Browsing by Subject "Stem cells"

Now showing 1 - 8 of 8
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    Advancing cardiac tissue engineering via multicellular complexity
    (2024-04) Givens, Sophie
    Here I advance the field of tissue engineering through the additions of epicardial (EPC) and epicardial-derived cells to 2D and 3D in vitro ventricular models derived solely from human induced pluripotent stem cells (hiPSC). Through the singular addition of EPCs, EPCs undergoing EMT into epicardial-derived cells (EPC->DC), and fully differentiated epicardial-derived fibroblasts (EPD-FB) I was able to advance our understanding of the effect each of these cells on driving the maturation of hiPSC-derived cardiomyocytes (CM). First, I determined that EPCs and EPC->DC both spur CM proliferation while simultaneously promoting electrochemical maturation with no changes in cellular or tissue level force generation. In the presence of EPCs, CM maintain expression of fetal-like myofilament isoforms while the EPC->DCs had the opposite effect on CM, driving myofilament maturation in a subpopulation of CM. Next, to promote force generation in parallel with electrochemical maturation spurred by EPCs, fully differentiated EPD-FBs were added to engineered heart tissues (EHTs). These tissues showed robust electrochemical maturation, force generation, as well as more mature myofilament isoforms. Intrigued by this finding, we explored the effects of EPD-FBs alone on CM phenotype in 2D and 3D co-cultures. EPD-FBs served to drive aspects of CM maturation that EPCs did not such as CM alignment, hypertrophy, multinucleation, and tissue level force generation. However, the electrochemical functionality lagged behind the EPC co-cultures. These results suggest that the developmentally informed addition of cardiac multicellularity drives advanced cardiac tissue functionality via a sequential transition from CM proliferation to maturation. These studies benefitted from a new cohort of hiPSC-lines that I reprogrammed from the aLVCF of three female and three male donors such that outcomes could be validated on multiple lines including those of male and female biologic sex. Going forward this cohort will serve as a valuable resource for evaluating the functional and genetic differences between female and male hiPSC- derived cell types in health and disease. Indeed, I have already used this cohort to identify global differences between male and female hiPSC-CM, even in the absence of hormonal signaling.
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    Cardiac repair using fibrin patch-based enhanced delivery of stem cells and novel strategies for fast examination of myocaridal energetics in vivo using 31P magnetic resonance spectroscopy.
    (2011-03) Xiong, Qiang
    The dissertation is related to heart disease in both basic scientific and pre-clinic respects. Cardiovascular disease is a leading cause of death in the developed countries even with optimal medical treatment. Recently, the emerging stem cell biology has shown great potentials for cardiac repair. Using both large (swine) and small (rodent) animal models with ischemic heart disease, we examined the functional improvement of enhanced delivery of combined human embryonic stem cell-derived endothelial cells and smooth muscle cells via a fibrin 3D porous scaffold biomatrix. The integration of stem cell biology and tissue engineering has resulted in significant improvement of both regional and global left ventricle function as compared to untreated animals, demonstrating a promising therapeutic strategy of using this cell type and the novel mode of delivery. As a most energy demanding organ, the heart energetic status is tightly associated with the organ's physiological and pathological conditions. Based on the ultra-high-field magnetic resonance imaging/spectroscopy techniques, we developed a noninvasive modality for rapid examination of cardiac energetics in vivo. The new platform will offer unprecedented understanding into the relationship between the cardiac energetic status and the organ's physiological/pathological conditions, and thus it is of great potential in both basic scientific research and clinical diagnosis.
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    Engineering a three-dimensional culture system for the directed differentiation of pluripotent stem cells toward a hepatocyte-like cell fate
    (2012-11) Owens, Derek Jason
    Because stem cells have the ability to self-renew and differentiate into more specialized cell types, they hold enormous potential in the fields of regenerative and personalized medicine as well as providing a model system for studying development in vitro. Stem cells with the capacity to differentiate to hepatocytes, the functional cells of the liver, have potential applications in the pharmaceutical industry in high-throughput drug toxicity screening and in clinical settings in bioartificial liver devices or as candidates for transplantation to treat end-stage liver disease. However, these applications rely on the ability to generate and differentiate the stem cells to functionally mature hepatocytes in a robust and reproducible manner. We have recently optimized a multistage directed differentiation protocol for guiding human embryonic stem (hES) cells and rat multipotent adult progenitor cells (rMAPCs), among other stem cell types, toward a hepatic fate. We also recently showed that rMAPCs, which are isolated from the bone marrow of post-natal rats and exhibit the ability to self-renew and differentiate to all three lineages, can be cultured as three-dimensional aggregates without losing their potency or self-renewal capacity. In this study, we report three-dimensional aggregate-based culture systems that enhance the differentiation of rMAPCs and hES cells to hepatocyte-like cells. rMAPCs were allowed to self-assemble into undifferentiated aggregates before being differentiated via the four-step directed differentiation protocol. Compared to adherent monolayer cultures, differentiation as aggregates resulted in significantly higher expression of liver-specific transcripts, including albumin, and increased secretion of albumin and urea. The differentiated cell aggregates also demonstrated functional activities of primary hepatocytes, as demonstrated by pentoxyresorufin O-dealkylation (PROD) and ethoxyresorufin O-dealkylation (EROD), and ultrastructural features of hepatocytes by electron microscopy. A similar three-dimensional culture system likewise enhanced the differentiation of hES cells. HSF6 cells differentiated as a monolayer culture were dissociated and allowed to self-assemble into three-dimensional spheroids in an extended differentiation culture. Compared cells maintained in the monolayer culture, cells within the spheroids exhibited significantly higher expression levels of liver-enriched transcripts and proteins, including Albuming, PEPCK, and ASGPR-1. Cells in the spheroids demonstrated hepatic functions EROD, PROD, and biliary accumulation of fluorescein diacetate metabolite and ultrastructural characteristics of hepatocytes by electron microscopy. Finally, whole-genome transcriptome analysis was performed to investigate the expression profile of liver-specific sets of genes, including the hepatocyte nuclear factors (HNFs), cytochrome P450s (CYP450s), and UDP-glucuronosyltransferases (UGTs), during differentiation. Cells in the spheroid were shown to have overall increased expression levels of most of the genes in these families, although the expression levels were still lower than in adult liver. The transcriptome analysis was also used to identify genes that change during establishment of the spheroid culture that may play a role in the enhanced differentiation status of the cells; multiple members of the aldo/keto reductase (AKR) and metallothionein (MT) families were found to have much higher expression in spheroids than in monolayer culture. These studies demonstrate the ability of three-dimensional, scalable culture systems to enhance the differentiation of pluripotent stem cells toward a hepatic fate and to maintain the differentiated phenotype for extended culture. With modifications to further enhance the maturity of stem cell-derived hepatocyte-like cells, these systems may facilitate the translation of stem cell generated tissues to technology.
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    Exploration Of Primitive Endoderm Cells With Transcriptome Analysis
    (2016-11) Cho, Dong Seong
    Stem cells hold great clinical potentials for regenerative medicine. Pluripotent stem cells can give rise to any adult cell type. However, pluripotent stem cells have risk of tumor formation and have limitations because of their growth properties. Despite their potentials for therapeutic application, pluripotent stem cells may not be the most suitable for manufacturing process. The derivation of multipotent adult progenitor cells (MAPCs) from rodent bone marrow suggested that these cell types have more amenable properties for manufacturing process than pluripotent stem cells. Especially, rat MAPCs (rMAPCs) can differentiate into multiple lineages including hepatocyte-like cells, β-cell like cells, and mesodermal cells. However, they are not pluripotent and have less risk of tumor formation than pluripotent stem cells. In addition, because MAPCs can be cultivated as single cells, they are less vulnerable than pluripotent stem cells for robust expansion for potential clinical application. Although rMAPCs were derived from rat bone marrow, they resemble the cells in primitive endoderm (PrE) of mouse embryos. Here I showed that a similar population of nascent PrE cells can be derived from pluripotent rat embryonic stem cells (rESCs) by culturing in rMAPC medium conditions, and term these cells converted (c)HypoSCs. Like rMAPCs, cHypoSCs can readily differentiate towards cells with hepatic and mesoderm features in vitro, and generate extraembryonic endoderm in vivo. Transcriptome analysis and mass cytometry further showed that cHypoSCs exhibit nascent PrE gene expression properties that are similar to those of rMAPCs. Cross-species meta-analysis with single-cell transcriptome data confirmed that cHypoSCs are similar to early PrE cells of the mouse blastocyst, while rodent ESCs are similar to early epiblast (EPI) cells. Along with this finding, transcriptome analysis of human in vivo PrE cells and EPI cells uncovered potential culture conditions to derive human nascent in vitro PrE cells from human pluripotent stem cells. We demonstrated the potential of differentiation capacity of human pluripotent stem cells to nascent PrE cells using the identified culture conditions. This study will elucidate the underlying mechanisms of PrE differentiation, and will facilitate the derivation of potentially suitable type of cells for therapeutic application.
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    Hematopoietic development from adult human embryonic stem cells and induced pluripotent stem cells
    (2012-12) Adlakha, Mitali
    The central goal of the project is to understand the role of non-coding RNAs in mediating cell fate decisions in both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Two specific aims are pursued in order to understand the process of hematopoiesis and erythropoiesis better. The first aim comprises of understanding the regulatory networks of hematopoiesis better, we plan to profile the microRNA and mRNA expression in hESC/iPSC-derived CD34+ CD45+ hematopoietic stem/progenitor cells compared to CD34+ cells isolated from umbilical cord blood (UCB). The second aim deals with understanding the erythropoietic development, by profiling the long non coding RNAs (lncRNAs) from erythroid cells derived from hESCs and iPSCs. CD34+ UCB derived erythroid cells are used as a positive control for comparing with hESC/iPSC derived erythroid cells. The overall significance of studying the role of non-coding RNA lies in the fact that it will help modulate the hematopoietic pathways and eventually be used for therapeutic purposes.
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    Oct4 lineage tracing of spermatogonial stem cells in the adult mouse testes
    (2013-12) Haider, Kerri Tana
    Spermatogenesis in mammals occurs in the testes to produce mature sperm for reproductive purposes. Spermatogonia are derived from primordial germ cells. Undifferentiated spermatogonia are thought to arise from a small population of cells arranged around the basal membrane of the seminiferous tubule, called spermatogonial stem cells (SSCs). SSCs divide either symmetrically or asymmetrically to maintain the stem cell pool and give rise to daughter cells. The identity of the spermatogenic stem cell remains unclear. Numerous researchers have attempted to isolate the SSC population but in most cases they also labeled progeny of the SSCs, so a definitive population of SSCs has yet to be isolated. Oct4 is a protein that is expressed in pluripotent cells and has been described as being expressed in undifferentiated SSCs. In adult male Oct4:CreER mTmG transgenic mice GFP expression indicating Oct4 driven tamoxifen inducible CreER recombination is observed only in the testes. Here we describe using this transgenic mouse to identify and lineage trace the progeny of Oct4:CreER expressing cells in the adult mouse testes using multiple tamoxifen pulse and chase experiments. In 4-day chase experiments single GFP expressing cells along the edge of the basal lamina are observed. Longer 10-day chase experiments show aligned spermatogonia expressing GFP in chains of 2, 4 and 8 cells linearly placed on the basal lamina of the tubule. Fallowing extended chase periods after tamoxifen administration GFP expressing cells are seen in all stages of spermatogenesis. Our results are consistent with the cells labeled immediately after tamoxifen addition being the spermatogenic stem cell.
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    Side Population Cells in the Adult Heart
    (2018-06) Yellamilli, Amritha
    The clinical outcomes for heart failure remain poor because current therapies do not address a critical feature of heart failure – loss of functional cardiomyocytes. To decrease the morbidity and mortality of patients with heart failure, multiple strategies are being developed to replace dead cardiomyocytes with new, functional ones. Adult stem cell transplantation studies have had modest clinical benefits primarily attributed to paracrine effects on several endogenous processes including cardiac regeneration. Many cardiac progenitor cell populations have been isolated from the adult mammalian heart and studied in cell culture or after transplantation; however, their roles in endogenous cardiac regeneration are highly contested. In the thesis work presented here, we used the side population phenotype as an unbiased approach to determine whether an endogenous progenitor cell population exists in the adult mammalian heart. We generated a new Abcg2-driven, lineage-tracing mouse model that efficiently labels side population cells in multiple tissues throughout the body, including the heart. With this mouse model, we first showed that the side population phenotype enriches for endogenous stem cells in the bone marrow and small intestine under homeostatic conditions. In the adult heart, we showed that cardiac side population cells contribute to 21% of newly formed cardiomyocytes either through direct differentiation or fusion. Moreover, cardiac side population cells are responsive to different forms of cardiac injury. Further characterization of cardiac side population cells will help us understand how they can be targeted in vivo for the development of new heart failure therapies.
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    Transcriptional profiling of pluripotent and multipotent stem cells to decipher pluripotency and lineage specification
    (2012-04) Sharma, Shikha
    Stem cells hold great promise for the fields of regenerative medicine, gene therapy and disease modeling. Understanding the transcriptional machinery involved in their maintenance is critical to their successful isolation and experimentation. Careful statistical analysis of high throughput transcriptome data can provide novel insights into the gene networks and patterns active in these cells. Public repositories are a source of gene expression data from various studies involving stem cells. This expression data can be overlaid on functional interactions maps of the genome to predict functional association. Further on, comparison of stem cells of different potencies can help identify key genes involved in the maintenance of pluripotency. The hypotheses derived from such transcriptional profiling can be tested experimentally to confirm expression and interactions. Transcriptome data from studies involving human and mouse pluripotent stem cells was collected from repositories such as GEO and Arrayexpress (EBI). Non-negative matrix factorization was used as a dimensionality reduction tool to detect biological patterns and clusters in the data. Following the classification of data into biologically meaningful classes, a ‘metagene’ profile characteristics of pluripotent stem cells was determined in both species. Reverse engineering was performed for predicting gene networks and signaling ‘hubs’. An algorithm was also developed to overlay this predicted gene signature onto functional networks that combine a large amount of genetic and genomic iv data from various sources, for detecting small subnetworks that are conserved in expression in the pluripotent stem cells of both species. Embryonic and induced pluripotent stem cells are considered as in vitro counterparts of pluripotent cells seen in the early embryo, namely the inner cell mass (ICM) and the epiblast. Multipotent adult progenitor cells (MAPCs), although isolated from the bone marrow of an adult rat, bear a striking similarity with another cell types in early embryonic development-the primitive endoderm or nascent hypoblast cells. Due to the developmental proximity of the pluripotent cells and the primitive endoderm cells in the early embryo, MAPCs have been used as a model system for probing gene interactions in pluripotent cells. On the basis of comparative transcriptome analysis, as well as, experimental studies, a model of gene regulation in MAPCs has been developed. The study of dynamics of this gene network provides novel insights into the transcriptional regulation of key pluripotency-associated genes. Also, using the concepts of cellular reprogramming, these multipotent stem cells (MAPCs) have been reprogrammed to a pluripotent state. This represents a unique reprogramming system where an Oct4 expressing extraembryonic cell has been transformed to an embryonic stem cells (ESC) like state. While this was performed using the traditional reprogramming cocktail consisting of Oct4, Sox2, Klf4 and c-Myc, future studies are likely to narrow down this number to one or two genes.