Browsing by Author "Connell, Jennifer Petsche"

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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 demonstrate limited cardiac differentiation following small molecule-based modulation of Wnt signaling pathway
    (IOP Publishing, 2015) Connell, Jennifer Petsche; Ruano, Rodrigo; Jacot, Jeffrey G.; Bioengineering
    Amniotic fluid-derived stem cells (AFSC) are a promising cell source for regenerative medicine and cardiac tissue engineering. However, a non-xenotropic differentiation protocol has not been established for cardiac differentiation of AFSC. We tested a small molecule-based modulation of Wnt signaling for directed cardiac differentiation of AFSC. Cells were treated with inhibitors of glycogen synthase kinase 3 and Wnt production and secretion in a time-dependent and sequential manner, as has been demonstrated successful for cardiac differentiation of embryonic and induced pluripotent stem cells. Cells were then analyzed for gene and protein expression of markers along the cardiac lineage at multiple days during the differentiation protocol. At the midpoint of the differentiation, an increase in the percentage of AFSC expressing Islet-1, a transcription factor found in cardiac progenitor cells, and Nkx-2.5, a cardiac transcription factor, was observed. After a 15 d differentiation, a subpopulation of AFSC upregulated protein expression of smooth muscle actin, myosin light chain-2, and troponin I, all indicative of progression down a cardiac lineage. AFSC at the end of the differentiation also demonstrated organization of connexin 43, a key component of gap junctions, to cell membranes. However, no organized sarcomeres or spontaneous contraction were observed. These results demonstrate that small molecule-based modulation of Wnt signaling alone is not sufficient to generate functional cardiomyocytes from AFSC, though an upregulation of genes and proteins common to cardiac lineage cells was observed.
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    Amniotic Fluid-Derived Stem Cells Demonstrated Cardiogenic Potential in Indirect Co-culture with Human Cardiac Cells
    (Springer, 2014) Gao, Yang; Connell, Jennifer Petsche; Wadhwa, Lalita; Ruano, Rodrigo; Jacot, Jeffrey G.; Bioengineering
    Amniotic fluid-derived stem cells (AFSC) have been shown to be broadly multipotent and non-tumorogenic. Previous studies of direct mixing of AFSC and neonatal rat ventricle myocytes indicated evidence of AFSC cardiogenesis. In this study, we examined human AFSC cardiogenic potential in indirect co-culture with human cardiac cells in conditions that eliminated the possibility of cell fusion. Human AFSC in contact with human cardiac cells showed expression of cardiac troponin T (cTnT) in immunohistochemistry, and no evidence of cell fusion were found through fluorescent in situ hybridization. When indirectly co-cultured with cardiac cells, human AFSC in contact with cardiac cells across a thin porous membrane showed a statistically significant increase in cTnT expression compared to non-contact conditions but lacked upregulation of calcium modulating proteins and did not have functional or morphological characteristics of mature cardiomyocytes. This suggests that contact is a necessary but not sufficient condition for AFSC cardiac differentiation in co-culture with cardiac cells.
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    Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes
    (Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd, 2013) Connell, Jennifer Petsche; Augustini, Emily; Moise, Kenneth J. Jr.; Johnson, Anthony; Jacot, Jeffrey G.; Bioengineering
    Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC.
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    In situ vascularization of injectable fibrin/poly(ethylene glycol) hydrogels by human amniotic fluid-derived stem cells
    (Wiley, 2015) Benavides, Omar M.; Brooks, Abigail R.; Cho, Sung Kyung; Connell, Jennifer Petsche; Ruano, Rodrigo; Jacot, Jeffrey G.; Bioengineering
    One of the greatest challenges in regenerative medicine is generating clinically relevant engineered tissues with functional blood vessels. Vascularization is a key hurdle faced in designing tissue constructs larger than the in vivo limit of oxygen diffusion. In this study, we utilized fibrin-based hydrogels to serve as a foundation for vascular formation, poly(ethylene glycol) (PEG) to modify fibrinogen and increase scaffold longevity, and human amniotic fluid-derived stem cells (AFSC) as a source of vascular cell types (AFSC-EC). AFSC hold great potential for use in regenerative medicine strategies, especially those involving autologous congenital applications, and we have shown previously that AFSC-seeded fibrin-PEG hydrogels have the potential to form three-dimensional vascular-like networks in vitro. We hypothesized that subcutaneously injecting these hydrogels in immunodeficient mice would both induce a fibrin-driven angiogenic host response and promote in situ AFSC-derived neovascularization. Two weeks postinjection, hydrogels were sectioned, and the following was demonstrated: the average maximum invasion distance of host murine cells into the subcutaneous fibrin/PEG scaffold was 147 ± 90 µm after 1 week and 395 ± 138 µm after 2 weeks; the average number of cell-lined lumen per square millimeter was significantly higher in hydrogels seeded with stem cells or cocultures containing stem cells (MSC, 36.5 ± 11.4; AFSC, 47.0 ± 18.9; AFSC/AFSC-EC, 32.8 ± 11.6; and MSC/HUVEC, 43.1 ± 25.1) versus endothelial cell types alone (AFSC-EC, 9.7 ± 6.1; HUVEC, 14.2 ± 8.8); and a subset of these lumen were characterized by the presence of red blood cells. Select areas of cell-seeded hydrogels contained CD31+ lumen surrounded by α-smooth muscle cell support cells, whereas control hydrogels with no cells only showed infiltration of α-smooth muscle cell–positive host cells.