Browsing by Subject "Maturation"

The Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects more than >95% of the global adult population. In immunocompetent individuals, EBV infection is asymptomatic and takes place in the oral cavity, where EBV establishes a life-long latent infection in memory B cells by temporally regulating viral gene expression to mimic B cell maturation. In immunocompromised individuals, however, EBV infection can give rise to infectious mononucleosis, epithelial carcinomas, and lymphomas. To model EBV-mediated lymphomagenesis, infection of EBV in vitro generates growth-transformed and immortalized lymphoblastoid cell lines (LCLs), which allows for the characterization of dynamic viral and host gene expression. Our lab has found that the early phase after infection is transcriptionally distinct from the late phase when infected B cells are fully growth-transformed. We also found that apoptosis regulation in each phase of infection is uniquely regulated by a single viral nuclear protein that regulates host gene expression through epigenetic mechanisms. To determine if apoptosis regulation in EBV-infected B cells is virus-specific, I have characterized apoptosis regulation in uninfected maturing B cells and mitogen-stimulated B cells. For the upregulation of one anti-apoptotic protein, EBV infection promotes a chromatin structure resembling that in germinal center light zone B cells, indicating that EBV phenocopies germinal center chromatin regulation to promote apoptosis resistance. In addition to apoptosis regulation, EBV infection phenocopies various aspects of GC B cells and plasmablasts, where the inhibition of plasma cell differentiation increases the efficiency of immortalization and growth-transformation of B cells infected in vitro. The work outlined in this dissertation demonstrate that viral and host genes cooperate in mediating apoptosis regulation, differentiation, and ultimately fate-determination of EBV-infected B cells.

Over recent decades, the continued clinical burden of ischemic heart disease has created a need for alternative therapies to improve the function of diseased myocardium. While the existing clinical interventions are often aimed at restoring blood supply to infarcted myocardium, they have been unable to effectively replace damaged muscle with healthy new tissue. Transplantation of a pre-formed, tissue-engineered cardiac muscle has been proposed as a potential strategy to remuscularize the infarcted heart and prevent adverse remodeling leading to heart failure. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are currently the only robust cell source for engineering of functional cardiac tissues. Despite more than 15 years of research with hPSC-CMs, human engineered cardiac tissues still fall short of replicating the mature electrical and contractile properties of adult human myocardium important for safety and efficacy of the future therapies. Furthermore, most of engineered cardiac tissues are currently engineered to be avascular and are too small for use in clinical applications. As such, the primary goals of this thesis were to: 1) generate mature engineered cardiac tissues from human pluripotent stem cells, 2) scale-up engineered tissue size to clinically relevant dimensions without loss of function, and 3) pre-vascularize engineered cardiac tissues in vitro and test their survival and ability to remain functional in vivo.

To address these goals, we first utilized our established hydrogel-based method to engineer 3D human cardiac tissues (“Cardiopatches”) and determine the effects of cardiomyocyte purity and culture conditions on the tissue structure and function. After 2wks of culture, hPSC-CMs in 3D cardiopatches exhibited faster conduction velocity (CV), longer sarcomeres, and increased expression of genes involved in cardiac contractile function compared to hPSC-CMs cultured in age- and purity-matched 2D monolayers. Furthermore, higher hPSC-CM fraction in cardiopatches yielded faster CV, while maximum forces of contraction were achieved for a particular range of hPSC-CM purities (60-80%). Furthermore, engineered cardiopatches demonstrated a positive inotropic response to β-adrenergic stimulation and generated contractile stresses in excess of 10mN/mm2. These functional results were reproducibly achieved using 4 independent hPSC lines.

Through further optimization, we identified low seeding density and transition from serum-free to serum-containing media as critical factors for accelerated maturation in vitro. These modifications yielded cardiopatches with improved electrical and mechanical function, with average contractile stresses (>20 mN/mm2) and CVs (>28 cm/s) approaching values in adult myocardium. Ultrastructural analysis of cardiopatches revealed highly organized sarcomeric structures, characterized by consistent H-zone and I-bands, frequent intercalated discs, abundant mitochondria, and occasional appearance of T-tubules and central M-bands. Continual increase in functional output during 3-week cardiopatch culture was associated with significant upregulation of molecular maturation markers that, in many instances, reached near-adult levels of expression. Under these optimized conditions, we successfully scaled up engineered cardiopatches to clinically relevant dimensions (4x4cm), while preserving high CVs and contractile strength (with absolute forces exceeding 20 mN) and lacking spontaneous or pacing-induced arrhythmias.

Finally, we explored the ability of cardiopatches to survive and undergo vascularization in vivo using a dorsal skinfold window chamber model in immunocompromised mice and following epicardial implantation onto healthy rat hearts. Within 2 weeks post-implantation into window chambers, initially avascular cardiopatches underwent robust vascularization in vivo and maintained ability to fire Ca2+ transients, albeit at an expense of declined electrical function. In an attempt to improve the vascularization and function in vivo, we developed methods to pre-vascularize cardiopatches with highly branched vascular networks made of hPSC-derived endothelial cells (hPSC-ECs). Upon implantation in window chambers, hPSC-ECs in cardiopatches co-localized with host vasculature and formed hybrid microvessels, indicative of host-donor vascular integration. To further establish their translational potential, we implanted cardiopatches into the mechanically active environment of healthy rat hearts, and demonstrated their survival, vascular integration, and higher levels of electrical function relative to those in dorsal window chambers. Lastly, as a proof-of-concept study, we implanted cardiopatches into a porcine model of myocardial infarction and demonstrated evidence of hiPSC-CM survival, thus providing a foundation for future large animal studies aimed at clinical translation.

In summary, this thesis describes novel methodologies to engineer human cardiac tissues with near-adult levels of electrical and mechanical function, capacity for pre-vascularization, scalability to clinical dimensions, and ability to survive, vascularize, and remain functional in vivo. To the best of our knowledge, the reported functional parameters of cardiopatches are the highest in the field, and our scale-up and pre-vascularization of cardiopatches without loss of function are the first reported in the field. As such, this thesis represents a significant advancement in human heart tissue engineering research that will enable development of next generation cell-based therapies for cardiac repair.

This study identifies and elaborates on a theme in the Hebrew Bible (HB) that has largely gone unnoticed by scholars: the transition of a male adolescent from boyhood to manhood. Beyond identifying the coming-of-age theme in different HB texts, the project also describes how the theme is employed by biblical narrators and redactors to highlight broader messages and transitions in the historical narratives of the HB. It also considers how these stories provide insight into the varying representations of biblical masculinity.

The project begins by showing how the recent discussions on masculinity in the HB and biblical rites of passage are incomplete without an analysis of how a boy becomes a man in the biblical text. It then establishes important principles for recognizing the maturation theme in a given narrative. More foundational work is done in chapter 2, which describes the characteristic features of manhood and boyhood as depicted in the HB to facilitate the identification of narratives where a transition is made from boyhood to manhood.

The next two chapters identify five case studies of coming-of-age: David in 1 Sam 17; Solomon in 1 Kgs 1-2; an alternative tale of Solomon's maturation in 1 Kgs 3; Moses in Exod 2; and Samuel in 1 Sam 3. Chapter 5 discusses the converse of the coming-of-age theme by presenting stories of boys who fail to mature: Jether in Judg 8, and Samson in Judg 13-16. In each case study, the narrator's techniques for highlighting the maturation theme are identified. The ways that the narrator employs the theme to point to other significant plot points or narrative transitions are also identified. Most notably, the failure-to-mature theme in the Samson narratives typifies Israel's political immaturity in Judges, and the two alternative tales of Solomon's maturation highlight an important transition in the Deuteronomistic History from the uncertain and often bloody years of the monarchy's establishment to the peaceful, prosperous reign of Solomon.

The seven case studies are also examined for the image of masculinity that they present, and that presentation is compared to the general view of manhood in the HB. Five of the seven offer quite similar images of masculinity; and these also cohere to the general picture of biblical manhood. However, two narratives (Samuel's maturation in 1 Sam 3 and Solomon's in 1 Kgs 3) depart from this conception of masculinity, each in the same way: both depict a masculinity free of violence and the need for the constant, forceful defense of manhood and honor. Since these two texts have often been ascribed to the same author, the Deuteronomistic Historian, the study suggests that he may be offering a new view of masculinity more suited to his historical context.

The project ultimately proves that the theme of male coming-of-age, heretofore virtually unrecognized, is found in several biblical texts. Moreover, this theme is often used to indicate other important messages and transitions in Israel's historical narrative and can provide unique insight into biblical constructions of masculinity.