There is a huge unmet clinical need to treat damaged articular cartilage such as that caused by osteoarthritis (OA) with an estimated 8.75 million people in the UK having sought treatment for OA (ARUK 2013). Embryonic stem cells (ESCs) offer a promising alternative therapeutic approach, potentially providing an unlimited source of chondrocytes capable of regenerating the damaged cartilage however this is limited by the efficiency of the chondrogenic differentiation protocol. An improved understanding of the posttranscriptional regulation of chondrogenesis by microRNAs (miRNAs) may enable us to improve hESC chondrogenesis. Also the recent discovery that miRNAs are selectively packaged into exosomes which can then be transferred to and be functionally active within neighbouring cells suggests they may have a role in cell-cell communication. This project investigated the regulation of miRNA expression in relation to the transcriptome during hESCs-directed chondrogenesis and the possible role for exosomes during differentiation and in stem cell maintenance of hESCs. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis using the Directed Differentiation Protocol (DDP) developed in our lab. Also small RNA-seq was performed on exosomes isolated from hESCs and chondroprogenitors along with the donor cells that the exosomes originated from. This revealed significant changes in the expression of several miRNAs during hESC-directed chondrogenesis including: upregulation of miRNAs transcribed from the four Hox complexes, known cartilage associated miRNAs and the downregulation of pluripotency associated miRNAs. Overall miRome and transcriptome analysis revealed the two hESC lines exhibited slightly different miRome and transcriptome profiles during chondrogenesis, with Man7 displaying larger changes in miRNA and mRNA expression as it progressed through the DDP suggesting it may be more predisposed to undergo chondrogenesis. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified four key functionally related clusters of co-expressed miRNAs and protein coding genes: pluripotency associated cluster, primitive streak cluster, limb development cluster and an extracellular matrix cluster. Further investigation of these gene/miRNA clusters allowed the identification of several potential novel regulators of hESC-directed chondrogenesis. In accordance with the reported literature the exosomal miRNAs from hESCs and hESC-chondroprogenitors were enriched with a guanine rich motif. Notably, several of these were enriched with targets associated with embryonic skeletal system development suggesting they may play a role in regulating differentiation. Preliminary functional experiments examining pluripotency-associated exosomes suggests they may have a role in regulating hESC stem cell maintenance. However the molecular mechanism by which this is achieved has not been investigated. This research identified main miRome and transcriptome changes during hESC-directed chondrogenesis leading to the identification of several potential novel regulators of chondrogenesis and pluripotency which can be further investigated. This project has also highlighted the potential of exosomal miRNAs to regulate hESC stem cell maintenance and differentiation.

No description available.

<p>Stem cell technologies hold great promise in solving problems within fields such as drug development, regenerative medicine, and disease modeling. Stem cell engineering provides a mechanism that will help stem cells achieve this promise. Currently, many applications within tissue engineering are limited by a lack of ability to create accurate micro-physiological structures that recapitulate multicellular tissue patterns <i>in vivo</i>. Precise control of spatial and temporal signaling is desired to perform concurrent differentiation to multiple cell types intentionally. The OptoWnt construct, a novel optogenetic system activating the Wnt signaling pathway, achieves precise spatiotemporal regulation, in pursuit of greater control in stem cell differentiation. We utilize OptoWnt, to differentiate stem cells into cardiovascular cells: endothelial progenitor cells and cardiomyocytes, valuable cell types for designing microtissues. Endothelial cells comprise the luminal lining of blood and lymphatic vessels, providing the integral structure for distribution within the body, separating mobile and stationary tissues. Cardiomyocytes provide the force required to pump blood throughout the human body and are a highly desired cell type in regenerative medicine.</p> <p>In this project, we have applied an optogenetic induced signaling pathway, OptoWnt, to differentiate human pluripotent stem cells (hPSCs) into cardiovascular cells via light-induced activation of Wnt signaling pathway. In the analysis of these cells and comparison to previous small molecule approaches to cardiovascular cell differentiation, we demonstrate the robustness of the optogenetic approach and similar efficiency that it has with the small molecule approach. In short, we have further demonstrated the utility and potential of optogenetic induction of developmental pathways, via the OptoWnt construct.</p>

African American women are underrepresented in science, technology, engineering, and mathematics (STEM) fields throughout the United States. As the need for STEM professionals in the United States increases, it is important to ensure that African American women are among those professionals making valuable contributions to society. The purpose of this phenomenological study was to describe the experiences of African American young women in relation to STEM education. The research question for this study examined how experiences with STEM in K-10 education influenced African American young women's academic choices in their final years in high school. The theory of multicontextuality was used to provide the conceptual framework. The primary data source was interviews. The sample was composed of 11 African American young women in their junior or senior year in high school. Data were analyzed through the process of open coding, categorizing, and identifying emerging themes. Ten themes emerged from the answers to research questions. The themes were (a) high teacher expectations, (b) participation in extra-curricular activities, (c) engagement in group-work, (d) learning from lectures, (e) strong parental involvement, (f) helping others, (g) self-efficacy, (h) gender empowerment, (i) race empowerment, and (j) strategic recruitment practices. This study may lead to positive social change by adding to the understanding of the experiences of African American young women in STEM. By doing so, these findings might motivate other African American young women to pursue advanced STEM classes. These findings may also provide guidance to parents and educators to help increase the number of African American women in STEM.

Stem cell transplantation with rehabilitation therapy presents an effective stroke treatment. Here, we discuss current breakthroughs in stem cell research along with rehabilitation strategies that may have a synergistic outcome when combined together after stroke. Indeed, stem cell transplantation offers a promising new approach and may add to current rehabilitation therapies. By reviewing the pathophysiology of stroke and the mechanisms by which stem cells and rehabilitation attenuate this inflammatory process, we hypothesize that a combined therapy will provide better functional outcomes for patients. Using current preclinical data, we explore the prominent types of stem cells, the existing theories for stem cell repair, rehabilitation treatments inside the brain, rehabilitation modalities outside the brain, and evidence pertaining to the benefits of combined therapy. In this review article, we assess the advantages and disadvantages of using stem cell transplantation with rehabilitation to mitigate the devastating effects of stroke. / Revisión por pares

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

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23794号 / 医博第4840号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 小川 誠司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The underrepresentation of women of color in science, technology, engineering, and mathematics (STEM) remains a critical issue. Uneven academic preparation and lack of interest have been cited as common factors associated with low participation and degree attainment rates among women of color in STEM; however, these factors do not adequately explain why women with academic achievement and interest comparable to their white, male peers pursue and attain STEM degrees at disproportionately low rates. Identity has been found to be a useful lens to understand why and how women of color pursue and attain STEM degrees and subsequent career goals. Viewing the challenges women of color face as a result of being in a "double bind"-both female and non-white -can enable scholars and practitioners alike to better understand how they navigate trajectories towards their career and other personal goals. Specifically, forms of capital that women of color access because of, and not in spite of, their identity have been found to provide means for women of color to successfully achieve their goals. This study uses ethnographic methods to explore the experiences of early-mid career Latina engineers and the forms of capital they have accessed along their trajectories through undergraduate education in engineering and in the early stages of their engineering-related careers. Findings reveal that Latinas draw upon multiple forms of science-related social and cultural capital to overcome obstacles related to being female and non-white in a male-dominated field in a U.S. context. The challenge associated with being a Latina is exacerbated in the workplace where "bro" culture is more pervasive than in college; however, an awareness of one's social identity, the "Americanness" of the gender/race gap in STEM, and a desire to make the field of engineering more welcoming for subsequent generations of women motivates study participants to persist. Specifically, women in this study draw strength from personal or inherited experience of struggle along their trajectories through engineering. They face challenges with an awareness that obstacles are part of any journey and have developed both an ability to transform obstacles into inspiration for working harder and an understanding that overcoming them is crucial both to repaying the debt of sacrifice of those who came before and paving the way for those who will come after. "Struggle" is thus a form of capital that women in my study acquired and is worthy of exploration as a distinct theoretical framework for persistence. Overall, findings from this study bear implications for individual supports and institutional transformation required to foster the success of Latinas in engineering as a distinct group as well as women of color in STEM broadly speaking. / Urban Education

The Federal Government has called for an overhaul of STEM education, saying that we as a nation must increase "opportunities for young Americans to gain strong STEM skills" (Office of Science and Technology Policy, 2013, p.1). Economically, these skills expand beyond those that make good doctors, professors, and engineers; there is a world of jobs going unfilled because our students are graduating without the skills or knowledge that such opportunities exist. To increase the future STEM workforce, we first need to increase student awareness of a variety of STEM careers early on (Tai et al., 2006). Career decisions are being made by students as early as middle school (Tai et al., 2006); and very little if any STEM career exploration is occurring before high school. This lack of early exposure to STEM career options means that students are likely making decisions about career choices without accurate information; choosing a path before knowing about all the options. This research is broken into two manuscripts; the first of which examined the impacts of design-based learning and scientific inquiry curriculum treatments with embedded career content on the career interest of fifth-grade students as compared to traditional classroom methods. It found that there is an upward trend in career interest with the use of these curriculum treatments, but it is not a significant change, likely due to the short time period of the unit and/or small n. The second manuscript examined the effect of a design-based learning curriculum treatment implementation for a single unit on Radford City Schools fifth-grade students' STEM attitudes and interest in STEM careers through a pre/post design. The study showed statistically significant growth in overall STEM attitudes and within the science subtest specifically. Career interest in the general field of science showed a significant increase, while a change of interest in specific career areas was not statistically significant. Collectively, this research serves as a foundation for the effectiveness of having career awareness and career exposure opportunities built into active learning instruction, which does not occur currently. Built on secondary principles, but at a level appropriate for elementary students, using active learning opportunities with embedded career connections has the potential to be an effective solution to students' premature exclusion of STEM-related study and work options identified in the literature. Through preliminary exposure to this unique combination at the elementary level, a stronger foundation can be built for both ability and interest in STEM. / Ph. D. / The Federal Government has called for an overhaul of STEM education, saying that we as a nation must increase “opportunities for young Americans to gain strong STEM skills” (Office of Science and Technology Policy, 2013, p.1). Economically, these skills expand beyond those that make good doctors, professors, and engineers; there is a world of jobs going unfilled because our students are graduating without the skills or knowledge that such opportunities exist. To increase the future STEM workforce, we first need to increase student awareness of a variety of STEM careers early on (Tai et al., 2006). Career decisions are being made by students as early as middle school (Tai et al., 2006); and very little if any STEM career exploration is occurring before high school. This lack of early exposure to STEM career options means that students are likely making decisions about career choices without accurate information; choosing a path before knowing about all the options. This research is broken into two manuscripts; the first of which examined the impacts of design-based learning and scientific inquiry curriculum treatments with embedded career content on the career interest of fifth-grade students as compared to traditional classroom methods. It found that there is an upward trend in career interest with the use of these curriculum treatments, but it is not a significant change, likely due to the short time period of the unit and/or small n. The second manuscript examined the effect of a design-based learning curriculum treatment implementation for a single unit on Radford City Schools fifth-grade students’ STEM attitudes and interest in STEM careers through a pre/post design. The study showed statistically significant growth in overall STEM attitudes and within the science subtest specifically. Career interest in the general field of science showed a significant increase, while a change of interest in specific career areas was not statistically significant. Collectively, this research serves as a foundation for the effectiveness of having career awareness and career exposure opportunities built into active learning instruction, which does not occur currently. Built on secondary principles, but at a level appropriate for elementary students, using active learning opportunities with embedded career connections has the potential to be an effective solution to students’ premature exclusion of STEM-related study and work options identified in the literature. Through preliminary exposure to this unique combination at the elementary level, a stronger foundation can be built for both ability and interest in STEM.

Master of Science / Department of Anatomy and Physiology / Lei Wang / Jishu Shi / Transforming growth factor beta (TGF-β) and interleukin-1β (IL-1β) are both up-regulated in high grade gliomas and their elevated activities have been associated with prognosis in glioma patients. It is known that TGF-β is involved in proliferation and maintenance of glioma stem cells. In this study, I evaluated whether IL-1β also plays an important role in glioma stem cell development. Glioma stem cells are usually identified by using a sphere assay where glioma stem cells proliferate as neurospheres in serum free medium (SFM) in the presence of two growth factors: EGF and bFGF. However, LN229, a human glioblastoma cell line does not form neurospheres in SFM, suggesting that LN229 cells contain very few stem cells. I found that combination of IL-1β and TGF-β, but not IL-1β or TGF-β alone induced LN229 cells to grow as neurospheres in SFM. Furthermore, quantitative RT-PCR analyses show that the expression of stem cell markers (Nestin, Bmi1, Notch2, and LIF), cytokines (IL-1β, IL-6 and IL-8) and invasive genes (SIP1, β-integrin and N-Cadherin) are significantly enhanced in IL-1β /TGF-β induced spheres compared to the control. Using an invasion assay, drug resistance test, and colony assay, I found that LN229 sphere cells induced by IL-1β and TGF-β are more invasive, have increased drug resistant ability, and are more oncogenic in comparison to the control. Together, these results suggest that IL-1β cooperates with TGF-β to induce glioma stem-like cell phenotype.