Browsing by Subject "Stem cell"
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Item In vitro generation of hematopoietic progenitors and functional T cells from pluripotent stem cells(2010-08) Lin, Jian, 1980-; Roy, Krishnendu; Tucker, Philip W.; Stevens, Scott W.; Tian, Ming; Whiteley, MarvinThe 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.Item Ultrasound and photoacoustic imaging to monitor stem cells for tissue regeneration(2014-05) Nam, Seung Yun; Emelianov, Stanislav Y.; Suggs, Laura J; Pearce, John A; Dunn, Andrew K; Hall, Neal ARegenerative medicine is an interdisciplinary field which has advanced with the use of biotechnologies related to biomaterials, growth factors, and stem cells to replace or restore damaged cells, tissues, and organs. Among various therapeutic approaches, cell-based therapy is most challenging and exciting for both scientists and clinicians pursuing regenerative medicine. Specifically, stem cells, including mesenchymal stem cells and adipose-derived stem cells, are promising candidate cell types for cell-based therapy because they can differentiate into multiple cell types for tissue regeneration and stimulate other cells through neovascularization or paracrine signaling. Also, for effective treatment using stem cells, the tissue engineered constructs, such as bioactive degradable scaffolds, that provide the physical and chemical cues to guide their differentiation are incorporated with stem cells before implantation. Also, it was previously demonstrated that tissue-engineered matrices can promote tubulogenesis and differentiation of stem cells to vascular cell phenotypes. Hence, during tissue regeneration after stem cell therapy, there are numerous factors that need to be monitored. As a result, imaging-based stem cell tracking is essential to evaluate the distribution of stem cells as well as to monitor proliferation, differentiation, and interaction with the microenvironment. Therefore, there is a need for a stem cell imaging technique that is not only noninvasive, sensitive, and easy to operate, but also capable of quantitatively assessing stem cell behaviors in the long term with high spatial resolution. Therefore, the overall goal of this research is to demonstrate a novel imaging method capable of continuous in vitro assessment of stem cells as prepared with tissue engineered constructs and noninvasive longitudinal in vivo monitoring of stem cell behaviors and tissue regeneration after stem cell implantation. In order to accomplish this, gold nanoparticles are demonstrated as photoacoustic imaging contrasts to label stem cells. In addition, ultrasound and photoacoustic imaging was utilized to monitor stem cells and neovascularization in the injured rat tissue. Therefore, using these methods, tissue regeneration can be promoted and noninvasively monitored, resulting in a better understanding of the tissue repair mechanisms following tissue injury.