Pluripotent stem cells hold great promise in regenerative medicine. Theoretically, a variety of tissues can be generated from this progeny. The production of tailor-made stem cells for individualized patient treatment is the ultimate goal of stem cell based therapy. Human induced pluripotent stem cells (iPSCs) hold the precious key to success and promote the clinical application of stem cells. By reprogramming somatic cells, pluripotent stem cells can be generated in a patient-specific manner and subsequently differentiated into specific tissue for regeneration. Nonetheless exposure of hiPSCs to animal feeder cells and serum during generation and maintenance imposes a risk of transmitting animal pathogens to human subjects, thus hindering their potential therapeutic application. In addition, the efficacy of iPSC generation is < 1% of total somatic cells used. The first part of the study focused on the development of improved methods to produce a more efficient xenogen-free culture system to produce more clinically compatible iPSCs. Specific tissue or cells derived from stem cells may offer a solution and cell therapy using endothelial cells and their progenitors may be possible in treatment of severe cardiovascular diseases. In theory, endothelial cells can be generated from different sources of progenitor cells although no direct comparison of these various derived endothelial cells (ECs) has been reported. Thus in the second part of the study, the functional and physiological properties of BM, ESC and iPSC-ECs will be evaluated to determine their therapeutic potential in ischemic disease. A mouse hind limb ischemia model was used to assess and monitor neovascularization by the derived ECs. The results can provide further insight to evaluate the possibility of using iPSCEC as the cell source for patient-specific treatment. Use of pluripotent stem cells is a promising approach in therapeutic angiogenesis although numerous hurdles continue to hamper their widespread clinical use. Conditioned medium derived from progenitor cells may be another possible strategy in the treatment of ischemic diseases such that direct cell transplantation is avoided. Conditioned media produced from ex vivo culture of endothelial cells contain a combination of angiogenic factors that can be applied to promote neovascularization in ischemic tissue. Nonetheless the efficacy of this angiogenic application is unknown. The third part of the study focused on the potential application of EC-derived conditioned media in the treatment of ischemic disease using a mouse hind limb ischemia model. Some cardiovascular risk factors such as diabetes might affect endothelial cell function such that autologous application of ECs and their conditioned media is not feasible. A human embryonic stem cell line may offer and alternative means to obtain stable quality ECs and conditioned medium for therapeutic use. In summary, advances in stem cell technology hold great promise for the treatment of cardiovascular disease, further improved by the generation of patient-specific stem cells using iPSC technology. Vascular cells can be generated from different sources of stem cells with similar angiogenic properties and may be used in the treatment of ischemic diseases. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The fundamental principle of tissue engineering is to use appropriate cell source, combined with scaffolds and bioactive factors to develop tissue constructs which restore, maintain or improve tissue function. There is increasing data emphasizing the importance of mechanical signals and extracellular matrix (ECM) proteins presented by the scaffold in determining stem cell fate/functions which are critical to tissue construct maturation and success of stem cell-based therapies. Cell-matrix adhesions are one of the major mechanosensing machineries cells use to convert information provided by ECM ligands and mechanical signals presented by scaffolds into intracellular biochemical signaling cascades which lead to particular functional responses. Therefore, understanding how ECM ligands and mechanical signals regulate cell-matrix adhesion formation and activation of associated intracellular signaling proteins is fundamental to rational design of biomaterial and loading protocol for optimal cell functional responses in tissue constructs. In this study, we attempted to understand the regulatory effects of external mechanical signal and exogenous ECM protein on cell-matrix adhesion formation and associated intracellular signaling proteins of human mesenhymal stem cells, and in particular, to test the hypothesis that mechanical stimulation or exogenous ECM protein can lead to adhesion maturation into 3D-matrix adhesions in 3D collagen environment. We used microencapsulation technique to embed cells in 3D collagen environment, forming disc-shaped hMSC-collagen constructs. By immunofluorescent staining and confocal microscopy, we visualized changes in size, morphologies and molecular composition of the adhesions. First of all, 2D adhesions of hMSCs were characterized. We showed that hMSCs form well-organized αv integrin-based focal adhesions and fibrillar adhesions in 2D culture. To investigate the regulatory effects of mechanical signals on adhesion signaling and maturation, we used micromanipulator-based loading device to impose dynamic compression to hMSC-collagen constructs. We found that dynamic compression lead to enlargement of integrin αv adhesions which recruit focal adhesion kinase (FAK), vinculin and extracellular signal-regulated kinase (ERK). In addition, FAK was activated at enlarged integrin αv adhesions and translocated to peri-nuclear region after compression, suggesting that loading induces activation of FAK signaling pathways through increased integrin αv clustering. Moreover, we demonstrated that dynamic compression can induce 3D-matrix adhesion formation, indicating the role of external force in integrin α5-based adhesion maturation in 3D collagen environment. We explored the effect of exogenous ECM proteins on adhesion maturation of hMSCs by adding fibronectin into cell-collagen mixture during fabrication of collagen constructs. Our results demonstrated that the exogenous fibronectin can induce α5 integrin-based adhesion maturation into 3D-matrix adhesions in our collagen constructs in a dose-dependent manner. This study demonstrated that the effect of external mechanical signals and exogenous ECM ligands on adhesion signaling and maturation of hMSCs in 3D collagen environment. Our findings contribute towards mechanobiology of hMSCs in 3D context. In particular, our results showed that exogenous proteins or external loading can lead to 3D-matrix adhesion formation, which may serve as a potential way to enhance biological functions of hMSCs in collagen constructs, facilitating stem cell-based therapies. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Nasopharyngeal carcinoma (NPC), endemic in southern China and Southeast Asia, was ranked 7th as the most common new malignancy in Hong Kong. Metastatic and recurrent NPC have a poor prognosis despite recent advancement in medicine. Inactivation of tumor suppressor genes (TSGs) through the loss of chromosomal regions is frequently reported in NPC. With the recent discovery of cancer stem cells (CSCs), which are refractory to current therapies, a new paradigm shift in the perspective of cancer therapy development has emerged. For the first time, this study aims to unravel the complexity of NPC tumorigenicity for identifying more effective targets by studying the possible interplay between CSCs and TSGs. NPC cell lines had different expression profiles of CSC markers, confirming not all CSC markers are applicable to every tumor type. Although CD24/CD44 were expressed in NPC, however CD24+CD44+ NPC cells did not initiate tumor formation. By utilizing a cancer hybrid cell model with a transferred single copy of chromosome 3, physiological β-catenin up-regulated core stem cell markers through the activation of Wnt signaling pathway in NPC. Moreover, the down-regulation of β-catenin suppressed chemoresistance and inhibited cell proliferation, colony formation, angiogenesis, the epithelial-mesenchymal transition (EMT) process, and the tumor microenvironment factors. Amongst the tumor microenvironment factors, chemokine Rantes and matrix metalloproteinase were down-regulated when β-catenin was knocked down. Therefore, activation of Wnt signaling provide an alternative platform for identifying putative CSCs in NPC, leading to the identification of several prospective CSC markers in NPC. Down-regulation of IKBB, a NF-KB inhibitor, in the majority of NPC patients indicated that IKBB plays a prominent role as a TSG in NPC. In this study, IKBB was found to exert its tumor suppressive functions by abrogating tumor formation, cell migration, invasion and angiogenesis. Angiogenic factors, including Rantes, Upar, IL6 and IL8, were significantly down-regulated by IKBB. In addition, IKBB also suppressed the binding activity of NF-KB. The involvement of Akt/Gsk-3β pathways was also observed. Taken together, IKBB regulated NPC tumorigenesis through NF-KB/Akt/Gsk-3β and interaction with tumor microenvironment. Collectively, this study demonstrated that the progression of NPC is not simply initiated by a single signaling pathway, but a dynamic and complex interplay between multiple signaling networks and the tumor microenvironment. NPC tumorigenesis is hypothesized to be driven by orchestrated interaction between CSCs and TSGs through crosstalk with the tumor microenvironment. Amongst the major players in the tumor microenvironment, Rantes/CCL5, IL6, and the matrix metalloproteinase are envisaged to induce angiogenesis, EMT, and metastasis in NPC. This dynamic intercommunication between CSCs and tumor suppressor IKBB signaling networks may shed better insights on modulation of the major hallmarks of cancer in orchestrating NPC development. The modulation of the major hallmarks of cancer by CSCs and IKBB, a TSG, involves promotion of aberrant proliferation, enhancement of invasion and metastasis, induction of angiogenesis, circumvention of tumor suppressors, and prevention of cell death. Taken together, selective and synergistic co-targeting these signaling networks and the tumor microenvironment will provide a more effective new modality of treatments for NPC. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Skeletal system plays a crucial role in our life. Skeletal diseases and disorders unlike cancer, are not fatal, but affect the quality of our life. Cell-based therapeutic strategies to generate targeted desired cell types for repair or replacement of damaged skeletal tissues are ideal regenerative medicines. Because of the heterogeneous cell types generated from embryonic and mesenchymal stem cells, the ability of progenitor population to differentiate into a target cell type appear to be a better alternative for tissue regeneration. Osteo-chondroprogenitors uniquely co-expressing Sox9 and Runx2 with dual differentiation potential to become chondrocytes and osteoblasts is a progenitor cell which is suitable for cell based therapy of bone disease. Therefore, developing effective strategies to generate sufficient quantities of osteo-chondroprogenitors are essential. Toward this, we took advantage of two lineage conversion approaches. The first strategy was to interrogate the ability of osteoblasts to be reprogrammed into induced pluripotent stem (iPS) cells and another one was to use defined transcription factors to induce chondrocyte lineage from skin fibroblasts. The selection of osteoblasts is based on the fact that it is originally derived from osteo-chondroprogenitor lineage and the stochastic events of iPS induction might revert osteoblasts first to their progenitor state before becoming pluripotent. The second approach is based on a previous report using three transcription factors (Sox9, Klf4 and c-Myc) to reprogramme skin fibroblasts into chondrocyte lineage. Our aim is to examine whether osteo-chondroprogenitors would be formed during the two reprogramming processes using Sox9-EGFP knock-in mice as a reporter. We reasoned that osteoblasts can be reprogrammed into iPS cells by four Yamanaka’s factors with pluripotency as shown by their ability to form teratomas and contribute to chimeric embryos. However base on the limitation of selector marker of osteo-chondroprogenitor we still cannot capture this progenitor during iPS reprogramming. And because of the pluripotency potential, pluripotent reprogramming approach also brings high risk of teratoma formation. Therefore our second objective was performed to examine whether osteo-chondroprogenitors would be formed during lineage reprogramming. Transient appearance of Sox9-EGFP/Runx2+ve cells was observed in the intermediate stage of over 14 days of chondrocyte lineage induction from skin fibroblasts by Sox9, klf4 and c-Myc. Cells expressing Sox9-EGFP/Runx2+ve showed typical molecular markers of osteo-chondroprogenitors. In vitro and in vivo differentiation assays demonstrated that Sox9-EGFP/Runx2+ve cells can differentiate predominantly into osteoblasts and chondrocytes. Taken together our data indicate that cells with osteo-chondrogenic potential could be generated by defined transcription factors-mediated reprogramming processes. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Esophageal squamous cell carcinoma (ESCC), the major histological subtype of esophageal cancer, is one of the most common malignancies with poor prognosis in the world. Despite continued development of diagnosis and treatment, ESCC remains the sixth leading cause of cancer death worldwide. Current treatment regimens in ESCC are often characterized by ineffectiveness and poor selectivity. Therapeutic methods directed at cancer-associated genes or cancer stem cells (CSCs) may be effective approaches to cure this deadly cancer. Therefore, this study aims to identify specific ESCC-related genes and cancer stemness genes which help us to develop new targeted agents to achieving objective, long-lasting therapeutic responses in ESCC. To obtain an accurate overview of genetic changes occurring in ESCC patients, our group performed microarray-based mRNA expression profiling and high-throughout transcriptome sequencing (RNA-Seq) to compare differentially expressed genes between ESCC tumors and their corresponding non-tumorous tissues. Prostate stem cell antigen (PSCA) was considered to be a candidate of primary interest due to significantly reduced expression in both microarray and RNA-Seq data. In this study, we examined the role of PSCA on the pathogenesis of esophageal cancer. Our results showed that PSCA was frequently down-regulated in ESCC. Its expression was negatively regulated by transcription factor SOX5. Also, we provided evidence that down-regulation of PSCA was associated with poor clinical outcomes of patients with ESCC. Both in vitro and in vivo assays revealed that PSCA could arrest cell cycle progression and promote differentiation. To further elucidate the mechanism involved in biological function of PSCA, we performed co-immunoprecipitation and mass spectroscopy to identify proteins that associate with PSCA. This study found that RB1CC1, a key signaling node to regulate cellular proliferation and differentiation, interacted specifically with PSCA both in vitro and in vivo. Binding of PSCA and RB1CC1 in cytoplasm resulted in stabilization and translocation of RB1CC1 into nucleus and then further regulates the crucial cell cycle and differentiation genes. Furthermore, in order to identify the cancer stemness genes specifically expressed in CSCs of ESCC, we utilized gene expression analysis to profile 34 stemness-associated genes in ESCC specimens. Developmental pluripotency associated 4 (DPPA4), a well known pluripotent marker of stem cell, was considered as the best candidate. Our following histopathological study demonstrated that DPPA4 rigorously marked the rare CSCs, in contrast to core stemness factors (OCT4 and SOX2) and previous reported CSC markers (CD90 and CD44), which expressed in a large population of cancer cells. Moreover, the expression of DPPA4 was also found to have prognostic value in ESCC, as the appearance of DPPA4+ cells was significantly associated with poor differentiation, advanced stage and higher incidences of lymph node metastasis. Finally, our functional studies showed that ESCC cells expressing exogenous DPPA4 conferred an enhanced ability to initiate tumor, self-renew, resist chemotherapy and metastasize through lymphatic system. In summary, this study provide evidence indicating that novel tumor suppressor gene PSCA and cancer stemness gene DPPA4 may contribute to the development and progression of ESCC. Additionally, they may serve as potential targets for development of effective therapeutic strategies. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

The United States is currently facing a shortage of qualified Science Technology Engineering & Mathematics workers. As a result, many STEM jobs are being filled by technically-skilled foreign workers. American institutions of higher education, including community colleges, must identify potential factors that contribute to the lack of interest in STEM majors, as well as the low rate of success of students who enter STEM majors but struggle to finish their degrees. Community colleges perform a larger share of STEM training than is generally understood. As highlighted in the National Science Foundation’s National Survey of Recent College Graduates, a surprisingly large proportion (44% overall) of those earning a degree in science and engineering (bachelor’s and master’s) reported that they had attended a community college. The purpose of this study was to ascertain the perceptions of African American community college students who are currently pursuing degrees in STEM majors and to examine the factors that influence their success. Qualitative research method of in-depth phenomenological interviewing was used for this study. This tool provided a strong method of scientific inquiry for understanding the context of the lives of successful African American students in STEM majors. Findings in this study have African American students identifying the meanings of the experiences of participating in a STEM workforce development program at Minneapolis Community and Technical College. They found the experience of Prioritizing to be the most crucial for them as they prepared for their STEM careers. Students consider Professionalism, Building a Network, Internships & Planning. Students also found advisors to be essential to their overall academic success. Furthermore, faculty members can make a difference in how students perceive their academic journey and how well they perform academically. These findings are also supported by Astin’s theory of student involvement, Ladson-Billings Cultural Relevant Pedagogy and Validation. Many community colleges are in urban areas and serve a very diverse population of students. This study can inform our faculty on best practices to prepare lecture materials, through providing cultural relevant pedagogy that addresses issues African American students are currently facing, which make their academic journey significant. When the academic coursework is applicable to real world situations students reported an easier transition to STEM workforce industries. / text

Game construction learning approaches have seen increased interest for computational learning and digital literacy in K-12 education, but the paucity of research on game text-based scripting skill development identifies a gap in the literature. This case study investigated text-based scripting skill development and intrinsic motivation with a class of eighth grade students who were engaged in game construction. The study participants were 20 students and their teacher. The case involved the open-ended, project-based game construction class. Data sources included classroom observations, teacher and student interviews, survey responses, and student game scripts. The findings showed that engaging in game construction with peer collaboration and teacher support helped the students develop scripting skills. Game scripting skill development involved the use of language arts and mathematic skills. Challenges in game scripting included student debugging difficulties, as well as technology issues that distracted the students from their work with battery charging problems, Wi-Fi connectivity drops, and broken computers. The students showed moderate intrinsic motivation toward text-based scripting in game construction and appeared to prefer design artwork to scripting. Implications suggest that developing game scripting skills promoted the practice of language arts literacy and mathematics concepts. Game scripting was an engaging self-directed autonomous learning experience. Text-based scripting development is suggested to be a distinct digital literacy.

No description available.

The cellular microenvironment consists of soluble and insoluble factors that provide signals that dictate cell behavior and cell fate. Limited characterization has hindered our ability to mimic the physiological or pathophysiological environment. While stem cells have vast promise in the areas of regenerative medicine and disease therapy, harnessing this potential remains elusive due to our limited understanding of differentiation mechanisms. Similarly, many in vitro cardiac disease models lack the critical structure- function relationships of healthy and diseased cardiac tissue. The goal of this work is to induce cardiomyogenesis and pathogenesis in vitro by recapitulating features of the native microenvironment during development and disease. / Engineering and Applied Sciences

The use of both multipotent progenitors and fully differentiated cells has been demonstrated to be effective for cell-based immunotherapy. The goal of this thesis was to establish an in vitro hematopoietic differentiation system to generate hematopoietic progenitor cells (HPCs) and functional T cells from pluripotent stem cells. Generation of progenitor T cells by co-culturing stem cells on Notch ligand-expressing OP9 stromal cells (OP9-DL1) had been successfully employed previously. However, further differentiation of these cells in vitro into mature, antigen-specific, functional T cells, without retroviral transduction of T cell receptors (TcRs), had not been achieved. In the thymic niche, differentiation of T cells to a state of antigen specificity is controlled by the interaction of their developing TcRs with the Major Histocompatibility Complex (MHC) on thymic stromal cells. We hypothesized that, by providing exogenous antigen-specific MHC/TcR signals, stem and progenitor cells could be engineered into functional effector T cells specific for the same antigen. In Chapter 3 and 4, we demonstrate that both thymus-derived double positive (DP: CD4+CD8+) immature T cells and mouse Embryonic Stem (ES) cells can be efficiently differentiated into antigen-specific CD8+ T cells using either MHC tetramers or peptide-loaded stromal cells. DP cells, following MHC/TcR signaling, retained elevated RAG1 levels, suggesting continuing TcR gene rearrangement. Both DP and ES cell-derived CD8+ T cells showed significant Cytotoxic T Lymphocyte (CTL) activity against antigen-loaded target cells, indicating that these cells are functional. This directed differentiation strategy could provide an efficient method for generating functional, antigen-specific CTLs from stem cells for potential use in adoptive T cell therapies. The use of ES cells in the clinic has been hindered by the unavailability of patient-specific ES cells and the ethical issues surrounding the use of human embryos. Induced pluripotent stem (iPS) cells offer great hope to regenerative medicine as their use can circumvent both the patient-specific and ethical issues associated with ES cells. In Chapter 5, we have developed a feeder cell-free suspension culture system supplemented with OP9-DL1 secretary factors to efficiently generated HPCs from iPS and ES cells. The differentiation potential of these HPCs was demonstrated by generation of DCs in the presence of GM-CSF and IL-3. The DCs express the activation molecules, CD86 and CD80 in response to LPS stimulation and are able to stimulate T cell proliferation in a mixed lymphocyte reaction. We employed extensive quantitative RT-PCR analysis to identify a number of differentially expressed genes in HPCs generated from the feeder-free culture. / text