The stringent requirement for hemopoietic growth factors (HGF) in the induction of hemopoiesis in vitro has raised questions as to their possible role(s) in leukemogenesis. Several recent clinical studies have shown aberrant cell growth factor gene activation in patient derived leukemic cells. Assessment of growth factor activity is often based on in vitro bioactivity assays of conditioned media or body fluids. The specificity of this type of endpoint is, however, open to question due to the overlap in biological activities of many HGFs. In assessing the role of growth factor gene expression in a murine myeloid leukemia model I have used a sensitive RNA detection procedure coupled with a vector-probe system that enables the synthesis of uniformly labelled radioactive DNA probes to detect unambiguously the expression of particular growth factor genes. The Abelson murine leukemia virus (A-MuLV) derived myeloid transformants used in this study had previously been shown to produce a multi-lineage colony stimulating activity (CSA). While these A-MuLV transformants were shown to produce GM-CSF, it seemed likely that the multi-lineage CSA was due to another factor. In addition to confirming the expression of GM-CSF mRNA, I was able to show that the cells of all four A-MuLV transformed lines tested also expressed interleukin-3 mRNA. This finding was strongly corroborated by bio-activity data obtained using the CM from the A-MuLV myeloid transformants. Additional preliminary analysis by bioactivity assays have also shown the possible presence of interleukin-6 (IL-6) and a recently described pre-B cell factor suggesting perhaps a common mechanism underlying the activation of these various growth factor genes. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

O transplante de células-tronco hematopoéticas (CTHs) é o tipo mais bem-sucedido de terapia celular realizado até os dias atuais. No entanto, apesar do sucesso e da relevância clínica das CTHs isoladas a partir de fontes adultas, o uso destas células tem algumas limitações em relação à sua disponibilidade, compatibilidade imunológica e risco de contaminação. Desse modo, busca-se o desenvolvimento de soluções para as dificuldades apontadas para suprir a demanda de transplantes. Uma abordagem emergente para superar este problema é baseada na cultura e diferenciação de células-tronco embrionárias humanas (CTEhs). Estas são célulastronco pluripotentes e indiferenciadas com elevada capacidade de auto-renovação e diferenciação em todas as células derivadas dos três folhetos germinativos. No entanto, os métodos de diferenciação utilizados para a produção de CTHs a partir de células pluripotentes ainda não são eficientes. Os protocolos descritos até o momento têm gerado números variados e populações de células heterogêneas, e produz apenas CTHs muito primitivas e imaturas com baixa capacidade funcional in vivo. Parte desta dificuldade pode decorrer da ineficiência do microambiente de cultura para a diferenciação. Neste trabalho, nós demonstramos um eficiente protocolo de diferenciação hematopoética baseado em cocultivo de CTEhs com fibroblastos embrionários murinos com alto rendimento na geração de célulastronco/progenitoras hematopoéticas (CTPHs) que expressam os antígenos CD45, CD43, CD31 e CD34, e apresentam potencial clonogênico in vitro equivalente ao de células mononucleares isoladas de sangue de cordão umbilical. Nós fomos capazes de produzir todas as células das linhagens eritróide e mielóide em diferentes estágios de maturação, como também células positivas para marcadores linfóides. Demonstramos ainda que as células hematopoéticas surgem no sistema de cultura a partir de um endotélio-hemogênico constituído por células CD34+CD31+. No entanto, apesar das características maduras das CTPHs obtidas por tal método, os ensaios de reconstituição hematopoiética mostraram que estas células ainda possuem limitada capacidade funcional de enxertamento em camundongos imunocomprometidos quando transplantadas por via retro-orbital. / Hematopoietic stem cells (HSC) transplant is the most successful type of cell therapy carried out to date. However, despite the success and the clinical relevance of HSC isolated from adult sources, these cells have some limitations regarding its availability, immunological compatibility and risk of contamination. Thus, we seek to develop solutions to overcome these difficulties to supply the demand for transplants. An emerging approach to overcome this problem is based on human embryonic stem cells (hESCs) culture and differentiation. These are pluripotent and undifferentiated stem cells with high capacity for self-renewal and differentiation in all cells derived from the three embryonic germ layers. However, differentiation methods used for HSC production from pluripotent cells are not efficient yet. Protocols described so far have generated varying numbers and heterogeneous cell populations, and produce only very primitive and immature HSC with low in vivo functional capacity. Part of this difficulty may result from the inefficiency of the microenvironment of culture for differentiation. Here, we demonstrate an efficient protocol based on co-culture of hESCs with mouse embryonic fibroblasts for hematopoietic differentiation with high performance to generate in vitro hematopoietic stem/progenitor cells (HSPCs) that express CD45, CD43, CD31 and CD34 antigens with high purity of positive cells. We were able to produce all cells of erythroid and myeloid lineages at different stages of maturation. Lymphoid potential of hematopoietic cells was also evidenced. We demonstrated the primitive origin of hematopoietic cells through capillary-like structures constituted by hemogenic CD34+CD31+ cells. However, despite mature features of HSPCs obtained by our protocol, hematopoietic reconstitution assays showed that these cells have yet limited functional capacity for grafting into immunocompromised mice when exogenously transplanted by retro-orbital route.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13464号 / 論医博第2251号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 新, 教授 江藤 浩之, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / The innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.

Indiana University-Purdue University Indianapolis (IUPUI) / SIRT1 (Sirtuin 1) is a founding member of a family of seven proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1-/- mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1-/- mouse embryonic stem (mES) cells in vitro, and hematopoietic progenitors in SIRT1+/+, SIRT1+/-, and SIRT1-/- mice. SIRT1-/- ES cells exhibited markedly delayed/immature formation of blast colony-forming cells (BL-CFCs). When individual blast colonies were analyzed for hematopoietic and endothelial potential, replated SIRT1-/- BL-CFC possessed limited hematopoietic potential, whereas endothelial potential was essentially unaltered. The ability of SIRT1-/- ES cells to form primitive erythroid progenitors was not only delayed but greatly decreased. Moreover, after differentiation of SIRT1-/- mES cells, there were also significant decreases in granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5, decreased β-H1 globin, β-major globin, and Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ES cell commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm the requirement for SIRT1 for normal development of hematopoietic progenitor cells, we assessed embryonic and adult hematopoiesis in SIRT1+/+, SIRT1+/- and SIRT1-/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythroid precursors compared to wild-type (WT) and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow hematopoietic progenitor cells (HPCs) in 5 week and 12 month old mice, which was especially notable at lower (5%) O2 tension. In addition these progenitors survived less well in vitro under conditions of delayed growth factor addition. Taken together, these results demonstrate that SIRT1 plays a role in ES cell hematopoietic differentiation and mouse hematopoiesis.

幹細胞療法是近年的研究熱點之一，然而幹細胞在組織修復中的實際應用受到移植後幹細胞存活率低的制約，約80% 的幹細胞在移植至組織後不能存活。 人類臍帶血管周皮 (HUCPV) 幹細胞為多功能間充質幹細胞移植提供豐富的細胞來源。 在合適的誘導環境下，它們具有向多種間充質細胞系分化的能力。 與從骨髓或臍帶血中提取的間充質幹細胞比較，人類臍帶血管周皮幹細胞的體外增殖更為容易。 在本研究中，我們從人類臍帶血管周圍組織中分離人類臍帶血管周皮幹細胞，並採用流式細胞技術分選細胞表面標記物CD34、CD45呈陰性同時CD44 、CD90、 CD105、 CD146呈陽性的HUCPV細胞。HUCPV細胞在體外培養以及三維支架的環境下具有分化為骨和軟骨的能力。 / 在本研究中，我們主要研究腦和生殖器官表達基因（BRE）在HUCPV細胞中的功能。 BRE蛋白與其他已知蛋白的同源性均不高，目前尚未鑑定出任何功能性的結構域。 至今為止，BRE基因的已知功能大多數是通過對腫瘤模型的研究發現的。 據報導，BRE能夠提高DNA損傷的腫瘤細胞的存活率，但BRE在幹細胞中的作用仍不清楚。 我們發現，當HUCPV細胞分化後，其BRE的表達水平降低。 此外，利用BRE-siRNA降低HUCPV細胞中BRE基因的表達，能夠促進HUCPV細胞向骨和軟骨分化的進程。 因此，我們假設BRE對維持HUCPV細胞的幹細胞功能具有重要的作用。 由於經過BRE基因沉默處理的HUCPV細胞與對照組相比並無顯著的表型差別，我們採用微陣列（microarray）以及比較蛋白組學的方法研究兩者間的區別，從而找出BRE基因的功能以及可能涉及BRE的信號通路。 / 通過微陣列技術，我們深入地分析了BRE基因表達沉默後HUCPV細胞的轉錄組。 在經過BRE基因沉默處理的HUCPV細胞中，我們發現與維持幹細胞多向分化潛能有關的OCT4、 FGF5和FOXO1A等基因的表達顯著下調。 另外，BRE基因的沉默能夠影響表觀遺傳調控基因以及TGF-β 信號通路組成部件的表達，而TGF -β 信號通路是維持幹細胞自我更新的重要通路。 這些結果提示，BRE作為一個重要的調控因子，在維持HUCPV細胞的多向分化潛能的同時能夠防止細胞分化。 / 在比較蛋白組學的研究中，我們發現BRE基因的沉默能夠降低細胞骨架結合蛋白的表達，例如actin, annexin II 及 tropomyosin。 此外，我們利用免疫共沉澱的方法證明了BRE蛋白與actin及 annexin II蛋白直接結合。 細胞骨架的改變可能為HUCPV細胞的分化提供了一個有利的環境，因而BRE基因的沉默能夠促進HUCPV細胞向骨和軟骨分化。 支持這一推論的其中一個依據是Lim et al., 2000; Solursh, 1989; Zhang et al., 2006，文獻報導肌動蛋白多聚化抑製劑能夠促進軟骨形成的過程。 綜上所述，本研究為進一步研究BRE基因在HUCPV細胞中的功能以及與BRE直接作用的蛋白打下了基礎。 / Stem cells therapy has gained considerable attention in recent years. However, the practical use of stem cells for tissue repair has been hindered due to their low survival rate after grafting into tissues, for approximately 80% of the stem cells died after implantation. Human umbilical cord perivascular (HUCPV) stem cells offer a new and rich resource of multipotent mesenchymal stem cells. These cells possess the ability to differentiate into various mesenchymal cell lineages when induced. HUCPV cells can be more easily amplified in culture than mesenchymal stem cells extracted from bone marrow or umbilical cord blood. In this study, HUCPV cells were isolated from the perivascular regions of human umbilical cords. The HUCPV cells were sorted using flow cytometer for CD34⁻, CD44⁺, CD45⁻, CD90⁺, CD105⁺ and CD146⁺ surface markers. These HUCPV cells were found to be capable of differentiating into osteogenic lineage in monolayer culture and chondrogenic lineage in pellet culture. These cells were also found to be capable of differentiating into osteogenic and chondrogenic lineage in silk fibroin which acted as three-dimensional scaffolds for the cells to grow on. / The function of the Brain and Reproductive Organ-Expressed (BRE) gene in the context of HUCPV cells was investigated. The BRE protein shares no homology with any other known gene products and contains no known functional domain. To date, most of what we know about the function of this gene has been conducted in the tumor model. It has been reported that BRE can enhance the cellular survival of cancer cells following DNA damage. The role of BRE in stem cells has never been examined. We have established that BRE expression was down-regulated when HUCPV cells started to differentiate. In addition, silencing BRE expression, using BRE-siRNA, in HUCPV cells could accelerate osteogenic and chondrogenic differentiation. Hence, we hypothesized that BRE played an important role in maintaining the stemness of HUCPV cells. Because there was a lack of phenotypic difference between the BRE-silenced HUCPV cells and cells transfected with the control-siRNA, we decided to profile these cells using microarray and proteomic analyses. The aim was to elucidate the function of the BRE gene and establish whether BRE was involved in any signaling pathways. / In the microarray analysis, we examined the transcriptome of HUCPV cells in response to BRE-silencing in depth. Amongst the genes that we identified were significantly down-regulated by BRE-silencing and involved in the maintenance of pluripotency in ES cells were OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and also components of the TGF-β signaling pathway. This pathway is crucially involved in maintaining stem cell self-renewal. Therefore, we propose that BRE acts like a modulator that promotes stemness and at the same time inhibits the differentiation of HUCPV cells. / In the comparative proteomic study, BRE-silencing resulted in decreased expression patterns of cytoskeletal binding proteins such as actin, annexin II and tropomyosin. In addition, co-immunoprecipitation experiments revealed that the BRE protein can bind directly with actin and annexin II. It is possible that altering the cytoskeleton may provide a favorable environment for HUCPV cells to differentiate. This may explain why we were able to accelerate osteogenic and chondrogenic differentiation following BRE-silencing. In support of the view, it has been reported that chondrogenesis could be enhanced after cells have been treated with actin polymerization inhibitors (Lim et al., 2000; Solursh, 1989; Zhang et al., 2006). In sum, our studies provide an insight into the function of the BRE gene in HUCPV cells and the proteins that BRE can directly act on. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Elve. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 135-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.viii / List of Figures --- p.ix / List of Tables --- p.xiii / Table of Abbreviations --- p.xiv / Contents --- p.xviii / Chapter 1 --- p.1 / Literature Review --- p.1 / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.2 --- Embryonic stem cells (ESCs) --- p.2 / Chapter 1.3 --- Epiblast-derived stem (EpiS) cells --- p.2 / Chapter 1.4 --- Somatic stem cells (SSCs) --- p.3 / Chapter 1.5 --- Induced pluripotent stem (iPS) cells --- p.5 / Chapter 1.6 --- Human umbilical cord perivascular (HUCPV) cells --- p.7 / Chapter 1.7 --- CD146 --- p.8 / Chapter 1.8 --- Stem cell senescence --- p.9 / Chapter 1.9 --- Brain and reproductive organ-expressed (BRE) protein --- p.12 / Chapter 1.10 --- Stem cell self-renewal --- p.14 / Chapter 1.11 --- Apoptosis --- p.16 / Chapter 1.12 --- Stem cell niche --- p.21 / Chapter 1.13 --- Stem cell homing --- p.22 / Chapter 1.14 --- Objective --- p.22 / Chapter 2 --- p.24 / Accelerated osteogenic and chondrogenic differentiation of HUCPV cells by modulating the expression of BRE --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Rationale --- p.27 / Chapter 2.3 --- Materials and Methods --- p.27 / Chapter 2.3.1 --- Extraction of HUCPV cells from umbilical cord --- p.27 / Chapter 2.3.2 --- Cell culture condition --- p.28 / Chapter 2.3.3 --- Flow cytometry analysis and cell sorting --- p.28 / Chapter 2.3.4 --- In vitro osteogenic differentiation --- p.29 / Chapter 2.3.5 --- In vitro chondrogenic differentiation --- p.29 / Chapter 2.3.6 --- Alcian blue staining --- p.29 / Chapter 2.3.7 --- Alizarin red S staining --- p.30 / Chapter 2.3.8 --- Immunofluorescence analysis --- p.30 / Chapter 2.3.9 --- Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) --- p.31 / Chapter 2.3.10 --- Transfection with siRNA --- p.35 / Chapter 2.3.11 --- Microarray --- p.35 / Chapter 2.3.12 --- Cell lysis and immunoprecipitation --- p.36 / Chapter 2.3.13 --- SDS-PAGE and Western blot --- p.36 / Chapter 2.3.14 --- Isoelectric focusing and 2-dimensional gel electrophoresis --- p.37 / Chapter 2.3.15 --- Migration (wound healing) assay --- p.38 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- HUCPV cells were capable to differentiate into osteoblasts and chondrocytes --- p.38 / Chapter 2.4.2 --- BRE expression is down-regulated when HUCPV cells begins to differentiate --- p.40 / Chapter 2.4.3 --- Silencing of BRE expression accelerates induction of osteogenesis and chondrogenesis --- p.40 / Chapter 2.4.4 --- Microarray analysis of BRE-silenced HUCPV cells --- p.42 / Chapter 2.4.4.1 --- Stemness factors --- p.43 / Chapter 2.4.4.2 --- Epigenetic regulation --- p.43 / Chapter 2.4.4.3 --- Signaling pathways crucial for stemness maintenance --- p.44 / Chapter 2.4.4.4 --- TGF-β signaling --- p.44 / Chapter 2.4.4.5 --- FGF signaling --- p.44 / Chapter 2.4.4.6 --- NOTCH signaling --- p.45 / Chapter 2.4.4.7 --- WNT signaling --- p.46 / Chapter 2.4.4.8 --- Homeobox transcription factors (HOX) --- p.46 / Chapter 2.4.4.9 --- Cell cycle regulation --- p.47 / Chapter 2.4.4.10 --- Chemokines and cytokines regulation --- p.48 / Chapter 2.4.4.11 --- Apoptosis --- p.49 / Chapter 2.4.5 --- BRE-silencing alters the cellular proteome of HUCPV cells --- p.50 / Chapter 2.4.5.1 --- BRE-silencing alters the cytoskeletal binding proteins of HUCPV cells --- p.51 / Chapter 2.4.5.2 --- BRE-silencing alters the expressions of stemness-related proteins in HUCPV cells --- p.52 / Chapter 2.4.5.3 --- BRE-silencing alters the expressions of apoptosis-related proteins in HUCPV cells --- p.53 / Chapter 2.5 --- Discussion --- p.86 / Chapter 2.5.1 --- Microarray study discussion --- p.87 / Chapter 2.5.2 --- Proteomic study discussion --- p.89 / Chapter 3 --- p.93 / Replicative senescence alters the transcriptome and proteome of HUCPV cells --- p.93 / Chapter 3.1 --- Introduction --- p.93 / Chapter 3.2 --- Materials and methods --- p.93 / Chapter 3.3 --- Results --- p.93 / Chapter 3.3.1 --- Microarray analysis of aged HUCPV cells --- p.94 / Chapter 3.3.1.1 --- Stemness factors --- p.95 / Chapter 3.3.1.2 --- Epigenetic regulation --- p.96 / Chapter 3.3.1.3 --- Senescence associated markers --- p.96 / Chapter 3.3.1.4 --- Chemokines and cytokines regulation --- p.97 / Chapter 3.3.1.5 --- Matrix metalloproteinases regulation --- p.97 / Chapter 3.3.1.6 --- WNT signaling --- p.98 / Chapter 3.3.1.7 --- Toll-like receptor signaling pathway --- p.98 / Chapter 3.3.2 --- Proteomic profiling of aged HUCPV cells --- p.98 / Chapter 3.4 --- Discussion --- p.117 / Chapter 3.4.1 --- Aging alters the transcriptome of HUCPV cells --- p.117 / Chapter 3.4.2 --- Aging alters the proteome of HUCPV cells --- p.118 / Chapter 4 --- p.121 / Osteogenic and chondrogenic differentiation capacities of HUCPV cells in silk fibroin scaffold --- p.121 / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.2 --- Materials and methods --- p.121 / Chapter 4.2.1 --- Extraction of silk fibroin --- p.121 / Chapter 4.2.2 --- Fabrication of porous silk fibroin scaffold --- p.122 / Chapter 4.2.3 --- Scanning electron microscopy --- p.123 / Chapter 4.2.4 --- Cell culture --- p.123 / Chapter 4.3 --- Results --- p.124 / Chapter 4.4 --- Discussion --- p.132 / Chapter 5 --- p.133 / Conclusions --- p.133 / References --- p.135

Neonatal hypoxic-ischemic encephalopathy (HIE) is a common clinical problem. Tanshinone IIA is a compound purified from the Chinese herb Danshen ( Radix Salviae Miltiorrhiza Bge). Thrombopoietin (TPO) and Erythropoietin (Epo) are hematopoietic growth factors. The effects of tanshinone IIA, EPO and TPO on hypoxia-ischemia brain injury were investigated in this study, using in vitro model of neural cell culture and an in vivo model of hypoxic-ischemic brain damage. / Our observation provided the first evidence showing the expression of functional TPO receptor c-mpl in central nervous system. It revealed that novel agents TPO, EPO and tanshinone IIA have neuroprotection effects against brain injury induced by hypoxia-ischemia in neonatal rats, and these agents could be developed for clinical applications. / To investigate the effect of TPO, EPO and tanshinone IIA on in-vivo neural protection, a neonatal rat model of hypoxic-ischemic brain damage was established. Our results demonstrated significant and sustained brain injury in the hypoxic-ischemic and vehicle-treated group, measured by the reduction in relative weights of the ipsilateral (right) to the contralateral (left) brain at 1 and 3 weeks post-surgery, compared with those of sham-operated animals. At 3 weeks post-surgery, the hypoxic-ischemic animals had decreased cortical neuron density quantified by neuron-specific enolase (NSE) staining, and compromised sensorimotor functions in response to the postural reflex test. Treatment with TPO, EPO and tanshinone IIA significantly reduced the severity of brain injury, as indicated by the significantly increased ipsilateral brain weight and neuron density. Recoveries of sensorimotor functions (p < 0.05) and histopathology were also observed in animals that received TPO, EPO and tanshinone IIA. The plasma of tanshinone IIA-treated animals exhibited higher antioxidant activities (oxygen radical absorbance capacity assay) than those from vehicle-treated rats. / TPO and TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres, cerebellum, mouse neural progenitor cell line C17.2 and four neuroblastoma cell lines (SK-N-MC, MHH-NB-11, SK-N-AS and SH-SY-5Y) using RT-PCR methods. TPO proteins were detected in human cerebrospinal fluid (CSF) and plasma by ELISA. Furthermore, TPO receptor c-mpl was confirmed in human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immunohistostaining. TPO had a stimulating effect on the growth of neural progenitor cell C17.2 in culture via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway as demonstrated by Western blot. The anti-apoptotic effects of TPO, EPO on C17.2 cells were demonstrated by staining with Annexin-V and PI. EPO exerted a protective effect against SHSY-5Y cell damage induced by NMDA (N-methyl-d-aspartate), as demonstrated by the MTT and LDH assay. The anti-oxidative property of tanshinone IIA was studied in the C17.2 cell line. Tanshinone IIA increased the viability of these cells subjected to 2,2'-azobis (2-amidino propane hydrochloride) (AAPH)-induced oxidative stress. / by Xia Wen-Jie. / "May 2005." / Advisers: Kwok-Pui Fung, Tai-Fai Fok. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0126. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 126-146). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

When a multicellular organism suffers damages to tissues/organs it heals itself by either substituting the lost cellular matrix by scar formation or by regenerating the lost tissue. Regeneration likely occurs by a recapitulation of the developmental process that formed the organ. Many processes regulating organ development are based on epithelial-mesenchymal interactions and a strict control of organ specific stem/progenitor cells. Elucidation of the molecular basis of these processes is therefore vital in order to develop novel therapies in regenerative medicine. The LIM homebox gene Lhx2 is interesting in this context since Lhx2 has been shown to be important for the formation of several organs by regulating epithelial-mesenchymal interactions and progenitor cell function. Targeted inactivation of Lhx2 leads to a lethal anemia due to malformed liver and severe neural abnormalities such as hypoplasia of the forebrain and anophtalmia. Thus, elucidation of the mechanisms of the function of Lhx2 in different organ systems would give important insights into the molecular mechanisms regulating epithelial-mesenchymal interactions and stem/progenitor cell function. To elucidate the function of Lhx2 in the hematopoietic system Lhx2 was initially expressed in hematopoietic progenitor cells derived from ES cells differentiated in vitro using retroviral vectors. This approach led to the generation of hematopoietic stem cell (HSC)-like cell lines suggesting that Lhx2 could impact HSC function. However neither the specificity nor the efficiency of the Lhx2-induced phenotype could be determined using this approach. To be able to elucidate the function of Lhx2 in the hematopoietic system, an ES cell line with inducible Lhx2 expression was generated. Lhx2 expression induces self-renewal of a distinct hematopoietic progenitor cell from which HSC-like cell lines were established. Down-regulation of Lhx2 in these HSC-like cell lines leads to a rapid loss of stem cell character, providing a good model to study the molecular function of Lhx2 in hematopoietic stem/progenitor cells. A global gene expression analysis was performed comparing the Lhx2+ stem cell population to the Lhx2- differentiated progeny. This approach identified genes putatively linked to self-renewal/differentiation of HSCs. A considerable proportion of the genes showed an overlapping gene expression pattern with Lhx2 expression in tissue of non-hematopoietic origin suggesting that Lhx2 function in stem/progenitor cells partly overlap with Lhx2 function during organ development. In order to define other Lhx2-dependent progenitor cell populations and to generate a tool to analyze the function of Lhx2 in organ development a new transgenic mouse model was generated. By using a specific part of the Lhx2 promoter to drive expression of Cre recombinase in vivo (Lhx2-Cre mice) we have been able to define the first eye committed progenitor cells in the forebrain. By using the Lhx2-Cre mice it will be possible to distinguish the function of genes during eye development from their function in the patterning of the forebrain e.g. the eye field transcription factors. Conditional inactivation of Lhx2 in these eye specific progenitor cells causes an immediate developmental arrest. The transgene is also active in Lhx2-/- embryonic forebrain, but re-expression of Lhx2 in Lhx2-/- progenitor cells only promote formation of retinal pigment epithelium cells. Analysis of genes expressed by the Lhx2+ stem cell population allowed us to define novel genes putatively linked to Lhx2 function in eye development. Thus, we have defined the progenitor cells in the forebrain committed to eye development and the expansion and patterning of these progenitors are dependent on Lhx2. Although commitment to eye development is Lhx2-independent, Lhx2 might be important for the acquisition of the oligopotent fate of these progenitor cells.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

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