Browsing by Author "Jacot, Jeffrey G"

Now showing 1 - 7 of 7
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    A two-pronged approach towards the development of novel therapeutics for advanced endometrial cancer
    (2015-11-24) Engel, Brian Joseph; Carson, Daniel D; Jacot, Jeffrey G; McCrea, Pierre D; Stern, Michael; Wagner, Daniel S
    Endometrial cancer is the fourth most common cancer among women. The standard of care involves hysterectomy with adjuvant radiation and chemotherapy for advanced disease. Despite these efforts, treatment of advanced and metastatic disease is not very effective. This body of work describes a two-pronged approach to address lack of treatments for advanced endometrial cancer. The first approach was an in-depth study of the mechanism and physiological effect of mucin 1 (MUC1)-driven epidermal growth factor receptor (EGFR) expression and signaling. MUC1 is a large, heavily glycosylated transmembrane protein that functions to lubricate surfaces, provides protection from external insult and plays an important role in embryo implantation. EGFR is a receptor tyrosine kinase that influences cellular proliferation, migration and apoptosis. MUC1 increases EGFR gene expression, mRNA levels, protein levels and signaling in endometrial cancer cell lines. Consequently, MUC1 expression is associated with increased EGF-dependent cellular proliferation, survival and resistance to EGFR inhibitors. In addition, MUC1 and EGFR co-expression is associated with increased cellular proliferation in endometrial tumors. The second approach involved the development and characterization of an advanced three dimensional (3D) hyaluronic acid (HA)-based culture model that is compatible with existing high throughput drug screening methodologies. This system incorporates three layers: an acellular cushion layer; an encapsulated cancer cell layer for growth in 3D; and a collagen-containing layer that supports the growth of stromal cells on top of the hydrogel (2.5D). The robustness of this system was evaluated by incorporating endometrial or prostate cancer cells with associated stromal cells. Both culture systems provided high cancer and stromal cell viability and facilitated paracrine interactions. The response to cytotoxic drugs from cells cultured in 3D HA better matched clinical data than cells grown in 2D and 3D-alginate. These studies provide mechanistic evidence for regulation that occurs in advanced endometrial cancer, as well as an improved platform to screen for effective therapeutics. The 3D culture system could be leveraged to evaluate novel therapeutics for the treatment of advanced endometrial cancer which may include MUC1 and EGF-directed therapies.
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    Amniotic Fluid-derived Stem Cell Isolation, Maintenance, and Differentiation for Cardiac Tissue Engineering
    (2014-12-05) Connell, Jennifer Petsche; Jacot, Jeffrey G; Fraser, Charles D; Lwigale, Peter; Grande-Allen, K. Jane
    Cardiac tissue engineering is limited by the lack of a clinically relevant cell source. Amniotic fluid-derived stem cells (AFSC) are broadly multipotent and proliferate rapidly, making them a promising cell source for tissue engineering applications. AFSC can also be utilized autologously for congenital heart defects, the most severe of which are identified in utero, allowing for ample time to isolate and expand the cells to prepare a patch for implantation shortly after birth. This thesis focused on the characterization of AFSC and their potential to differentiate towards a cardiac lineage. For characterization studies, stem cells from amniotic fluid were sorted for c-kit protein expression at the first passage or left unfractionated and then expanded in 5 different media. Protein and gene expression of markers common to pluripotent stem cells were analyzed from passages 2 through 6, and differentiation capacity of the stem cells towards osteogenic, endothelial, and neurogenic lineages were compared at passages 5 and 6. The unfractionated AFSC maintained higher expression of stem cell markers but displayed a significant decrease in those markers at passage 6. Correspondingly, indicators of the lineages of interest were higher following differentiation at passage 5 compared to passage 6. To investigate the cardiac tissue engineering potential of AFSC, cells were differentiated in indirect co- cultures with neonatal rat ventricular myocytes (NRVM) and under a small molecule- based directed differentiation regime. NRVM induce AFSC to form functional gap junctions following indirect co-culture. AFSC undergoing directed differentiation also localized gap junctions to cell membranes and additionally demonstrated an up regulation in cardiac transcription factors and sarcomere proteins. In both co-culture and small molecule-based differentiation methods, however, no organized sarcomeres or spontaneously beating cells were observed. While AFSC hold great potential for regenerative medicine applications, particularly in congenital defect repair, functional cardiomyocytes have not yet been obtained, and it is likely that additional cues beyond chemical signaling and growth factors will be required. Overall, these studies led to a greater understanding of the cardiac potential of AFSC and the effect of sorting and culture conditions on maintenance of stem cell properties in AFSC.
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    Amniotic Fluid-Derived Stem Cells as a Source of In Situ Vascularization within Fibrin/Poly(Ethylene Glycol) Hydrogels
    (2014-12-04) Benavides, Omar M; Jacot, Jeffrey G; Fraser, Charles D; Moake, Joel L; Olson, John S
    One of the greatest challenges in regenerative medicine is providing a significant source of vascularization within engineered tissues. Successful vascularization requires both a scaffold that supports vessel formation and a reliable source of vascular cell types. Broad potential for differentiation, high proliferation rates, and autologous availability for neonatal applications make amniotic fluid-derived stem cells (AFSC) well suited for regenerative medicine strategies. We utilized chemical-mediated differentiation of AFSC into endothelial-like cells (AFSC-EC), which expressed key proteins and functional phenotypes associated with endothelial cells. Fibrin-based hydrogels were shown to stimulate AFSC-derived network formation in vitro but were limited by rapid degradation. Incorporation of poly(ethylene glycol) (PEG) provided mechanical stability while retaining key benefits of fibrin-based scaffolds – quick polymerization, high biocompatibility, and vasculogenic stimulation. AFSC-EC as a vascular cell source and AFSC as a perivascular cell source were compared to established sources of these cell types – human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), respectively. In vitro, cell-seeded hydrogels were assessed based on network formation, including parameters such as vessel thickness, length, and area. The development of robust vessels required the presence of both an endothelial and a perivascular cell source and was seen in AFSC co-cultures. Additionally, the co-culture of AFSC with AFSC-EC resulted in a synergistic effect on network parameters similar to MSC. Based on this data, we hypothesized that subcutaneously injecting similar hydrogels in immunodeficient mice would both induce a fibrin-driven angiogenic host response and promote in situ AFSC-derived neovascularization. Two weeks post-injection, AFSC-seeded hydrogels demonstrated significantly higher vascular lumen formation versus those without cells or those seeded with endothelial cells alone; a subset of these lumen were characterized by the presence of red blood cells, suggesting anastamosis with host vasculature. In support of the Pediatric Cardiovascular Bioengineering Lab’s global vision, this research demonstrates that AFSC-seeded fibrin/PEG hydroge In support of the Pediatric Cardiovascular Bioengineering Lab’s global vision, this research demonstrates that AFSC-seeded fibrin/PEG hydrogels have the potential to serve as a vascularized platform for the development of an engineered cardiac patch to be used in autologous repair of congenital heart defects.
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    Predicting Tissue Characteristics in Brain Tumors Using Radiological-Pathological Correlations
    (2016-08-26) Lin, Jonathan Sunwei; Hazle, John D; Jacot, Jeffrey G
    This thesis describes the development of prediction techniques for tissue characteristics in brain tumors, using both imaging and tissue information. Magnetic resonance imaging (MRI) serves as an aid in the clinical management of brain tumors, both as a tool for tumor characterization and monitoring, as well as guidance for biopsy sampling. However, the use of conventional MRI can lead to biopsy sampling errors and limited information for tumor analysis. The development of prediction techniques for tissue characteristics in brain tumors could provide valuable, additional information that would assist with both of these tasks. This work describes how such techniques were built using three key steps: data collection and curation, model construction, and model validation. Data collection involved patient screening and recruitment for an IRB-approved, HIPAA-compliant clinical trial protocol. Thirty-one (31) treatment-naïve, adult glioma patients were imaged using an extensive set of MR imaging techniques, from which multiple biopsy targets were specified using conventional and advanced MR image sequences. Biopsy tissue from these target sites was sampled under stereotactic guidance during craniotomy procedures, then stained and analyzed for World Health Organization (WHO) grade, cell proliferation markers, vascularity markers, and cell density. Images were normalized using biological reference regions, registered to the patient’s T2 volume, then sampled at the relevant biopsy sites by propagating a spherical volume of interest (VOI) through all the images. Model construction involved using both linear/logistic regression and random forest (RFT) machine learning techniques to relate 25 imaging parameters to 4 tissue parameters, all obtained from the same biopsy sites. Various statistical tests and analyses were also used to develop different sets of imaging parameters to serve as inputs to the regression and RFT techniques.
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    Regulation of the neurovascular patterning by growth factors and cytokines during anterior ocular development
    (2015-12-16) Ojeda Cardenas, Ana; Bennett, George N; Jacot, Jeffrey G; McNew, James A; Tao, Yizhi J
    The cornea is a transparent, avascular, and one of the most innervated tissue of the body. Corneal diseases including injuries, neovascularization, congenital eye defects and degenerations, represent a major public health burden. Although, studies have been focused on understanding the basis of transparency, innervation, and neovascularization of the adult cornea, little is known about the molecular mechanisms that lead to this specialized structure results in a highly innervated but avascular tissue during embryogenesis. The purpose of this work was to identify molecular regulators of the neurovascular patterning during cornea development. First, Sema3A, a well-known chemorepulser of axons, was identified as a key modulator in the establishment of cornea avascularity in both, avian and murine models. Moreover, I demonstrated that chemokines, initially described for their function in controlling immune cell migration, also play an important role in axon guidance and vasculogenesis during ocular development. Examination of the expression of the chemokine CXCL14 by in situ hybridization and immunohistochemistry revealed novel patterns of localization in the corneal stroma, iris, lens epithelium, retina and trigeminal ganglion. Comparison in the expression of CXCL14 and CXCL12 shows that they are expressed in complementary patterns in most tissues during ocular development, suggesting an interactive regulation of these chemokines. Visual examination of Retrovirus-mediated Knockdown of CXCL14 embryos revealed relatively smaller eyes compared to controls, and immunohistochemical analysis of ocular nerves indicated exacerbated projection of sensory nerves into the corneal stroma, corneal epithelium and iris, which subsequently elevated nerve density in these tissues. In vitro analyses revealed that CXCL14 has an inhibitory effect on CXCL12-induced axon growth of trigeminal ganglion sensory neurons. Furthermore, Knockdown of CXCL14 in Tg(tie1: H2B:eYFP) transgenic Japanese quail embryos resulted in ectopic migration of YFP fluorescently labeled angioblasts into the cornea and exogenous CXCL14 inhibits VEGF- and CXCL12-induced angioblast migration into the cornea. This is the first time that CXCL14 has been shown to have a critical function during embryogenesis that may be mediated through inhibition of CXCL12 signaling. Collectively, these results demonstrate that neurovascular patterning of the anterior eye during development depends on an intricate process and fine balance of growth factors and cytokines. These findings will contribute to a better understanding of the molecular mechanisms involved in pathological conditions such as cornea neovascularization, anterior segment ocular dysgeneses and wound healing, where angiogenesis and nerve regeneration are critically compromised.
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    Self-contained 3D Differentiation of Reprogrammed Amniotic Fluid Derived Stem Cells for Congenital Heart Repair
    (2017-11-28) Tsao, Christopher; Jacot, Jeffrey G
    Congenital heart defects (CHD) are the most common type of birth defect and the leading cause of infant death. The most severe defects, such as Tetralogy of Fallot and hypoplastic left heart syndrome, can require immediate surgical intervention soon after birth. Current repair strategies involve surgically implanting inactive patch materials which often require repeat surgeries. Since congenital heart defects can be detected as early as the first trimester, the time between diagnosis and surgery can effectively be used to engineer functioning cardiac tissue. The goal of this study was to create an implantable cardiac patch that could direct the differentiation of induced pluripotent stem cells (iPSC) reprogrammed from human amniotic fluid derived stem cells (AFSC). This differentiation would take place within a closed system, minimizing laboratory handling and maximizing clinical applicability. The resulting cardiac patch would overcome current patch deficiencies associated with arrhythmia, mechanical mismatch, or even heart failure. By creating a three dimensional system capable of temporally regulating the release of small molecules, autologous induced pluripotent stem cells could be directed to functional cardiomyocytes for use as an implantable cardiac patch for congenital heart defect repair. Further development of this system could also be used to develop repair strategies for ischemic heart repair. In order to obtain an autologous cardiomyocyte cell source for CHD, AFSC were readily isolated from amniotic fluid obtained through routine amniocentesis. These cells were classified by previous members in our lab as broadly multipotent, though not sharing the same pluripotency as embryonic stem cells. Attempts to directly differentiate AFSC into cardiac cells resulted in expression of early and late stage cardiac markers, but lack of classic cardiomyocyte contractility. Therefore this study investigated the reprogramming of AFSC to iPSC by modified mRNA transfection and the differentiation of these reprogrammed cells into cardiomyocytes through small molecule inhibitors of the GSK3 and Wnt signaling pathways. Reprogrammed cells were shown to express markers of pluripotency and formed teratomas in vivo. Cardiac differentiation resulted in immature spontaneously beating cells which were characterized through genetic expression, immunohistochemistry and electrophysiology. By encapsulating GSK3/Wnt small molecule inhibitors within porous silica particles (pSi), reprogrammed AFSC were differentiated into to cardiomyocytes with minimal intervention. The release of inhibitors from pSi was tuned by varying the thickness of polymer coatings to coincide with the temporal cues for cardiac differentiation. We evaluated the nanoparticle size, zeta-potential, and release profile in a 2D culture, as well as cell differentiation efficiency, phenotypic analysis and electrophysiology. Before translating the iPSC-derived cardiomyocyte (CM) differentiation into a three dimensional space, we first investigated an electrospun (ES) gelatin biomaterial and evaluated it for cardiac cell toxicity and the promotion of neovascularization in vivo. pSi containing vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) were conjugated to the ES gelatin and shown to have a sequential sustained release in vitro. Results showed a decrease in cellular toxicity in vitro due to reduced particle internalization and increased neovascularization in vivo. The results of this research could provide new insights into repair strategies for CHD that would be functional and able to grow with the patient. It can also provide an innovative platform for future tissue engineering constructs as well as help develop cardiac specific toxicity platforms.
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    Use of Human Pediatric Cardiac Progenitor Cells in an Engineered Heart Patch
    (2015-09-30) Gao, Yang; Jacot, Jeffrey G; Grande-Allen, Jane; Harrington, Daniel
    Congenital heart defects (CHD) are the most common birth defects in the US and the leading cause of death in newborns. Some of the most prevalent CHD require surgical interventions with patch materials. However, current commercial patch materials are acellular, non-conductive, and non-contractile; they can induce arrhythmias and require reoperations. We envision an engineered cardiac patch seeded with autologous cells from the patient. However, mature cardiomyocytes (CM) rarely proliferate. This research examined the ability of primary pediatric cardiac cells (PPCC) isolated from pediatric CHD biopsy samples supplied by Texas Children’s Hospital to differentiate into CM or induce CM differentiation in stem cells. Previous studies indicated evidence of cardiogenesis in Amniotic fluid-derived stem cells (AFSC) when directly mixed with neonatal rat ventricle myocytes. We hypothesized that co-culturing with human PPCC will induce cardiac differentiation in human AFSC (hAFSC). hAFSC co-cultured in contact with PPCC showed a statistically significant increase in cTnT expression compared to non-contact conditions but did not have functional or morphological characteristics of mature cardiomyocytes. This result suggests that contact is a necessary but not sufficient condition for AFSC cardiac differentiation in co-culture with PPCC. Cardiac progenitor cells (CPC) are proliferating cells with the ability to differentiate into cardiac cells. CPC can be identified from cardiac cells by the expression of Isl-1, SSEAs, and c-Kit. We hypothesized that there are potential CPC in PPCC. We found a small subpopulation (1%-4%) of the primary cells expressing Isl-1, SSEA-4, and c-Kit. However, when exposed to oxytocin, PPCC did not differentiate into functional CM as shown with murine CPC in literature. Extracellular matrix proteins isolated from adult cardiac tissue have been shown to promote CM maturation in vitro. PPCC can be expanded in vitro and cultured in PEGylated fibrin hydrogels. PPCC conditioned gels can then be decellularized. We found that PPCC could be cultured in fibrin hydrogels and that stem cell derived CM were viable when cultured on these conditioned gels. Overall, this research demonstrated that PPCC are a potential tool for CM differentiation and maturation in the development of a tissue engineered cardiac patch for repair of CHD.