metabolism is a major source of reactive oxygen species (ROS) which is regulated by a cytoprotective transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, this study investigate relationship between metabolism, ROS, and Nrf2 during mouse ES cell differentiation. In vitro models representing early lineage differentiation were used. By measuring metabolic profiles, ROS, and Nrf2 levels from the models, Nrf2 was found related to pluripotency and ROS. However, relationship among metabolism and Nrf2 or ROS could not be detected. Gain- and loss-of-function experiments by pharmacological activator, short hairpin RNA knockdown, and CRISPR-Cas9 genome editing showed that Nrf2 could promote pluripotency and inhibit differentiation, especially during early differentiation toward neural lineage. This study suggested a new player in transcription control that governs pluripotency and differentiation.

In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to 'reprogram' differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.

Lam Audrey Carmen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 166-195). / Abstracts in English and Chinese. / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.x / Abstract --- p.xiii / Chapter Chapter One - --- Introduction --- p.1 / Chapter Section 1.1 --- Hematopoietic Stem Cells --- p.1 / Chapter 1.1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.2 --- Hematopoietic Stem and Progenitor Cells --- p.1 / Chapter Section 1.2 --- Stem Cell Transplantation --- p.4 / Chapter 1.2.1 --- Stem Cell Transplantation --- p.4 / Chapter 1.2.2 --- Sources of Hematopoietic Stem Cells for Transplantation --- p.4 / Chapter 1.2.3 --- Cord Blood as a Source of Hematopoietic Stem Cells --- p.6 / Chapter 1.2.3.1 --- Advantages of Cord Blood Transplant --- p.6 / Chapter 1.2.3.2 --- Disadvantages of Cord Blood Transplant --- p.7 / Chapter Section 1.3 --- Ex Vivo Expansion --- p.8 / Chapter 1.3.1 --- Optimization of Expansion Conditions --- p.10 / Chapter 1.3.1.1 --- CD34+ Cell Selection --- p.10 / Chapter 1.3.1.2 --- Cytokines --- p.11 / Chapter 1.3.1.2.1 --- Thrombopoietin --- p.12 / Chapter 1.3.1.2.2 --- Stem Cell Factor --- p.14 / Chapter 1.3.1.2.3 --- Flt-3 Ligand --- p.15 / Chapter 1.3.1.2.4 --- Granulocyte-Colony Stimulating Factor --- p.16 / Chapter 1.3.1.2.5 --- Interleukin-3 --- p.17 / Chapter 1.3.1.2.6 --- Interleukin-6 --- p.18 / Chapter 1.3.1.2.7 --- Comparison of Flt-3 Ligand and Stem Cell Factor --- p.20 / Chapter 1.3.1.3 --- Culture Medium --- p.20 / Chapter 1.3.2 --- Mannose-Binding Lectin --- p.22 / Chapter 1.3.3 --- Ex Vivo Expansion for Clinical Transplantation --- p.23 / Chapter Section 1.4 --- Non-Obese Diabetic/Severe Combined Immunodeficient Mouse Transplantation Model --- p.29 / Chapter Chapter Two - --- Objectives --- p.32 / Chapter Chapter Three - --- Materials and Methodology --- p.34 / Chapter Section 3.1 --- Collection of Cord Blood Samples / Chapter Section 3.2 --- Cryopreservation and Thawing of Cord Blood --- p.34 / Chapter Section 3.3 --- Enrichment of CD34+ Cells --- p.35 / Chapter Section 3.4 --- Ex Vivo Expansion --- p.38 / Chapter 3.4.1 --- Effects of Flt-3 Ligand and stem Cell Factor on the Expansion of Megakaryocytic Progenitor Cells --- p.39 / Chapter 3.4.1.1 --- Ex Vivo Expansion of Cord Blood CD34+ Cells with Flt-3 Ligand or Stem Cell Factor --- p.39 / Chapter 3.4.1.2 --- Flt-3 Receptor Assay --- p.40 / Chapter 3.4.2 --- Effects of Mannose-Binding Lectin on the Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells --- p.41 / Chapter 3.4.2.1 --- Ex Vivo Expansion of Cord Blood CD34+ Cells with Mannose-Binding Lectin --- p.41 / Chapter 3.4.2.2 --- Effects of Mannose-Binding Lectin on the Preservation of Early Stem and Progenitor Cells --- p.41 / Chapter 3.4.2.3 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.42 / Chapter 3.4.3 --- "Optimization of Culture Duration, Culture Media, Autologous Plasma and Cytokine Combinations for the Preclinical Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells" --- p.42 / Chapter 3.4.3.1 --- "Comparison of Culture Duration, Culture Media and Cytokine Combinations" --- p.42 / Chapter 3.4.3.2 --- Effects of Autologous Cord Blood Plasma --- p.43 / Chapter 3.4.3.3 --- Effects of Flt-3 Ligand and Dosage of Thrombopoietin and Stem Cell Factor --- p.43 / Chapter 3.4.3.4 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.44 / Chapter Section 3.5 --- Progenitor Colony-Forming Assays --- p.44 / Chapter 3.5.1 --- Colony-Forming Unit Assay --- p.44 / Chapter 3.5.2 --- Colony Forming Unit Megakaryocyte --- p.46 / Chapter 3.5.3 --- Calculations of CFU --- p.46 / Chapter Section 3.6 --- Flow Cytometry Analysis --- p.47 / Chapter Section 3.7 --- Transplantation of Non-Obese Diabetic/Severe Combined Immunodeficient Mice --- p.48 / Chapter Section 3.8 --- Assessment of Human Cell Engraftment in Transplanted NOD/SCID Mice --- p.49 / Chapter 3.8.1 --- Flow Cytometry Analysis --- p.49 / Chapter 3.8.2 --- PCR Analysis --- p.50 / Chapter Section 3.9 --- Statistical Analysis --- p.52 / Chapter Chapter Four - --- Effects of Flt-3 Ligand and Stem Cell Factor on the Expansion of Megakaryocytic Progenitor Cells --- p.53 / Chapter Section 4.1 --- Results --- p.53 / Chapter 4.1.1 --- Ex Vivo Expansion of CD34+ Cells --- p.53 / Chapter 4.1.2 --- Identification of Flt-3 Receptors --- p.55 / Chapter Section 4.2 --- Discussion --- p.55 / Chapter Chapter Five- --- Effects of Mannose-Binding Lectin on the Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells --- p.68 / Chapter Section 5.1 --- Results --- p.68 / Chapter 5.1.1 --- Ex Vivo Expansion of CD34+ Cells with Mannose-Binding Lectin --- p.68 / Chapter 5.1.2 --- Effects of Mannose-Binding Lectin on the Preservation of Early Stem and Progenitor Cells --- p.72 / Chapter 5.1.3 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.75 / Chapter Section 5.2 --- Discussion --- p.76 / Chapter Chapter Six - --- "Optimization of Culture Duration, Culture Media, Autologous Plasma and Cytokine Combinations for the Preclinical Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells" --- p.111 / Chapter Section 6.1 --- Results --- p.111 / Chapter 6.1.1 --- Kinetics of Expansion --- p.111 / Chapter 6.1.2 --- Assessment of Culture Media --- p.113 / Chapter 6.1.3 --- Effects of Autologous Cord Blood Plasma --- p.115 / Chapter 6.1.4 --- Effects of Granulocyte-Colony Stimulating Factor --- p.117 / Chapter 6.1.5 --- Effects of Interleukin-6 --- p.118 / Chapter 6.1.6 --- Effects of Increased Dosage of Thrombopoietin and Stem Cell Factor --- p.119 / Chapter 6.1.7 --- Effects of Flt-3 Ligand --- p.120 / Chapter 6.1.8 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.121 / Chapter Section 6.2 --- Discussion --- p.123 / Chapter Chapter Seven- --- General Discussion and Conclusion --- p.163 / Bibliography --- p.166