Opportunistic infection is always a potentially life threatening complication in pediatric oncology patients and hematopoietic stem cell transplant recipients. With the advances in various disease treatment protocols, the overall and event-free survivals of this high risk population improve significantly. In this thesis, the author reported a number of original studies to discuss different strategies in prevention of this serious complication. Firstly, the author demonstrates that pediatric oncology patients are still vulnerable to various vaccine-preventable infectious diseases up to 18 months after stopping chemotherapy. For those vaccine-preventable infectious diseases, pediatric oncology patients can mount a significant and persistent immune response to common inactivated vaccine (namely diphtheria-tetanus-pertussis vaccine). For non-vaccine preventable infectious diseases, regular monitoring of plasma viral load and strategic use of antiviral agents as pre-emptive or prophylactic agent is an effective approach to prevent infection. In hematopoietic stem cell transplant setting, adoptive transfer of acquired immunity from donor to recipient and incorporation of this parameter in donor selection process can be considered. The findings of the studies can be applied to clinical setting. The future direction of our studies includes the immune responses of other common vaccines namely pneumococcal vaccine and pandemic influenza vaccine in high risk population. The role of transfer of donor's varicella zoster immunity in prevention of herpes zoster infection in transplant recipient can be further explored. With the advances in supportive care of our vulnerable patients, the survival rate is expected to be further improved in the future. / by Frankie Wai Tsoi, Cheng. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 193-208). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Embryonic stem (ES) cells, which possess proliferating and differentiating abilities, are a potential source of cells for regenerative medicine. Nowadays, the challenge in using ES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential towards the derivation of a specific cell fate. This study is aimed to establish a simple and efficient method to derive ES cells into neural lineage cells and examine the safety and efficacy of derived cells in a mouse ischemic stroke model. To explore the underlying mechanisms responsible for lineage commitment of stem cells, Notch signaling and serotonin responses are also studied. / In a non-contact coculture system, mouse ES cells (D3 and E14TG2a) were cocultured with the stromal cells MS5 for eight days. On the other hand, human ES cells (H9 and H14) were directly cocultured with MS5 in a contact manner for two weeks. Derived cells were further propagated in a serum-free medium and selected subsequently in a differentiating medium. The cell viability, numbers, phenotypes and lineage-specific gene expression profile were evaluated at stages of induction, propagation and selection. / In vivo, behavioral assessments of ischemic mice after transplantation of mouse ES cell derivatives revealed a significant improvement in spatial learning and memory ability as compared to ischemic mice without cell therapy. Histology of brain sections of transplanted mice demonstrated the migration of BrdU+ cells to the CA1 region of the hippocampus, which was evident of both an increase of pyramidal neuron density and normalized morphology. Teratoma development was found in one out of 17 transplanted mice. / MS5 was noted to express genes encoding neurotrophins and neuroprotective factors. Functional tests showed that MS5 exerted neurotrophism on neuroblastoma cell lines (SK-N-AS, SH-SY5Y, and SK-N-MC) and ES cells. The numbers of viable cells and the proportion of neural subtypes derived from ES cells at three stages of the culture system were significantly higher than those of the control cultures without MS5 induction, respectively. MS5 cocultures generate a relatively higher yield of neural lineage cells but select against the mesodermal and endodermal lineage derivatives. Together with non-contact MS5 coculture, serotonin could further increase the proportion of neural precursors and accelerate maturation of neural progenitor cells in a synergistic manner. During the induction phase with non-contact MS5 coculture, the Notch inhibitor could significantly decrease the number of derived neural precursors and instigate non-neural differentiation. With the supplement of the Notch inhibitor, serotonin could neither promote the expression of neuroectodermal genes nor enhance the proportion of neural precursors in MS5-cocultured ES cells. Notably, in the propagation of undifferentiated human ES cells, Notch signaling was also found to play an active role in maintaining cell survival. / The Notch inhibitor (gamma-secretase inhibitor) and serotonin were supplemented into induction cultures to investigate the roles of Notch signaling and the neurotransmitter serotonin in neural differentiation. For in vivo study, mouse ES cell-derived cells were labeled with BrdU and implanted onto the caudate putamens of mice having undergone transient occlusion of bilateral common carotid arteries and reperfusion to induce cerebral ischemia. Spatial learning and memory ability of transplanted mice were assessed in a water maze system. Histological assessment was also conducted on brain sections of mice three weeks post transplant to examine the migration and homing of implanted cells. / This study describes a simple and efficient differentiation protocol to derive mouse ES cells and human ES cells into neural lineage cells. Derived cells appear to significantly improve cognitive functions in a mouse ischemic stroke model. Data of the study suggest that MS5 cells may exert a neurotrophic effect on ES cells. With MS5 coculture, serotonin synergistically promotes neural commitment and facilitates maturation of derived neural precursors in ES cell cultures. In contact coculture with MS5, Notch signaling is shown to play a role in the directed neural differentiation of human ES cells, whereas in maintenance culture, Notch signaling is also important to cell survival of human ES cells. Thus, Notch signaling through cell-cell interaction may explain, at least partially, the difference between mouse ES cells and human ES cells in cell growth ability when seeded at low cell densities. / Yang Tao. / Adviser: Ho Keung Ng. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: November 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 161-194). / 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.

Homing of hematopoietic stem cells (HSC) to their bone marrow (BM) niches is crucial to clinical stem cell transplantation. However, the molecular mechanism controlling this process remains not fully understood. In this study, we aimed to explore novel regulators of HSC homing through investigating downstream signals and effector molecules of the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis. We further characterized specific functions of targeted regulators by in vitro and in vivo migration/homing assays on human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. / In summary, we have provided the first transcriptome profile of CB CD34 + cells downstream of the SDF-1/CXCR4 axis. We also reported the first evidence that HSC homing was regulated by the tetraspanin CD9. By comparing the homing-related responses of CD34+ to SDF-1 and CTCE-0214, we identified RGS13 as another potential regulator of HSC homing. It is anticipated that strategies for modulating the expressions and functions of CD9 and RGS13 might improve HSC homing to their hematopoietic niches. / To investigate the transcriptional regulation provided by the SDF-1/CXCR4 axis, we performed the first differential transcriptome profiling of human CB CD34+ cells in response to a short-term exposure of SDF-1, and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family proteins, was expressed in CD34 +CD38-/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously downregulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expressions were modulated via the CXCR4, G-protein, PKC, PLC, ERK and JAK2 signals. Pretreatment of CD34+ cells with anti-CD9 mAb ALB6 significantly inhibited SDF-1-mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells were enhanced. Infusion of CD34+ cells pretreated with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID mice. There also appeared a preferential homing/retaining of untreated CD34+CD9+ cells to these niches. Our results indicate that CD9, as a downstream member of SDF-1/CXCR4 signals might possess specific and important functions in HSC homing. / We first investigated the effects of SDF-1 and its analog, CTCE-0214 (a small cyclized peptide analog of the SDF-1 terminal regions), on homing-related properties (chemotaxis, transwell migration, adhesion and actin polymerization) of CB CD34+ cells. Our results demonstrated that both SDF-1 and CTCE-0214 induced a robust actin polymerization response and improved adhesion of CD34+ cells to fibronectin. Unlike SDF-1, CTCE-0214 did not induce a chemotactic response when added to the lower chamber of the transwell system. Addition of CTCE-0214 to the upper chamber significantly improved migration of CD34+ cells to a SDF-1 gradient, but there was no preferential enhancement in the migration of specific colony-forming unit (CFU) progenitors or the more primitive CD34+CD38 -/lo subpopulation. Pre-exposure of CD34+ cells to CTCE-0214 for 4 hours promoted cell migration, whereas SDF-1 pretreatment retarded migration. To dissect the molecular mechanisms leading to the observed functional differences mediated by SDF-1 and CTCE-0214, we investigated whether the two compounds differentially regulated the expression of several known regulators of HSC migration. Flow cytometric analysis revealed that the cell surface expression of CD26, CD44, CD49d, CD49e and CD164 was not changed by either compounds. Exposure to SDF-1, but not CTCE-0214, decreased membrane expression of CXCR4 on CD34+ cells. Addition of CTCE-0214 to the upper chamber inhibited the SDF-1-induced CXCR4 downregulation in both migrated and non-migrated cell population in the transwell setting. Notably, SDF-1 and CTCE-0214 had an opposite effect on the expression level of regulator of G-protein signaling 13 (RGS13), a negative regulator of chemokine-induced responses. Treatment of CD34+ with SDF-1 for 4 hours resulted in a significant increase in RGS13 expression, whereas CTCE-0214 induced a time-dependent decrease in RGS13 expression. Our results provide the first evidence that SDF-1 and CTCE-0214 differentially regulate migration of CD34 + cells, and we speculate that this might be attributed to their differential regulation of CXCR4 and RGS13 expression. / Leung, Kam Tong. / Adviser: Karen Kwai Har Li. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 146-167). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Li Chi-kong. / "September 2002." / Thesis (M.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 223-251). / 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.

Background and purpose. Recent studies have shown that the children with severe nocturnal enuresis, or bedwetting, often have underlying bladder dysfunction as well as various types of brainstem disorders, including arousal inability, a deficient response to startle sounds, or prepulse inhibition. Since the pontine micturition centre is anatomically very close, even overlapping with the nuclei responsible for sleep arousal, one may speculate that there may be close inter-relationships between abnormal bladder function, brainstem dysfunction and sleep-arousal disturbance. We hypothesize that the brainstem function would be changed in response to alteration of bladder function. Using conventional-fill cystometric study, functional magnetic resonance imaging (fMRI) scanning and immunohistochemistry approaches, we propose to characterize the functional changes in the brainstem in response to altered bladder function (i.e. surgically reduced bladder capacity). / Conclusions. Our results showed that bladder dysfunction elicited by surgical reduction in bladder capacity can induce functional changes in the central nervous system. In response to surgical reduction in bladder capacity, deactivation in the vlPAG was detected suggesting that the vlPAG plays a role in the biofeedback of bladder dysfunction. / Data are expressed as the mean +/-1SD unless otherwise specified. Appropriate statistical tests were used for parametric and non-parametric testing between the groups by using the SPSS computer program. In all comparisons a statistical significance level of 95% (p<0.05) was chosen. / Immunohistochemistry study showed a significant decrease in the reaction of dopaminergic neuron in the correspondent regions, suggesting a dopaminergic dependent change in the vlPAG in response to the bladder dysfunction. / In addition, we will explore the use of electroacupuncture (EA), a traditional Chinese therapy that has been broadly used for treatment of bladder functional disorders, to modulate functional changes in the central nervous system. We hypothesize that functional change in various nuclei in the central nervous system that are responsible for micturition control can also be affected by acupuncture treatment. A further aim of this study was to identify brain areas involved in EA to acupoint Chiliao (BL 32), a special acupoint broadly used for the treatment of bladder disorders. / Materials and methods. The study was divided into four parts. Seventy-five male New Zealand white rabbits (14-16 weeks, mean body weight: 3.0-3.5 kg) were used. / Moreover, in this study, we also found a notable activation in the vlPAG and dlPONS in response to the acupuncture stimulations to acupoint Chiliao (BL 32). The changes were identical to that induced by the bladder distension, suggesting a neuromodulation in central nervous system in response to acupuncture therapy. / Results. Study I: Bladder dysfunction elicited by surgical reduction in bladder capacity. Compared to sham animals, the maximum cystometric capacity in animals with RBC operation was markedly decreased at week 4 (35.3+/-8.2 ml vs. 71.6+/-12.9 ml, p<0.05), and week 8 (46.2+/-12.1ml vs. 82.7+/-20.1 ml, p<0.05) groups respectively; however, the maximum voiding detrusor pressure was significantly increased at week 4 (24.4+/-7.0 vs.16.5+/-7.2 cm water, p<0.05) and week 8 (27.7+/-8.p vs. 16.8+/-7.5 cm water, p<0.05) groups respectively, and their corresponding vesical pressure was also enhanced. Other parameters including maximum flow rate, and bladder emptying efficiency did not change significantly in between the sham and RBC subgroups. / Study I: Establishment of the dysfunctional bladder animal model with small bladder capacity. Forty rabbits underwent either sham operation (n=20) or operation for reduced bladder capacity (RBC) (n=20). The sham-operated and the RBC animals were further divided into two groups, i.e. four, and eight weeks after operation (n=10 in each sham and RBC subgroup). A conventional-fill cystometric study was performed on these animals whilst awake in order to evaluate the functional changes (if any) in response to surgical bladder capacity reduction, compared to sham subgroup. / Study II: Detection of functional changes in the brainstem in response to bladder dysfunction. FMRI scanning was performed at the brainstem region in sham-operated and RBC rabbits (12 in each group) at four weeks postoperatively. Bladder stimulation was provided by warm saline (37°C) infusion through a urethral catheter until bladder distended to 70% of the maximum capacity. Area(s) of brainstem activation were assessed by comparing the fMRI scans performed before and after warm saline infusion. / Study II: Functional changes in brainstem in response to bladder dysfunction. FMRI scanning results demonstrated that for the sham animals, there were two activated regions in the brainstem in response to bladder distention, one in the ventrolateral region of periaqueductal gray (vlPAG, 83.3%, 10/12), and the other in the dorsolateral region of pons (dlPONS, 91.7%, 11/12). In animals with RBC operation, only 25% (3/12) showed vlPAG activation compared to 83.3% (10/12) in sham group (p<0.05); however, 83.3% (10/12) of animals showed similar dlPONS activation compared to 91.7% in sham group (p>0.05). / Study III: Catecholaminergic neurotransmitters changes in the brainstem affected areas in response to bladder dysfunction. Brainstem immunohistochemistry results showed that a large number of dopaminergic neuron scattered throughout the whole vlPAG, rarely appeared in dlPAG (dorsolateral region of periaqueductal gray) (lambda +6.0 to +3.0 mm); and an abundant noradrenergic neurons were also accumulated in a restricted region of dlPONS (lambda +3.0 to 0 mm). Compared with the sham group, the density of TH-positive neurons in the vlPAG was significantly decreased in RBC group (38.38+/-4.71 vs.51.57+/-8.38/field, p<0.05); for the another region of dlPONS, although the density of TH-positive neurons decreased slightly in RBC group compared to sham group, the results showed no statistical difference between groups (106.89 +/- 21.61 vs.120.61 +/- 17.03/field, p>0.05). / Study III: Investigation of the catecholaminergic neurotransmitters changes in brainstem affected areas in response to bladder dysfunction. After fMRI examination, all animals were euthanized, and their brainstems were collected for immunohistochemistry study with tyrosine hydroxylase, dopamine-beta-hydroxylase assays to investigate the changes of catecholaminergic neurotransmitters including dopaminergic, noradrenergic and adrenergic in response to bladder dysfunction elicited by surgical reduction in bladder capacity. / Study IV: Electroacupuncture modulation via acupoint Chiliao (BL 32) on bladder and the brainstem activated sites. FMRI study showed that the two brainstem micturition centers of the vlPAG (72.7%, 8/11) and dlPONS (82.8%, 9/11) can be activated by EA on BL 32, and there were no significant difference compared with stimulation of bladder distention (72.7% vs. 83.3% in vlPAG and 82.8% vs. 91.7% in dlPONS respectively, p>0.05). Urodynamic results showed that, bladder contraction obviously evoked in response to EA on BL 32 (ON-EA state) compared to before EA state (OFF-EA state), displaying a significantly increased detrusor pressure (14.04+/-3.17 vs. 8.19+/-0.69 cm water, p<0.05) and vesical pressure (13.48+/-1.61vs. 7.90+/-0.81 cm water, p<0.05). In addition, dissection of BL 32 showed that the stem of S1 and S2 pass through the region 0.5 cm around the acupuncuture needle. / Study IV: Investigation of electroacupuncture modulation via acupoint Chiliao (BL 32) on bladder and the brainstem activated sites. FMRI scanning and urodynamic evaluation were performed respectively during ON/OFF EA on acupoint Chiliao (BL 32) on sham-operated animals (n=12) at four weeks post-operation. At last, dissection of acupoint BL 32 was performed on seven sham animals. / We also found that surgically induced bladder dysfunction, mainly displaying as reduced bladder capacity and maximum voiding detrusor pressure enhanced, was elicited at four to eight weeks after the surgical reduction in bladder capacity in rabbit. / Xiang Bo. / "August 2006." / Adviser: Chung Kwong Yeung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1551. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 161-196). / 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. / School code: 1307.

Human pluripotent stem cells (hPSCs) such as, human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS) are a valuable resource to generate bespoke cell types for a number of therapeutic applications involving cell therapy, drug screening and disease modelling. The overarching goal of this project was to generate a set of transgenic tools by gene targeting and genetic modification of hESCs for applications in stem cell biology such as the in vitro isolation, analyses and derivation of lineage specific cell types. The transgenic tools generated in this study were designed and tested in particular for the human 3rd pharyngeal pouch epithelium (3PPE) like cells and its derivatives, namely the thymus and parathyroid, which are key organs involved in T-cell development and calcium homeostasis respectively. The forkhead transcription factor FOXN1 is considered a master regulator of the development of the thymic epithelium (TEC), the major functional component of the thymic stroma, which is intimately involved in T-cell differentiation. So, to facilitate the prospective isolation of FOXN1 expressing TECs, gene targeting was employed to place a fluorescent reporter and a lineage selection antibiotic resistance gene under the direct control of the endogenous FOXN1 promoter. To date, I have not been able to detect either the fluorescent reporter, or FOXN1 expression using published directed differentiation protocols, but only what can be deemed as precursors expressing the cytokeratin K5 and other markers associated with the development of the thymus and parthyroid from 3PPE. The lack of endogenous FOXN1 activation was observed in both the unmodified parent and the targeted FOXN1 knock-in human ES lines. Further, over-expression of FOXN1 cDNA during the differentiation protocol did not result in the activation of endogenous FOXN1. So, the results evinced in this study could be due to a number of reasons such as, technical issues associated with transference of the published protocols to the cell lines used in this study, differences in hESC lines, and effects of different hESC culture methods and practices. The homeobox gene HOXA3 is expressed in the 3PPE during development. So, a HOXA3 transgenic reporter hESC line could be an invaluable tool for prospective isolation of in vitro derived 3PPE like cells. The reporter was generated by Piggy Bac transposase mediated transposition of a HOXA3 containing Bacterial Artificial Chromsome (BAC) in the FOXN1 knock-in human ES line. To date, this is biggest reported cargo that has been successfully transposed in human ESCs. Moreover, this is the first lineage specific double reporter transgenic hESC line that has been reported for this lineage. This HOXA3 reporter line was then used to isolate and enrich for HOXA3 expressing 3PPE like cells with very high efficiencies during the directed differentiation of hESCs, thus demonstrating the key objective of this transgenic hESC line for this study. In a novel parallel approach, I have conceived, designed and generated transgenic hESCs lines capable of inducible and constitutive over-expression of key transcription factors involved in the development of 3PPE and its derivatives, the thymus and parathyroid. The objective of the said over-expression hESC lines was to interrogate if such a system could elicit morphological and gene expression changes in hESCs following over-expression. By testing the chosen panel of transcription factors in hESCs, I was able to detect cells expressing FOXN1 and GCMB, which are key markers of TECs and PTECs. Further, I have isolated an expandable population of cells expressing markers analogous to their in vivo counterpart found in the 3PPE of a developing mouse embryo around E9.0. The in vivo potency of these in vitro derived 3PPE like cells is yet to be ascertained. Nevertheless, transgenic constructs generated in this experiment could also be tested during future attempts at the differentiation of hESCs to TECs and PTECs, and also used as a basis for future studies involving the direct conversion of patient specific fibroblasts to 3PPE like cells and its derivatives. In summary, several transgenic tools developed in this project, namely the FOXN1 knock-in transgenic hESC line, FOXN1-HOXA3 double transgenic hESC line, over-expression 3PPE transgenes and hESC transgenic lines, and results from the deployment of these tools provide a foundation, from which protocols to generate functional TECs and PTECs can be refined and optimised. These transgenic hESC lines also provide a tractable model, which could be used to interrogate the development of human TECs and PTECs from human 3PPE, and identify hitherto unknown early events in their development in an in vitro reductionist setting.

Kwok, Po Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-130). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 中文摘要 --- p.v / Acknowledgements --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Table of Abbreviations --- p.xii / Contents --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- The Biology of Human Umbilical Cord Perivascular Cells (HUCPVs) and Their Potential Applications in Tissue Regeneration / Chapter 2.1 --- INTRODUCTION --- p.5 / Chapter 2.1.1 --- Stem cells --- p.5 / Chapter 2.1.2.1 --- Embryonic stem cells --- p.6 / Chapter 2.1.2.2 --- iPS cells --- p.7 / Chapter 2.1.2.3 --- Somatic stem cells --- p.8 / Chapter 2.1.3 --- Mesenchymal stem cells --- p.9 / Chapter 2.1.4 --- Pericytes --- p.11 / Chapter 2.1.5 --- CD146 positive MSCs --- p.12 / Chapter 2.1.6 --- Human umbilical cord perivascular cells (HUCPVs) --- p.13 / Chapter 2.1.7 --- The biology of stem cell microenvironment (niche) --- p.14 / Chapter 2.1.8 --- Current applications of HUCPVs --- p.17 / Chapter 2.1.9 --- Regenerative medicine --- p.17 / Chapter 2.1.10 --- Applications of stem cells in bone regeneration --- p.19 / Chapter 2.2 --- MATERIALS AND METHODS --- p.22 / Chapter 2.2.1 --- Cell culture --- p.22 / Chapter 2.2.2 --- Preparation of Human Umbilical Cord Perivascular (HUCPV) cells --- p.22 / Chapter 2.2.2.1 --- Isolation of Human Umbilical Cord Perivascular (HUCPV) cells from human umbilical cord --- p.22 / Chapter 2.2.2.2 --- Purification of HUCPV cells --- p.23 / Chapter 2.2.3 --- Immunocytochemsitry --- p.24 / Chapter 2.2.4 --- Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) --- p.25 / Chapter 2.2.4.1 --- Isolation of total cellular RNA --- p.25 / Chapter 2.2.4.2 --- Complementary DNA (cDNA) synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase chain reaction (PCR) --- p.26 / Chapter 2.2.5 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.30 / Chapter 2.2.6 --- In vitro differentiation assays --- p.33 / Chapter 2.2.6.1 --- Osteogenic differentiation --- p.33 / Chapter 2.2.6.2 --- Adipogenic differentiation --- p.33 / Chapter 2.2.6.3 --- Chondrogenic differentiation --- p.34 / Chapter 2.2.6.4 --- In vitro chondrogenic differentiation on gelfoam® --- p.34 / Chapter 2.2.7 --- Cytochemistry staining --- p.35 / Chapter 2.2.7.1 --- Alkaline Phosphatase staining --- p.35 / Chapter 2.2.7.2 --- Alizarin Red S staining --- p.35 / Chapter 2.2.7.3 --- Oil Red O staining --- p.36 / Chapter 2.2.7.4 --- Alcian Blue staining --- p.36 / Chapter 2.2.8 --- Scanning electron microscopy (SEM) --- p.37 / Chapter 2.2.9 --- Transmission electron microscopy (TEM) --- p.37 / Chapter 2.2.10 --- Paraffin tissue embedding --- p.38 / Chapter 2.2.10 --- Haematoxylin and Eosin staining --- p.38 / Chapter 2.3 --- RESULTS --- p.40 / Chapter 2.3.1 --- Isolation and purification of HUCPVs --- p.40 / Chapter 2.3.2 --- Osteogenic differentiation of HUCPVs under normoxia --- p.41 / Chapter 2.3.3 --- Osteogenic differentiation of HUCPVs under hypoxia --- p.42 / Chapter 2.3.4 --- Adipogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.5 --- Chondrogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.6 --- Chondrogenic differentiation of HUCPVs on gelfoam® --- p.44 / Chapter 2.4 --- DISCUSSION --- p.59 / Chapter Chapter 3 --- Calcium and Calcium-sensing Receptor (CaSR) in osteogenesis / Chapter 3.1 --- INTRODUCTION --- p.62 / Chapter 3.1.1 --- Metabolism of calcium --- p.62 / Chapter 3.1.2 --- Calcium-sensing receptor --- p.64 / Chapter 3.1.2.1 --- The molecular structure of calcium-sensing Receptor (CaSR) --- p.64 / Chapter 3.1.2.2 --- The expression pattern of calciumsensing receptor (CaSR) --- p.67 / Chapter 3.1.2.3 --- The physiological function of calcium-sensing receptor in different tissues or organs --- p.68 / Chapter 3.1.2.4 --- Regulatory role of calcium-sensing receptor in calcium sensing and homeostasis --- p.71 / Chapter 3.1.2.5 --- The role of calcium-sensing receptor in diseases --- p.72 / Chapter 3.1.2.6 --- Genetic animal models targeting calciumsensing receptor --- p.73 / Chapter 3.1.2.7 --- Calcium-sensing receptor in mesenchymal lineage Differentiation --- p.76 / Chapter 3.1.2.8 --- The role of calcium-sensing receptor in the skeleton --- p.76 / Chapter 3.1.3 --- Calcium-sensing receptor related pathway --- p.78 / Chapter 3.1.3.1 --- Cyclic AMP pathway --- p.78 / Chapter 3.1.3.2 --- Cyclic AMP response element-binding protein (CREB) --- p.80 / Chapter 3.2 --- MATERIALS AND METHODS --- p.83 / Chapter 3.2.1 --- Preparation of primary mouse osteoblasts (MOB) from long bone --- p.83 / Chapter 3.2.2 --- Preparation of primary mouse osteoblasts (CMOB) from calvaria --- p.84 / Chapter 3.2.3 --- Immunocytochemistry --- p.84 / Chapter 3.2.4 --- Osteogenic differentiation --- p.85 / Chapter 3.2.3 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.85 / Chapter 3.2.4 --- Cell proliferation measurement by BrdU ELISA (colorimetric) assay --- p.85 / Chapter 3.2.5 --- Western blotting analysis --- p.86 / Chapter 3.2.5.1 --- Preparation of the protein lysate --- p.86 / Chapter 3.2.5.2 --- Protein quantitation --- p.86 / Chapter 3.2.5.3 --- SDS-PAGE --- p.87 / Chapter 3.2.5.4 --- Protein transfer --- p.87 / Chapter 3.2.5.5 --- Immunodetection --- p.88 / Chapter 3.2.6 --- cAMP EIA assay --- p.89 / Chapter 3.3 --- RESULTS --- p.91 / Chapter 3.3.1 --- "Expression of CD 146 and CaSR in HUCPVs, primary mouse long bone osteoblasts and MC3T3-E1 cell line" --- p.91 / Chapter 3.3.2 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under normoxia --- p.91 / Chapter 3.3.3 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under hypoxia --- p.92 / Chapter 3.3.4 --- The effect of calcium treatment on cell proliferation in primary mouse long bone osteoblasts --- p.93 / Chapter 3.3.5 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse long bone osteoblasts --- p.94 / Chapter 3.3.6 --- The effect of calcium treatment on calcium-sensing receptor expression in HUCPVs --- p.95 / Chapter 3.3.7 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse calvarian osteoblasts --- p.96 / Chapter 3.3.8 --- The effect of calcium treatment on cyclic AMP levels in primary mouse long bone osteoblasts --- p.97 / Chapter 3.4 --- DISCUSSION --- p.117 / Chapter Chapter 4 --- General Discussions --- p.121 / References --- p.124 / Appendices --- p.131

肌腱修復一直是一個難題，因為依靠現在的治療很難將肌腱功能恢復到正常水平，近年來肌腱幹細胞的分離和發現為肌腱修復提供了新的策略。但是在利用肌腱幹細胞修復肌腱之前，我們應該瞭解肌腱幹細胞的哪些方面呢？ / 不同來源的成體幹細胞雖然具備相似的幹細胞特性，但是他們仍然具有組織特異性和功能的差異。這就意味選擇合適的細胞來源對於肌腱再生和肌腱組織工程有特殊意義。所以我們認為與骨髓間充質幹細胞相比，肌腱幹細胞具備特殊的幹細胞特性。迄今為止，還沒有研究比較肌腱幹細胞和骨髓間充質幹細胞的幹細胞特性。臨床應用要求幹細胞在體外增殖培養，體外的微環境也會影響幹細胞的幹性和治療潛能，所以我們還並不清楚肌腱幹細胞的幹性在體外培養中維持多久。成功的幹細胞治療需要深入理解組織特異性幹細胞的體內特徵和他們在組織修復中的作用。肌腱幹細胞的体内特徵还有没详细研究过，而且也不知道這些內源性幹細胞是否參與肌腱修復。 / 所以為了更好地利用肌腱幹細胞進行肌腱修復，本研究的總體目標是比較肌腱幹細胞和骨髓間充質幹細胞的幹細胞特性，同時從臨床角度考慮研究肌腱幹細胞體外幹性的維持。進一步研究鑒定肌腱幹細胞的體內特徵，並且探索他們在肌腱癒合中的作用。本研究將會探討我們應該瞭解關於肌腱幹細胞的體內和體外特性。 / 在第一部分研究中， 我們從同一隻GFP大鼠中分離出肌腱幹細胞和骨髓間充質幹細胞。經過比較，我們發現肌腱幹細胞与骨髓間充質幹細胞相比具备更高的克隆形成能力，增殖速度，更強的多向分化能力和更高的肌腱相关的基因表达。所以肌腱幹細胞表現出更好的幹性，可能是比骨髓间充质干细胞更好的用于肌腱再生的细胞来源。 / 在第二部分研究中，我們發現肌腱幹細胞伴隨體外傳代培養細胞衰老β-半乳糖苷酶活性增高，而同時間充質幹細胞標誌物和多向分化能力降低，所以研究人員和臨床醫生在利用肌腱幹細胞進行組織工程時需要考慮在體外傳代培養中他們的幹性的變化。 / 在第三部分研究中，IdU標記滯留細胞方法用於在體內標記幹細胞。我們發現休眠的幹細胞以IdU標記滯留細胞的形式存在於肌腱中，相比肌腱本體更多標記滯留細胞位於和肌腱腱鞘和肌腱骨結合部位。其中我們發現在肌腱腱鞘中的標記滯留細胞位於血管周圍的微環境血管，所以血管周圍的微環境可能是肌腱幹細胞來源之一。肌腱損傷后，位於損傷區域的標記滯留細胞的數量，增殖標誌物，肌腱相關標誌物， 多能性標誌物，和微血管相關標誌物都有明顯增加，意味著標記滯留細胞可能通過遷移，增殖和分化參與肌腱修復。 / 綜上所述，我們的結果為理解肌腱幹細胞的體外幹性特徵和在體外培養中的幹性變化以及体内肌腱幹細胞的鑒定提供了新的解釋，這有利于未來促進肌腱幹細胞的組織工程應用於肌腱修復。 / Tendon repair remains a great challenge due to current therapies cannot restore normal tendon function. Tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and characterized in vitro in recent studies and provide new strategies for tendon repair. But what should we know about tendon stem cells before we use them to repair injured tendon? / Although stem cells that originate from different tissues share some common stem cell characteristics, they might also exhibit some tissue unique properties and hence functional differences. Therefore, we hypothesized that TDSCs have unique stemness properties compared with bone marrow-derived stem cells (BMSCs). There has been no study to compare the stemness properties of TDSCs and BMSCs. Clinical applications often require the in vitro expansion of stem cells. In vitro microenvironment also affects the stemness properties and therapeutic potential of stem cells. It is not clear if the stemness properties of TDSCs can be maintained and how long that they can be preserved during in vitro expansion. Moreover, successful stem cell-based repair therapies will require an understanding of tissue specific stem cells in vivo and their roles in the tissue repair. Tendon stem cells have not been described in details in vivo and it is unknown whether these endogenous stem cells participate in the tendon healing. / Therefore, in order to better make use of TDSCs for tendon repair, the objective of this study is to characterize the stemness properties of TDSCs compared with BMSCs and also to investigate the stemness limitation of TDSCs during culture in vitro for clinical use purpose. Furthermore, this study aims to identify the putative tendon stem cells in vivo and their role in tendon healing. This study would tell how much we should know about tendon stem cells in vitro and in vivo. / In the first part of the study, TDSCs and BMSCs were isolated from the same GFP Sprague-Dawley rat. TDSCs showed higher mensenchymal and pluripotent stem cell makers; clonogenicity; proliferative capacity; and tenogenic, osteogenic, chondrogenic, and adipogenic differentiation markers and multi-lineage differentiation potential than BMSCs. Compared with BMSCs, TDSCs shows great stemness properties and might be an alternative cell source for tendon regeneration. / In the second part of this study, the senescence-associated β-galactosidase activity of TDSCs increased while their stem cell-related marker expression and the multi-lineage differentiation potential decreased during in vitro passaging. It suggests that researchers and clinicians need to consider the changes of stemness properties of TDSCs when multiplying them in vitro for tissue engineering. / In the third part of the study, IdU label-retaining method was used for the labeling of stem cells in vivo. We have identified quiescent stem cells as IdU label retaining cells (LRCs) at the peritenon, tendon mid-substance and tendon-bone junction. More LRCs were found at the peri-tenon and tendon-bone junction compared to the mid-substance. Some LRCs could be identified in the peri-vascular niche in the peri-tenon, suggesting that peri-vascular niche is one source of tendon stem cells. After injury, The LRC number and the expression of proliferative, tendon-related, pluripotency and pericyte-related markers in LRCs in the window wound increased, indicating that LRCs might be involved in tendon repair via cell migration, proliferation and differentiation. / In conclusion, our results have provided new findings about the understanding of tendon-derived stem cells including their stemness properties and their changes during the in vitro culture, as well as in vivo identity of tendon stem cells, which might facilitate the application of TDSCs in tissue engineering for tendon repair in the future. / 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. / Tan, Qi. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 130-162). / Abstracts also in Chinese.

The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

Mouse embryonic stem (ES) cells have played a crucial role in studying developmental processes and gene function in vivo. They are extremely useful in the generation of transgenic animals as they can be genetically manipulated and subsequently microinjected into blastocyst stage embryos, where they combine with the inner cell mass and contribute to the developing embryo. Some of the resulting pups are chimaeric, consisting of a mixture of cells derived from the host blastocyst and the injected ES cells. We have identified several ES cell clones arising from gene targeting experiments with an impaired capacity to generate viable chimaeras. When injected into blastocysts, these clones cause embryonic death during mid to late gestation, suggesting that the cells are able to contribute to the embryo but interfere with normal embryonic development. The aim of this work was to identify the underlying changes in the transcriptome, epigenome or cell surface markers that have occurred in these compromised ES cells and to further define the developmental phenotype of the chimaeric embryos. Different stages during development were analysed and whereas there was little difference in embryonic death at gestational day e13.5, there was a significant decrease in embryos surviving to gestational day e17.5. Additionally, severe haemorrhaging was observed in all the dead embryos and small foci of haemorrhaging could also be seen in a number of embryos that were still alive. This was also observed at e13.5, albeit to a less severe extent. Using RNA sequencing to discover differences in the transcriptome between control ES cells and the compromised ES cells, five genes were identified that were downregulated in the compromised cells. Four of these, Gtl2, Rtl1as, Rian and Mirg are all located in the imprinted Dlk1-Dio3 region on chromosome 12 and are normally expressed from the maternal genome. This pattern was also validated in tissues from e17.5 chimaeric embryos. The expression of this locus is to a large extent regulated by a differentially methylated region located approximately 13kb upstream of the Gtl2 promoter, the IG-DMR. Whereas this is usually only methylated on the paternal copy, in the compromised ES cells both the paternal and the maternal copy were fully methylated, likely causing the silencing of Gtl2, Rtl1as, Rian and Mirg. Using the DNA methyltransferase inhibitor 5-azacytidine, expression of Gtl2 could be rescued. Injection of those 5-azacytidine treated cells into blastocysts did partially rescue the embryonic lethal phenotype. Additionally, cell surface markers were analysed in a phenotypic screen using phage display. NGS analysis of the phage outputs indicates that there may be additional differences in cell surface markers between the control and compromised ES cell clones, but their specific details remain to be identified. Overall, we have identified the maternally expressed genes of the Dlk1-Dio3 region as markers that can distinguish between ES cells with normal or compromised developmental potency and propose to include these genes in the pre-blastocyst injection screening routine for experiments involving the production of chimaeras or genetically modified mouse strains.