"Recent evidence suggests that delivering human mesenchymal stem cells (hMSCs) to the infarcted heart reduces infarct size and improves ventricular performance. However, cell delivery systems have critical limitations such as inefficient cell retention and poor survival, and lack targeted localization. Our laboratories have recently developed a method to produce discrete fibrin microthreads that can be attached to a needle and delivered to a precise location within the heart wall. We hypothesize that fibrin microthreads will support hMSC proliferation, survival and retention of multipotency, and may therefore facilitate targeted hMSC delivery to injured tissues such as infarcted myocardium. To test this hypothesis, we bundled 100 μm diameter microthreads to provide grooves to encourage initial cell attachment. We seeded hMSCs onto the microthread bundles by applying 50,000 cells in 100 μL of media. The number of cells adhered to the microthreads was determined up to 5 days in culture. Cell density on the fibrin microthreads increased over time in culture, achieving an average density of 730 ± 101 cells/mm2. A LIVE/DEAD assay confirmed that the cells were viable and Ki-67 staining verified the increase in cell number over time was due to proliferation. Additionally, functional differentiation assays proved that the hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that delivering 1 to 4 cell seeded microthread bundles to the infarcted rat myocardium has the potential to produce a positive improvement in mechanical function and these microthreads support hMSC proliferation and survival. Additionally these findings suggest that cell-seeded microthreads may serve a platform technology to improve localized delivery of viable cells to infarcted myocardium to promote functional tissue regeneration. "

The longstanding belief that women are born with a finite ovarian reserve has been debated for over a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs) from adult mammalian ovaries. This rare cell population has now been reported in the mouse, rat, pig, rhesus macaque monkey and humans and, although a physiological role for the cells has not been proven, they do appear to generate oocytes when cultured in specific environments, resulting in live offspring in rodents. The primary aim of this thesis was to verify independently the existence of OSCs in human ovary and determine whether they could be isolated from a large animal model, the cow. The secondary aim was to investigate the cells’ in vitro potential, both to undergo neo-oogenesis and as a model for germ cell development. Putative bovine and human OSCs were isolated from disaggregated adult ovarian cortex using a previously validated fluorescence-activated cell sorting (FACS)-based technique, with cells sorted for externally expressed DDX4 (VASA). Freshly isolated and cultured cells were characterised by analysing their expression of pluripotency and germline markers, using RT-PCR, immunocytochemistry and Western blotting. The in vitro neo-oogenesis potential of the cells was explored by injecting fluorescently labelled cells into fragments of adult ovarian cortex and by forming aggregated artificial “ovaries” with putative OSCs and fetal ovarian somatic cells. Germ cell model experiments comprised treatment of cultured cells with BMP4 and/or retinoic acid (RA), with subsequent quantitative RT-PCR and immunocytochemistry analysis for downstream BMP4- and RA-response genes, and liposomal-mediated transfection of cells with a DAZL overexpression plasmid to assess their meiosis-related gene response. Scarce populations of putative OSCs were retrieved from 5 human samples (aged 13- 40 years) and 6 bovine samples. The cells were cultured long-term for up to 7 months and demonstrated consistent expression of several pluripotency-associated and germline markers at the mRNA and protein level, including LIN28, NANOG, POU5F1 (OCT4), IFITM3 (fragilis), STELLA, PRDM1 (BLIMP1), and C-KIT, indicating their early germline nature. Investigation of neo-oogenesis potential revealed that putative human OSCs were associated rarely with fetal somatic cells in primordial follicle-like structures, but could not be confirmed to have undergone oogenesis. However, like early germ cells, putative bovine and human OSCs were BMP4 and RA responsive, with both species demonstrating significant upregulation of expression of ID1 and bovine cells exhibiting a significant increase in MSX1, MSX2 and the meiotic marker SYCP3 in response to BMP4 and/or RA treatment. Cells could be successfully transfected to overexpress DAZL; however, no significant downstream gene expression changes were observed. This is the first report of putative bovine OSC isolation and corroborates a previous report showing putative human OSC isolation. Although the expression of both stem cell and germline markers indicates the cells have characteristics of OSCs, their capacity to enter meiosis and form functional oocytes has yet to be determined. Putative bovine OSCs, however, show promise as a novel model for investigating germ cell development. If their potential can be harnessed, then OSCs may have a role in clinical applications, for example in fertility preservation, in the future. Future experiments will examine the neo-oogenesis capabilities of the cells further and explore novel cell delivery systems for clinical use.

Allogeneic haematopoietic stem cell transplantation (HSCT) has been used successfully to treat children and adults with high-risk or relapsed hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. When initially developed, allogeneic HSCT was conceived as a method of rescuing patients from the toxic side effects of dose-intensive chemoradiotherapy. Due to transplant-related toxicities, the application of myeloablative allogeneic HSCT has been limited to younger patients without organ dysfunctions. Since the early 1990s many groups of investigators have explored strategies using less intensive preparative regimens that would allow engraftment of hematopoietic progenitor cells from either identical or non-identical donors. These reduced-intensity conditioning (RIC) regimens result in less tissue damage, less inflammatory cytokine secretion, and possibly lower rates of graft-versus-host disease (GVHD) and non-relapse mortality (NRM). Such non-myeloablative approach, or "mini-transplant", has been suggested to benefit older patients as well as in conditions in which traditional myeloablative conditioning regimens are associated with high rates of non-relapse mortality. / Allogeneic HSCT is the only curative therapy for many patients with myeloid malignancies or myelodysplastic syndrome (MDS). The development of reduced-intensity preparative regimens may allow the extension of this form of treatment to older and patients with coexisting medical illness. On the other hand, relapse after transplantation remains the most important cause of treatment failure in patients with refractory acute myeloid leukemia (AML) or MDS, and is associated with poor survival. Evaluation of prognostic factors may help to improve the results of myeloablative and RIC allogeneic HSCT in this group of patients. Furthermore, the impact of comorbidities on outcomes of RIC allogeneic HSCT in this group of patients with refractory AML or MDS needs to be defined. / The application of embryonic and adult stem cells in regenerative and reparative therapies of non-hematopoietic diseases is emerging rapidly. Human umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and mesenchymal progenitor cells. Although clinical experience to date with UCB has focused on hematological application, early preclinical studies support the hypothesis that multipotential stem cells derived from UCB exhibit functional characteristics similar to that observed in adult marrow-derived stem cells in mediating vascular and organ regenerative capabilities. However, the application of these preclinical findings in clinical setting needs to be further studied. Mini-transplant of human UCB may be an effective approach to repair organ damage in patients with non-hematological diseases. / Wong Siu Ming Raymond. / Adviser: Joseph J.Y. Sung. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 187-223). / 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.

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.

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.

The governance of cell fate decisions during development is a fundamental biological problem. An important aspect of this is how cells exit a multipotent state and choose their fates in a correct manner and proportion. To tackle an aspect of this problem, I have focused on 2 multipotent models: one infinite self-renewal pluripotency in an artificial environment, and the other, bipotent progenitors in the context of the mouse embryo. The first model aimed to explore the effects of chromatin-associated factors on the ability of pluripotent mouse Embryonic Stem Cells (ESCs) to self-renew, via monitoring gene expression heterogeneity of key genes. The second model focused on Neural Mesodermal Progenitors (NMPs), a bipotent cell population found in the Caudal Lateral Epiblast (CLE) of mammalian embryos, which contributes to the spinal cord and paraxial mesoderm. The aim here was to derive NMPs in vitro which exhibit similar gene expression patterns and function like their mouse embryo counterpart and study their renewal and differentiation in detail. The first multipotent model explores the effects of chromatin remodelling on cell fate decisions, specifically investigating the consequences of inhibiting the histone acetyltransferase Kat2a on the ESCs fate. I found first, that the effect of Kat2a inhibition depends on the pluripotent state of the cells; cells in a ground state exhibit a resistance to Kat2a inhibition and maintain their pluripotency, whereas cells in a naïve state experience destabilization of their pluripotency gene regulatory network and shift towards differentiation. Second, that Kat2a inhibition in the naïve state results in a decline in the gene expression noise strength contributed by the promoter activation operation, which suggests that when ESCs become lineage-primed their transcriptional noise is constrained. In the bipotent model, the NMPs are identified as cells coexpressing Sox2 and T/Brachyury, a criterion used to derive NMP-like cells from ESCs in vitro. Comparison between the different NMPs protocols stresses that Epiblast Stem Cells (EpiSCs) are an effective source for deriving a multipotent population resembling the embryo Caudal Epiblast (CE), that generates NMPs. Furthermore, self-organization of this CE-like population, resulted in axially organized aggregates. Exploiting the mouse embryo CLE as a reference shows that EpiSCs derived NMPs, monolayers and aggregates, consist of a high proportion of cells with the embryo's NMP signature. Importantly, studying this system in vitro sheds light on the sequence of events which lead to NMP emergence in vivo. On this basis, I conclude that understanding the initial state of cells at a crossroads is important to reveal the limitations it imposes on the cells fate exploration, hence makes it possible to mimic more precisely the fate decision process in vitro.