Huang, Li. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 162-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Hematopoietic cell populations exhibiting detectable telomerase activity and elongated telomere lengths display strong engraftment survivability in humans during transplants. We investigated telomerase activity and telomere length in umbilical cord blood hematopoietic cell populations obtained from ViaCell Inc. at various intervals of a two-week ex vivo stem cell amplification process. Telomerase activity is increased with time in ViaCell's amplification process, perhaps in response to the removal of differentiated cells and expansion of primitive hematopoietic stem cell populations in tissue culture media containing a mixture of growth factors. Two of ViaCell's cell culture fractions were analyzed for telomere length using a TLA. Our results showed relatively long telomere lengths for day-0 and day-14 cord populations, and that despite an upregulation of telomerase activity in Day-14 samples, a loss of about 2 kb of telomeric DNA occurs. Our data are consistent with a model in which the increase in telomerase activity in day-14 ex vivo amplified cord blood hematopoietic cells relative to fresh cord is sufficient to reduce, but not prevent, telomere shortening caused by cell proliferation. Lastly, we investigated various culture conditions that could potentially upregulate telomerase activity in the Day-14 amplified cells. However none of the treatments tested altered telomerase activity. Our detection of increased telomerase activity and relatively long telomere lengths in ViaCell's Day-14 ex vivo amplified cord blood stem cell fraction provides support for ViaCell's Selective Clonogenic AmplificationTM indicating a high engraftment potential for these cells.

Amplification of hematopoietic stem cells (HSCs) from human cord blood has applications for a variety of cell therapy protocols. The purpose of this thesis (performed in collaboration with ViaCell, Inc.) was to analyze differential gene expression (especially related to cytokines) during the process of human HSC amplification in vitro. When applied to markers previously shown to be specific for HSC's and/or progenitor cells, the analysis validates ViaCell's cellular product. Total cellular RNA was isolated from cord blood samples at various stages of amplification and used to synthesize cDNAs as probes for hybridization arrays. mRNA candidates increased in cell populations enriched for stem cells were first identified using hybridization arrays, then confirmed by RT-PCR. Restriction mapping confirmed RT-PCR amplicons. The results identified several developmentally interesting cytokines (CD117, Jagged-2, Manic Fringe, and Notch) upregulated in stem cell enriched fractions. Analysis of one candidate previously shown to be a marker for HSCs and progenitors, CD117, was extended using Western blots to show a CD117-related protein upregulation. The observed upregulations did not contain many inflammatory cytokines, which could hinder survival of HSC grafts. The future hope for the non-CD117 candidates is as potential growth modifiers for stem cell samples isolated by clonogenic amplification.

C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling via stromal cell-derived factor-1 (SDF-1) has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis-resistant (AR) cells were collected from mammosphere culture from 2 immortalised (MCF10A, 226L) and 3 malignant (MCF7, T47D, SKBR3) breast cell lines. For all cell lines, AR cells had a significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR cells of the normal cell lines also demonstrated increased formation of 3D structures using the Matrigel assay. In vivo, MCF7 and T47D AR cells demonstrated increased capacity to form tumours compared with unsorted cells. This suggests that AR cells are enriched for normal and malignant breast stem cells. We performed an Agilent custom gene microarray and demonstrated up-regulation of CXCR4 mRNA expression in AR cells. CXCR4 protein expression was also higher in AR cells, shown by flow cytometry. The effects of AMD3100 (CXCR4 antagonist) and SDF-1 (CXCR4 ligand) on stem cell activity were investigated in the mammosphere assay. In the normal cell lines, SDF-1 significantly reduced MFE and this decrease was rescued by AMD3100. Incubation with AMD3100 increased MFE in the estrogen receptor positive breast cancer cell lines (MCF7 and T47D) and patient-derived metastatic tumour samples. AMD3100 reduced the self-renewal of T47D cells, as assessed by second generation mammospheres. MCF7 cells were retro-virally transfected to over-express CXCR4 or sorted for CXCR4 cell surface expression. Mammosphere formation was significantly increased in CXCR4+ and CXCR4 over-expressing cells compared with CXCR4- and parental cells. There was a greater reduction in self-renewal following AMD3100 treatment in the CXCR4 over-expressing cells compared with parental cells. AMD3100 has been shown to have an agonistic effect on the novel chemokine receptor CXCR7, a scavenging receptor for SDF-1. All cell lines demonstrated cell surface expression of CXCR7, measured by flow cytometry and mRNA expression. Potential interactions between CXCR4, CXCR7 and SDF-1 must be considered in future investigation of the role of CXCR4 signalling. Our data establish that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. CXCR4 influences self-renewal of malignant stem cells which may account for its role in tumorigenesis. CXCR4 signalling may be important in tumour formation at the sites of metastases as well as in cell migration. Its role in stem cell migration merits further investigation. In conclusion, CXCR4-targeted therapy, alongside current standards of care, may improve breast cancer outcomes.

丙戊酸 (VPA) 是一種在臨床上被用來抗驚厥和穩定情緒的藥物。除了治療神經功能紊亂的功效以外, 最近的研究發現VPA可以導致癌細胞的凋亡及分化，增加胚胎幹細胞的分化，並在提升由成體細胞誘導成多功能幹細胞的效率。至今，VPA在幹細胞發育的作用並不十分清楚。在這項研究中，我們使用體外培養體系，來研究VPA在小鼠神經幹細胞，神經前體細胞和胚胎幹細胞的生物學效應。 / 實驗發現VPA可以抑制神經前體細胞株C17.2和神經幹細胞的增生。另一方面，VPA可以增加這兩類細胞分化形成神經元，但同時並沒有增加神經膠質細胞和少突膠質細胞的分化。另一方面，VPA抑制胚胎幹細胞向神經類細胞分化。實驗顯示這種抑制作用源自於VPA對胚胎幹細胞早期譜系分化的抑制。 / 胚胎幹細胞分化實驗顯示，VPA可以抑制胚胎幹細胞向神經外胚層譜系分化，並促進對中胚層和內胚層譜系的分化。經VPA處理後的胚體不能夠產生β-微管蛋白III陽性樹突狀突起。我們還發現，在含血清的培養液中分化的胚體會出現跳動的細胞群。此類細胞群在VPA組裡面的比例較高。因此可以推斷VPA有區增強心肌分化的功能。免疫熒光染色也顯示，VPA處理組中較多細胞為內胚層蛋白標記 FOXA2和甲胎蛋白(AFP)陽性。經VPA處理六天後的胚體胚層標誌基因表達量顯示，神經外胚層標記 Pax6和Noggin在VPA處理組中表達量降低。VPA處理組的中胚層標記brachyury和Mef2c以及內胚層標記GATA4和FOXA2胚層標記的表達量均有上調。 / 在神經幹細胞, 神經前體細胞以及胚胎幹細胞的分化中，可以從VPA處理組觀察到經典 wnt信號通路上調。定量逆轉錄聚合酶鏈反應檢測顯示在VPA處理組裡面, 神經幹細胞, 神經前體細胞以胚體的 wnt信號分子的表達水平的上調。免疫墨點法也檢測到VPA處理組中活化β-catenin較對照組為高。 / Wnt信號通路抑製劑 IWP-2可以破壞棕櫚酰化wnt信號，從而抑制信號分子的成熟和分泌。IWP-2抵銷了VPA 在pNSC中促進神經元的分化的作用。另一方面，抑制wnt信號通路增加了VPA處理組胚體中β-微管蛋白III陽性樹突狀突起的生成, 並且減少跳動細胞群以及內胚層標記蛋白FOXA2和甲胎蛋白陽性的細胞比例。定量逆轉錄聚合酶鏈反應檢測顯示，IWP-2處理組的胚體和對照組相比，神經外胚層標記Pax6和Noggin被上調，中胚層標記 brachyury和Mef2c和內胚層標記 GATA4和FOXA2均被下調。IWP-2 VPA雙重處理組和VPA處理組相比也呈現相同趨勢。這些數據表明，VPA對於神經幹細胞的神經元分化和胚胎幹細胞的胚層譜系分化的影響是通過wnt信號通路介導的。 / 體內研究可以觀察到VPA處理會導致小鼠胚胎中出現的一系列畸形，其中出現有關神經管閉合，異常出血和小腸生長畸形的症狀。胚層標誌分析指出，從VPA注射組孕鼠體內取出的早期階段胚胎具有和在胚胎幹細胞實驗中類似的胚層標記基因表達模式。中胚層和內胚層標記上調而外胚層標記下調。另一方面，VPA在全腦缺血小鼠的治療方面有幫助改善認知功能的作用。 / 這項研究為說明 VPA的致畸作用提供了一個潛在的機制。同時也能夠顯示VPA對全腦缺血患者的潛在治療效果。 / Valproic acid (VPA) is commonly used as a mood stabilizer and anticonvulsant. Despite the clinical relevance to neural disorders, recent studies of VPA revealed the apoptotic and differentiating effects on cancer cells, enhancement of lineage commitments of embryonic stem cells and augmentation of the efficiency in the induction of somatic cells into pluripotent stem cells. Up to date the roles of this small molecule in stem cell development are not well understood. In this study, in vitro culture system was used to elucidate the biological effects of VPA on mouse primary neural stem cells (pNSC), neural precursor cells C17.2 and embryonic stem cells, D3 and E14Tg2a. / VPA was found to inhibit the proliferation of NPC C17.2 and pNSC. On the other hand, it enhanced the differentiation into neurons but not astrocytes and oligodendrocytes. The differentiation of ESC revealed that VPA treatment inhibited the differentiation of ESC into the neuro-ectodermal lineage but promoted the commitment towards the mesodermal and endodermal lineages. / Embryoid bodies (EB) derived from VPA-treated ESC displayed less cell foci with β-tubulin III+ protrusions, but an increase of beating cell clusters, suggesting that VPA enhanced cardio differentiation. Immunofluorescence staining demonstrated that a higher portion of the cells in the VPA-treated group were positive for endodermal markers, FOXA2 and α-fetoprotein. Quantitative RT-PCR (q-PCR) for dermal markers in EB differentiated and treated with VPA for six days showed that neural-ectodermal markers, Pax6 and Noggin, were down-regulated, whereas mesodermal markers, brachyury and Mef2c, and endodermal markers, GATA4 and FOXA2, were all up-regulated. / In studies of the molecular signalling mediated by VPA, quantitative RT-PCR revealed an up-regulation in the gene expression level of wnt molecules in VPA-treated pNSC, NPC and EB. Western blotting also envisaged a higher level of activated β-catenin proteins in VPA treated cells. / The wnt pathway inhibitor IWP-2 was employed to disrupt the palmitoylation of wnt and block the maturation and secretion of the signalling molecule. It was noted that the application of IWP-2 negated the VPA-enhanced neuronal differentiation of pNSC. On the other hand, wnt inhibition increased the incidence of EB having β-tubulin III⁺ protrusions and reduced the numbers of beating EB, FOXA2⁺ and α-fetoprotein⁺ cells in EB-derived cultures of both controls and VPA-treated group. Besides, IWP-2 enhanced the gene expression of neural-ectodermal markers, Pax6 and Noggin, but repressed the gene expression of mesodermal markers, brachyury and Mef2c, and endodermal markers, GATA4 and FOXA2, in EB cultures treated with and without VPA. Data suggest that VPA modulated the neuronal differentiation of pNSC and the dermal commitment of ESC via the wnt pathway. / In vivo study demonstrated that VPA mediated malformations in mouse embryos including deficits in neural tube closure, abnormal bleeding and intestine outgrowth. Dermal marker analysis of VPA-treated embryos at the early stage of development displayed a similar pattern of gene expression noted in in vitro ESC study. Mesodermal and endodermal genes were up-regulated while the ectodermal genes were down-regulated. In global brain ischemic mice, VPA helped restore the cognitive impairment, suggesting the potential therapeutic effect of VPA in global brain ischemia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lau, Shong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 182-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of publications --- p.ii / Abstract --- p.iii / 綜述 --- p.vi / Table of content --- p.viii / Lists of figures --- p.xii / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.2 --- Embryonic stem cells --- p.2 / Chapter 1.3 --- Application of stem cells for therapeutic purpose --- p.2 / Chapter 1.3.1 --- Application of stem cells in neurological disorders --- p.3 / Chapter 1.3.2 --- Application of stem cells in cardiac repair --- p.6 / Chapter 1.3.3 --- Application of stem cells in diabetes --- p.8 / Chapter 1.3.4 --- Application of haematopoietic stem cells --- p.9 / Chapter 1.3.5 --- Application of messenchymal stem cells --- p.10 / Chapter 1.4 --- Reprogramming --- p.11 / Chapter 1.4.1 --- Somatic cell nucleus transfer (SCNT) --- p.11 / Chapter 1.4.2 --- Altered nucleus transfer --- p.11 / Chapter 1.4.3 --- Induced pluripotent stem cells --- p.12 / Chapter 1.5 --- Propagation of ESC --- p.15 / Chapter 1.6 --- Molecular mechanism of ESC pluripotency and self-renewal --- p.16 / Chapter 1.6.1 --- Oct3/4 and Sox2 --- p.16 / Chapter 1.6.2 --- Nanog --- p.17 / Chapter 1.6.3 --- LIF/JAK/STAT3 --- p.17 / Chapter 1.6.4 --- Wnt --- p.18 / Chapter 1.6.5 --- Basic fibroblast growth factor (bFGF) --- p.18 / Chapter 1.6.6 --- TGF-β/Activin/Nodal --- p.18 / Chapter 1.7 --- Wnt signalling pathway --- p.19 / Chapter 1.7.1 --- Canonical wnt pathway --- p.20 / Chapter 1.7.2 --- Planar cell polarity pathway --- p.21 / Chapter 1.7.3 --- Wnt/Ca²⁺ Pathway --- p.22 / Chapter 1.7.4 --- Signal specificity --- p.23 / Chapter 1.7.4.1 --- Wnt 1 --- p.24 / Chapter 1.7.4.2 --- Wnt 2 --- p.25 / Chapter 1.7.4.3 --- Wnt3/wnt3A --- p.25 / Chapter 1.7.4.4 --- Wnt4 --- p.27 / Chapter 1.7.4.5 --- Wnt5 --- p.28 / Chapter 1.7.4.6 --- Wnt7 --- p.29 / Chapter 1.7.4.7 --- Other wnts --- p.31 / Chapter 1.8 --- Valproic acid --- p.33 / Chapter 1.8.1 --- Effect of VPA on histone modification --- p.34 / Chapter 1.8.2 --- Interaction of VPA with extracellular signal-regulated kinase pathway --- p.35 / Chapter 1.8.3 --- Effect of VPA on PI3K pathway --- p.37 / Chapter 1.8.4 --- Effect of VPA on wnt pathway --- p.37 / Chapter 1.8.5 --- VPA on stem cells --- p.38 / Chapter 1.8.6 --- VPA on brain ischemia animal model --- p.39 / Chapter 1.9 --- Overall aim and design of the Study --- p.41 / Chapter 2.1 --- Cells /cell lines --- p.53 / Chapter 2.1.1 --- Neural precursor cell line C17.2 --- p.53 / Chapter 2.1.2 --- Primary neural stem cells --- p.53 / Chapter 2.1.3 --- Primary mouse embryonic fibroblast --- p.53 / Chapter 2.1.4 --- Embryonic stem cell line D3 --- p.54 / Chapter 2.1.5 --- Embryonic stem cell line E14TG2a --- p.54 / Chapter 2.2 --- Cell cultures and assays --- p.54 / Chapter 2.2.1 --- Medium and solutions --- p.54 / Chapter 2.2.2 --- Isolation of primary neural stem cells --- p.58 / Chapter 2.2.3 --- Maintenance and passaging of primary neural stem cells --- p.60 / Chapter 2.2.4 --- Cryopreservation and thawing of primary neural stem cells --- p.60 / Chapter 2.2.5 --- Coating of coverslips using ornithine and laminin (O&L) --- p.61 / Chapter 2.2.6 --- Differentiation of primary neural stem cells --- p.61 / Chapter 2.2.7 --- Isolation of mouse embryonic fibroblasts --- p.62 / Chapter 2.2.8 --- Maintenance and passaging of mouse embryonic fibroblasts --- p.63 / Chapter 2.2.9 --- Cryopreservation and thawing of mouse embryonic fibroblasts --- p.63 / Chapter 2.2.10 --- Preparation of gelatin-coated culture wares and cover slips --- p.64 / Chapter 2.2.11 --- Preparation of irradiated mouse embryonic fibroblast feeder layer --- p.64 / Chapter 2.2.12 --- Maintenance and passaging of neural precursor cell C17.2 --- p.64 / Chapter 2.2.13 --- Cryopreservation and thawing of neural precursor cell C17.2 --- p.64 / Chapter 2.2.14 --- Differentiation of neural precursor cell C17.2 --- p.65 / Chapter 2.2.15 --- Maintenance and passaging of mouse embryonic stem cell line --- p.65 / Chapter 2.2.16 --- Cryopreservation and thawing of mouse embryonic stem cell line --- p.65 / Chapter 2.2.17 --- Spontaneous differentiation of embryonic stem cells --- p.66 / Chapter 2.2.18 --- Neural differentiation of embryonic stem cells --- p.66 / Chapter 2.2.19 --- WST-1 proliferation assay --- p.67 / Chapter 2.2.20 --- Colony formation assay --- p.67 / Chapter 2.2.21 --- Two-stage neural differentiation assay --- p.68 / Chapter 2.3 --- Molecular analysis --- p.68 / Chapter 2.3.1 --- In-house prepared solutions and reaction mixes --- p.68 / Chapter 2.3.2 --- RNA extraction --- p.72 / Chapter 2.3.3 --- Synthesis of complementary DNA (cDNA) by reverse transcription --- p.73 / Chapter 2.3.4 --- Polymerase chain reaction (PCR) --- p.74 / Chapter 2.3.5 --- Quantitative polymerase chain reaction (qPCR) --- p.74 / Chapter 2.3.6 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting --- p.75 / Chapter 2.4 --- Microscopy and Immunofluorescence staining --- p.77 / Chapter 2.5 --- In vivo Study --- p.77 / Chapter 2.5.1 --- Embryo malformation study --- p.77 / Chapter 2.5.2 --- Establishment of a mouse global brain ischemia model --- p.78 / Chapter 2.5.3 --- Behaviour study --- p.79 / Chapter 2.5.4 --- Histological analysis --- p.79 / Chapter 2.5.4.1 --- Animal sacrifice for brain harvest --- p.79 / Chapter 2.5.4.2 --- Paraffin sectioning --- p.80 / Chapter 2.5.4.3 --- Haematoxylin and eosin staining --- p.80 / Chapter 2.5.4.4 --- Cresyl violet staining --- p.81 / Chapter 2.5.4.5 --- TUNEL assay --- p.81 / Chapter 2.6 --- Statistical analysis --- p.82 / Chapter 2.7 --- Equipments used in this study --- p.82 / Chapter Chapter 3 --- Effects of VPA on proliferation and differentiation of mouse NPC, pNSC and ESC --- p.88 / Chapter 3.1 --- Effects of VPA on mouse ESC proliferation --- p.88 / Chapter 3.1 --- Effects of VPA on mouse ESC proliferation --- p.88 / Chapter 3.2 --- Effects of VPA on differentiation and proliferation of neural lineage cells --- p.89 / Chapter 3.2.1 --- Effects of VPA on differentiation and proliferation of NPC C17.2 --- p.89 / Chapter 3.2.2 --- Effects of VPA on differentiation of pNSC --- p.89 / Chapter 3.2.3 --- Effects of VPA on proliferation and stemness of pNSC --- p.90 / Chapter 3.2.4 --- Effects of VPA on neural differentiation of mouse ESC --- p.91 / Chapter 3.2.5 --- Two-stage neural differentiation assay of mouse ESC --- p.91 / Chapter 3.3 --- VPA-mediated lineage commitment of mouse ESC --- p.92 / Chapter 3.3.1 --- Cardio differentiation of ESC --- p.92 / Chapter 3.3.2 --- Endodermal commitment of mouse ESC --- p.93 / Chapter 3.3.3 --- Germ layer marker expression in mouse ESC --- p.93 / Chapter 3.3.4 --- Up-regulation of histone acetylation in VPA-treated mouse ESC --- p.94 / Chapter 3.6 --- Summary --- p.94 / Chapter Chapter 4 --- Wnt signalling played as a mediator of VPA in the differentiation of ESC, NPC and pNSC differentiation. --- p.118 / Chapter 4.1 --- VPA treatment up-regulated wnt pathway in mouse ESC, NPC and pNSC --- p.118 / Chapter 4.2 --- Effect of wnt inhibitor IWP-2 on stem cells in differentiation cultures supplemented with VPA. --- p.119 / Chapter 4.2.1 --- Effects of wnt inhibitor IWP-2 on the neural differentiation of NPC C17.2 --- p.119 / Chapter 4.2.2 --- Effect of wnt inhibitor on differentiation of pNSC --- p.120 / Chapter 4.2.3 --- Effect of IWP-2 on the differentiation culture of EB --- p.121 / Chapter 4.2.4 --- Effect of wnt inhibitor on the lineage commitment of mouse ESC --- p.121 / Chapter 4.2.5 --- Effects of IWP-2 on VPA-induced lineage markers expressed by mouse ESC --- p.123 / Chapter 4.3 --- Effects of wnt inhibitor IWP-2 on ESC proliferation. --- p.124 / Chapter 4.4 --- Summary --- p.125 / Chapter Chapter 5 --- In vivo effects of VPA --- p.154 / Chapter 5.1 --- Effects of VPA on mouse embryo development --- p.154 / Chapter 5.1.1 --- Effects of VPA at the daily dose of 300mg/kg body weight --- p.154 / Chapter 5.1.2 --- Effects of VPA at the daily dose of 600 mg/kg body weight --- p.155 / Chapter 5.1.3 --- Effects of VPA at twice daily dose of 600 mg/kg body weight --- p.155 / Chapter 5.2 --- Effects of VPA on ischemic mice --- p.156 / Chapter 5.2.1 --- Behaviour test --- p.156 / Chapter 5.2.2 --- Histological and molecular assessment --- p.157 / Chapter 5.3 --- Summary --- p.157 / Chapter Chapter 6 --- Discussion --- p.171 / Chapter 6.1 --- Modulation of ESC fate at early developmental stage --- p.171 / Chapter 6.2 --- Neural differentiation of pNSC and NPC upon VPA treatment --- p.172 / Chapter 6.3 --- VPA-mediated wnt signaling on the differentiation of mouse ESC --- p.173 / Chapter 6.4 --- VPA-mediated wnt signalling in the neuronal differentiation of mouse neural stem/progenitor cells --- p.175 / Chapter 6.5 --- Effects of VPA on the proliferation of mouse ESC, pNSC and NPC --- p.176 / Chapter 6.6 --- Effects of VPA on mouse embryo --- p.177 / Chapter 6.7 --- Therapeutics of VPA in ischemic stroke --- p.178 / Chapter Chapter 7 --- Conclusion --- p.180 / References --- p.182

Lau, Yuen Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-163). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.iv / Table of content --- p.V / List of figures --- p.ix / List of tables --- p.xiii / List of abbreviations --- p.xiv / Chapter 1 --- INTRODUCTION --- p.Page / Chapter 1.1 --- Embryonic stem cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.2 --- Promising use of ESCs in drug development and regenerative medicine --- p.1 / Chapter 1.1.3 --- Maintenance of self-renewal and pluripotent properties of ESCs --- p.3 / Chapter 1.2 --- Cell cycle in ESCs --- p.5 / Chapter 1.2.1 --- Cell cycle --- p.5 / Chapter 1.2.2 --- Characteristics of cell cycle of ESCs --- p.6 / Chapter 1.3 --- Telomere --- p.8 / Chapter 1.3.1 --- Telomere structure and the telomeric proteins --- p.8 / Chapter 1.3.2 --- End replication problem --- p.10 / Chapter 1.3.3 --- Telomere dysfunction in cancer and cellular aging --- p.11 / Chapter 1.4 --- Telomerase --- p.12 / Chapter 1.4.1 --- Telomerase and stem cell characteristics --- p.13 / Chapter 1.4.1.1 --- Telomerase and cell proliferation --- p.13 / Chapter 1.4.1.2 --- Telomerase and stem cell differentiation --- p.14 / Chapter 1.4.2 --- Regulation of telomerase expression/ activity --- p.15 / Chapter 1.4.2.1 --- Regulation of telomerase at different levels --- p.15 / Chapter 1.4.2.2 --- Regulation of telomerase activity by cellular components in ESCs --- p.16 / Chapter 1.5 --- PinXl --- p.18 / Chapter 1.5.1 --- Expression of PinXl --- p.18 / Chapter 1.5.2 --- Effects of PinXl on the activities and the sub-cellular localization of telomerase --- p.19 / Chapter 1.5.3 --- Structure-function relationship of PinXl --- p.19 / Chapter 1.5.4 --- Effect of PinXl on the growth rate of normal and cancer cells --- p.21 / Chapter 1.5.5 --- Other functions of PinX 1 V --- p.22 / Chapter 1.5.6 --- Mouse homolog of PinXl and its function in mESCs --- p.23 / Chapter 1.6 --- Aims of this study --- p.24 / Chapter 2 --- METERIALS AND METHODS --- p.Page / Chapter 2.1 --- mESC culture and differentiation --- p.25 / Chapter 2.1.1 --- Cell line --- p.25 / Chapter 2.1.2 --- Irradiation of MEF --- p.25 / Chapter 2.1.3 --- mESC culture --- p.26 / Chapter 2.1.4 --- Differentiation of mESCs --- p.26 / Chapter 2.1.5 --- Establishment and' culture of feeder-free mESCs --- p.28 / Chapter 2.1.6 --- Culture of feeder-free mESCs --- p.28 / Chapter 2.2 --- Trypan Blue Exclusion Assay --- p.29 / Chapter 2.3 --- Sub-cloning --- p.29 / Chapter 2.3.1 --- Amplification of the insert gene by PCR --- p.29 / Chapter 2.3.2 --- Purification of PCR products --- p.31 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.32 / Chapter 2.3.4 --- Ligation of digested insert and vector --- p.33 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.34 / Chapter 2.3.6 --- Confirmation of positive clone by colony PCR --- p.34 / Chapter 2.3.7 --- Small scale preparation of the recombinant plasmid DNA --- p.35 / Chapter 2.3.8 --- Confirmation of positive clone by restriction digestion --- p.36 / Chapter 2.3.9 --- DNA sequencing of the recombinant plasmid DNA --- p.36 / Chapter 2.3.10 --- Large scale preparation of the recombinant plasmid DNA --- p.37 / Chapter 2.4 --- Design of siRNA targeting mPinXl and mPinXlt --- p.38 / Chapter 2.5 --- Transient transfection --- p.38 / Chapter 2.6 --- Cloning of siRNA into shRNA insert in Lentiviral Vector pLVTHM --- p.39 / Chapter 2.7 --- Lentiviral vector-mediated gene transfer to mESCs --- p.42 / Chapter 2.7.1 --- Lentivirus packaging --- p.42 / Chapter 2.7.2 --- Checking of successful transduction by lentivirus in HEK cells --- p.43 / Chapter 2.7.3 --- Multiple transductions to mESCs --- p.43 / Chapter 2.7.4 --- Selection of positive clones --- p.44 / Chapter 2.7.5 --- Monoclonal establishment --- p.44 / Chapter 2.8 --- "Total RNA preparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.45 / Chapter 2.9 --- Immunocytochemistry --- p.46 / Chapter 2.10 --- Western Blotting --- p.48 / Chapter 2.10.1 --- Total Protein Extraction vi --- p.48 / Chapter 2.10.2 --- Measurement of Protein Concentration --- p.48 / Chapter 2.10.3 --- SDS-PAGE and chemiluminescent detection --- p.49 / Chapter 2.11 --- Co-immunoprecipitation --- p.51 / Chapter 2.12 --- Telomere Repeat Amplification Protocol (TRAP) Assay --- p.52 / Chapter 2.13 --- Cell cycle analysis --- p.54 / Chapter 2.14 --- MTT assay --- p.54 / Chapter 2.15 --- Statistical analysis --- p.55 / Chapter 3 --- RESULTS --- p.Page / Chapter 3.1 --- mPinXlt was discovered in mESCs --- p.56 / Chapter 3.2 --- mPinXl and mPinXlt were expressed at transcriptional level in the inspected mouse tissues --- p.61 / Chapter 3.3 --- Expression of mPinXl and mPinXlt changed upon differentiation --- p.64 / Chapter 3.4 --- mPinXl and mPinXlt were both located in the nucleolus and the nucleoplasm in undifferentiated mESCs --- p.69 / Chapter 3.5 --- Co-immunoprecipitation (Co-IP) of mPinXl and mPinXlt with mTERT --- p.73 / Chapter 3.6 --- Transient knockdown of mPinXl in mESCs --- p.78 / Chapter 3.6.1 --- Knockdown of mPinXl decreased proliferation but did not change cell viability --- p.79 / Chapter 3.6.2 --- Knockdown of mPinXl decreased telomerase activity --- p.79 / Chapter 3.6.3 --- Knockdown of mPinXl did not change pluripotency --- p.80 / Chapter 3.6.4 --- Knockdown of mPinXl did not affect cell cycle progression --- p.80 / Chapter 3.7 --- Transient knockdown of mPinXlt using siRNA against mPinXlt in mESCs --- p.88 / Chapter 3.8 --- Transient over-expression of mPinXl and mPinXlt in mESCs --- p.90 / Chapter 3.8.1 --- Over-expression of mPinXl and mPinXlt decreased cell proliferation but didn't affect cell viability --- p.91 / Chapter 3.8.2 --- Over-expression of mPinXl increased telomerase activity --- p.92 / Chapter 3.8.3 --- Over-expression of mPinXl and mPinXlt did not affect pluripotency --- p.93 / Chapter 3.8.4 --- Over-expression of mPinXl and mPinXlt did not affect cell cycle progression --- p.93 / Chapter 3.9 --- Stable over-expression and knockdown of mPinXl and mPinXlt in mESCs --- p.103 / Chapter 3.9.1 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in all over-expression stable cell lines --- p.108 / Chapter 3.9.2 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in mPinXl knockdown stable cell lines --- p.113 / Chapter 3.9.3 --- Proliferation of all stable cell lines --- p.116 / Chapter 3.9.4 --- Telomerase activity of all stable cell lines --- p.121 / Chapter 3.9.5 --- Cell cycle distribution of all stable cell lines --- p.123 / Chapter 3.9.6 --- Pluripotency of all stable cell lines --- p.127 / Chapter 3.9.7 --- Differentiation of the stable cell lines --- p.130 / Chapter 3.9.7.1 --- Size of EBs formed from stable cell lines at Day 7 --- p.130 / Chapter 3.9.7.2 --- Beating curves of the stable cell lines derived EBs --- p.130 / Chapter 4 --- DISCUSSIONS --- p.Page / Chapter 4.1 --- mPinXlt gene was detected in mESCs --- p.137 / Chapter 4.2 --- "Presence of mPinXl and mPinXlt in mouse tissues, mESCs and their differentiation derivatives" --- p.138 / Chapter 4.3 --- Differences in expressions of mPinXl and mPinXlt in undifferentiated mESCs and their differentiation derivatives --- p.139 / Chapter 4.4 --- mPinXl and mPinXlt are pre-dominantly localized in the nucleolus --- p.141 / Chapter 4.5 --- mPinXl and mPinXlt interacted with mTERT --- p.143 / Chapter 4.6 --- "Transient knockdown of mPinXl slightly inhibited, while over-expression of mPinXl slightly promoted telomerase activity" --- p.143 / Chapter 4.7 --- Both transient knockdown and over-expression of mPinXl inhibited the growth of mESCs --- p.146 / Chapter 4.8 --- Both stable knockdown and over-expression of mPinXl did not affect cell proliferation and telomerase activity of mESCs --- p.148 / Chapter 4.9 --- Involvement of mPinXl and mPinXlt in the differentiation process of mESCs --- p.149 / Chapter 4.10 --- Regulation of mPinXl gene expression by mPinXlt --- p.151 / Chapter 4.11 --- Future perspectives --- p.152 / Chapter 5 --- CONCLUSION --- p.154 / Chapter 6 --- REFERENCES --- p.156

TGF-β/Activin/Nodal 信號通路調節了許多重要的細胞生物學過程,例如,細胞分裂,增殖,分化,遷移和衰老凋亡。此外,它也在胚胎髮育,損傷修復,腫瘤發生,組織纖維化,糖尿病發生及其免疫方面也扮演了重要的角色。TGF-β家族信號分子,包括TGF-β, Activin 和 Nodal,通過結合到它們各自的受體從而啟動它們,而啟動後的受體又可以通絡磷酸化作用進一步激活Smad2 和Smad3 蛋白,激活後的Smad2 和Smad3 蛋白可以和Smad4 蛋白形成複合體,一起從細胞膜轉移到細胞核內調節下游基因的表達。 / 在人的胚胎幹細胞中,TGF-β/Activin/Nodal signaling 做為最關鍵的信號分子,調節了人胚胎幹細胞的自我更新以及胚胎幹細胞多能性的維持。而在小鼠胚胎幹細胞中,該信號通路的功能並沒有清楚的研究。在本論文中,我們分離以及建立了Smad3 突變體的小鼠胚胎幹細胞系(Smad3-/-),該突變體細胞能夠維持正常小鼠胚胎幹細胞的形態,在自我增殖更新方面也沒有缺。此外,幹細胞多能性相關的標記基因以及組織標記基因表達水準也與野生型細胞非常相似,但是,在擬胚體的生長過程中,Smad3 被敲除後導致了組織發育相關的標記基因出現了差異性的表達。與野生型相比,中胚層標記基因（T 和GSC）的表達明顯受到了抑制。另外令人驚奇的是,將Smad3 基因敲除的胚胎幹細胞皮下注射裸鼠後長出了惡性的未完全成熟的畸胎瘤,而野生型的幹細胞則更傾向于長成成熟的良性畸胎瘤。進一步的分析發現,Smad3 功能性缺失後,細胞的增殖速率明顯增加了;紫外(UV)誘變後,相對於野生型,突變體細胞的抗凋亡能力也明顯增強了;並且在分化過程中,突變體細胞的遷移能力也要明顯強于野生型細胞。所有以上的細胞特徵可以解釋為什麼Smad3 基因敲除後會長出惡性的畸胎瘤。 / Microarray 分析結果發現,一個DNA 損傷修復基因Rif1,在Smad3 突變體細胞中呈現出了很高的上調,這個基因的上調現象已經發現是和侵蝕性腫瘤的發生是相關的,而且該基因的上調水準也與乳腺癌的浸潤程度是非常相關的。染色質免疫共沉澱和螢光素酶活性實驗進一步證實了Smad3 可以結合到Rif1 的啟動子領域從而直接抑制該基因的表達。這些實驗進一步說明了Smad3 可能通過下調Rif1 基因的表達,從而抑制了小鼠胚胎幹細胞長出惡性畸胎瘤的發生。 / 總之,我們建立了Smad3-/-基因敲除的小鼠胚胎幹細胞系,並且發現該突變體細胞傾向于長出惡性的畸胎瘤。我們推測,在正常的情況下,Smad3 正是通過抑制了DNA 損傷修復因數基因Rif1 的表達,從而阻止了惡性畸胎瘤的發生。這些研究的結果不僅開闊了我們對於惡性腫瘤發生的認識,而且為我們在幹細胞或誘導多能幹細胞治療應用中防止畸胎瘤的發生提供了新的思路和策略。 / TGF-β/Activin/Nodal signaling controls many important biological procedures in cells, such as cell division, proliferation, differentiation, migration and apoptosis in mammalian cells. It also plays a critical role in embryo development, wound healing, tumorigenesis, tissue fibrosis, diabetes and immunity. TGF-β superfamily ligands, such as TGF-β, Activin and Nodal bind to their respective ligand receptors and activate them, which in turn activate the receptor related SMAD proteins by phosphorylation, including Smad2 and Smad3. Once phosphorylated, they can cooperate with Smad4 and enter nucleus to bind promoter DNA sequence and regulate the target gene expression. / In human embryonic stem (ES) cells, TGF-β/Activin/Nodal signaling has been demonstrated to be the most critical pathways for ES cell self-renewal and maintenance of undifferentiated state. However, in mouse ES cells, its role is yet to be clearly exploited. In this study, we reported the derivation and establishment of mouse Smad3 knockout embryonic stem cell lines (Smad3-/- ES cells). Smad3-/- ES cells maintain normal ES cell morphology and express higher level of mouse ES cell markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1(SSEA1), and display no defect on self-renewal capacity. In addition, both of them show similar expression profiles of pluripotent and lineage marker genes compared to wild type ES cells. However, Smad3 ablation results in transient different expression of germ layer markers during embryoid body (EB) development. The expression of mesoderm lineage marker, like T and GSC, is significantly reduced in the EBs developed by Smad3-/- ES cells compared to EBs formed by wild type ES cells. More interestingly, to investigate the differentiation potential of Smad3-/- ES in vivo, we subcutaneously injected both wild type and Smad3-/- ES cells into nude mice, and observed that Smad3-/- ES cells are prone to grow malignant immature teratomas, while wild type ES cells develop normal mature teratomas. Further characterization of Smad3-/- ES cells demonstrates that depletion of Smad3 increases ES cell proliferation; Smad3-/- ES cells show higher capacity of the anti-apoptosis after UV irradiation and the migration potential of Smad3-/- ES cell differentiated cells is enhanced compared to wild type ES cells in the wound healing assay. Therefore, Smad3-/- ES cells exhibit enhanced malignancy, which may underlie their teratoma malignancy. / Microarray analysis shows that Rif1, a DNA repair factor is highly upregulated in Smad3-/- ES cells. Upregulation of DNA repair factor is found to be associated with invasive tumor. And the expression level of Rif1 is linked to the invasive degree of breast cancer at certain level. Chromatin immunoprecipitation (ChIP) assay and luciferase assay confirm that Smad3 binds to Rif1 promoter region and directly represses its expression; knockdown Rif1 in Smad3-/- ES cells rescues the expression level of Ccnd2 and migration potential to wild type ES cell level. Taken together, all these data suggests that Smad3 may suppress the malignancy of mouse embryonic stem cell formed teratoma through downregulating Rif1 expression in normal condition. / In summary, we reported the establishment of Smad3-/- ES cells and characterization of these cells. We discovered that Smad3-/- ES cells are prone to grow malignant teratomas compared to wild type ES cells. We hypothesized that Smad3 may suppress the malignancy of teratoma through repressing a DNA repair factor, Rif1. This information will not only broaden our general knowledge of malignant teratomas, but also help us to develop strategies to prevent malignant teratoma formation in ES/iPS cell therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Peng. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 149-181). / Abstracts also in Chinese.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Neural stem cells (NSCs) in the adult forebrain are thought to comprise a subpopulation of cells that express glial fibrillary acidic protein (GFAP), termed B cells. These GFAP+ cells generate proliferating neuroblasts that migrate from the lateral ventricle subependyma along the rostral migratory stream to become olfactory bulb interneurons. Based on this lineage, we set out to create a NSC deficient mouse through targeted ablation of dividing GFAP+ cells in vivo. We successfully depleted the GFAP+ cells as seen using an in vitro colony forming assay in multiple kill paradigms, however we were unable to permanently eliminate the multipotent, self-renewing colony forming cells. Instead, the targeted ablation of GFAP+ cells revealed an upstream, GFAP- cell that was induced to proliferate in the presence of leukemia inhibitory factor (LIF). These findings support the hypothesis that a population of GFAP-, LIF responsive cells are the definitive adult NSC upstream of GFAP+ cells.

Neural stem cells (NSCs) in the adult forebrain are thought to comprise a subpopulation of cells that express glial fibrillary acidic protein (GFAP), termed B cells. These GFAP+ cells generate proliferating neuroblasts that migrate from the lateral ventricle subependyma along the rostral migratory stream to become olfactory bulb interneurons. Based on this lineage, we set out to create a NSC deficient mouse through targeted ablation of dividing GFAP+ cells in vivo. We successfully depleted the GFAP+ cells as seen using an in vitro colony forming assay in multiple kill paradigms, however we were unable to permanently eliminate the multipotent, self-renewing colony forming cells. Instead, the targeted ablation of GFAP+ cells revealed an upstream, GFAP- cell that was induced to proliferate in the presence of leukemia inhibitory factor (LIF). These findings support the hypothesis that a population of GFAP-, LIF responsive cells are the definitive adult NSC upstream of GFAP+ cells.