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Wallace H. Coulter Department of Biomedical Engineering

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Now showing 1 - 10 of 17
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    Investigating the role of intercellular communication on spatial differentiation through agent-based modeling
    (Georgia Institute of Technology, 2018-07-30) Glen, Chad Michael
    The initiation of heterogeneity within a population of phenotypically identical progenitors is a critical event for the onset of morphogenesis and differentiation patterning. Information flow between adjacent cells informs cell fate decisions and can occur by a number of mechanisms. Gap junction communication within multicellular systems produces complex networks of intercellular connectivity that result in heterogeneous distributions of intracellular signaling molecules. In this work, an agent-based computational model of ESC collective behavior was designed to prompt the state change of individual cells through intracellular accumulation of molecular differentiation cues throughout a colony. The model yielded complex, dynamic transport networks for delivery of differentiation cues between neighboring cells, reproducing the distribution and variety of observed morphogenic trajectories that result during retinoic acid–induced mouse ESC differentiation. Furthermore, the model correctly predicted the delayed differentiation and preserved spatial features of the morphogenic trajectory that occurs in response to perturbation to intercellular communication. The relationship between intercellular communication and neural differentiation was further interrogated through the CRISPRi-mediated knockdown of connexin43 (Cx43), the predominant gap junction protein in pluripotent cells. The selective removal of Cx43 during the differentiation of human induced pluripotent cells (hiPSCs) reiterated the role of intercellular communication in the temporal control of differentiation by delaying neural commitment. These findings suggest an integral role of gap junction communication in the temporal coordination of emergent patterning during early differentiation and neural commitment of pluripotent stem cells. To facilitate future studies of emergence in multicellular systems, a multiscale communication agent-based model generator (MsCAMgen) was developed in Python. MsCAMgen provides a framework for modeling various spatial aspects of a multicellular network without requiring explicit programming by the user. Each model is capable of accounting for cell division and growth, state changes between different cell types, extracellular diffusion of molecules that are secreted and consumed by cells, intercellular communication of small molecules between neighboring cells, and intracellular gene/protein networks. The ability to quickly add and remove these features at the discretion of the user makes MsCAMgen an ideal platform for investigating emergence in biological systems. Furthermore, the ease of simulating diverse morphological structures that can include and integrate each of these processes distinguishes MsCAMgen as a uniquely suited tool for optimizing the design of engineered living systems. In summary, this thesis interrogated the intercellular network within pluripotent colonies, described the spatiotemporal trajectory of early neural differentiation using an agent-based intercellular transport model, and developed an adaptable application to facilitate accelerated design of engineered living systems such as organoids by enabling the analysis of multiscale communication within cell populations of any morphology or organization.
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    Heparin microparticle-mediated delivery of BMP-2 and pluripotent stem cell morphogens for bone repair
    (Georgia Institute of Technology, 2016-11-08) Hettiaratchi, Marian Hirushika
    The delivery of bone morphogenetic protein-2 (BMP-2) offers a promising means of stimulating endogenous repair mechanisms to heal severe bone injuries. However, clinical application of growth factor therapy is hindered by the lack of adequate biomaterials to localize BMP-2 delivery. Glycosaminoglycans, such as heparin, have the capacity to strongly bind BMP-2 and other growth factors implicated in bone regeneration, and present the opportunity to locally deliver growth factors to injury sites. Moreover, pluripotent stem cells (PSCs) secrete many potent heparin-binding growth factors that have been implicated in tissue regeneration following cell transplantation and may provide cues for repair. Thus, heparin can also be used to concentrate and deliver PSC-derived morphogens to tissue injury sites, thereby overcoming challenges associated with PSC transplantation. The goal of this work was to improve growth factor delivery for bone repair by both (1) creating an effective biomaterial for BMP-2 delivery and (2) investigating PSC morphogens as a novel source of therapeutic growth factors. We developed heparin-based microparticles that could bind and retain large amounts of bioactive BMP-2 in vitro and improve BMP-2 retention in vivo, resulting in spatially localized bone formation in a critically sized rat femoral defect. Furthermore, heparin microparticles could also sequester and concentrate complex mixtures of bioactive PSC-secreted proteins, which may be developed into cell-free therapies in the future. Overall, this work broadens current understanding of bone tissue engineering, biomaterial delivery strategies, and stem cell-based therapeutics, and provides valuable insight into developing affinity-based biomaterials for clinical applications.
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    Heparin and PEG-based hydrogels to modulate and interrogate dynamic cell behavior
    (Georgia Institute of Technology, 2016-10-14) Rinker, Torri Elise
    Hydrogel-based biomaterials are often used for biomolecule delivery or encapsulation of cells for tissue engineering and regenerative medicine applications. However, utilizing hydrogels in dynamic cell systems can be challenging, as hydrogels must be engineered to account for changes in cellular behavior. For example, the hydrogel cell culture platforms and analyses techniques employed to investigate cell response to disease conditions should account for variations in cellular communication. In addition, hydrogels used to modulate cellular differentiation, either through protein delivery or direct interactions with cells, should account for evolving cell phenotype. Thus, in this work, hydrogel-based technologies were developed and utilized to interrogate and modulate dynamic cellular behavior. A PEG-based platform was designed and utilized to interrogate MSCs, adipocytes, and osteoblasts under hyperglycemic conditions via multivariate analyses, as these three cell types are implicated in abnormal deposition of marrow adipose tissue and bone in diabetes and osteoporosis. Then, as heparin binds many growth factors involved in cellular differentiation processes, heparin-based MPs were used to temporally modulate endochondral ossification in ATDC5 cells, possibly through heparin-mediated protein sequestration. To further modulate the timing of protein sequestration, heparin-PEG core-shell MPs were designed to enable sequestration and temporally controlled redelivery of protein. Finally, hydrolytically degradable heparin-PEG-based MPs were engineered with tunable heparin content and degradation rate, to enable temporally controlled protein release. Overall, this work demonstrates the ability of PEG and heparin-based hydrogels to investigate and regulate evolving cellular processes.
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    Engineering mesenchymal stromal cell constructs to enhance immunomodulation
    (Georgia Institute of Technology, 2016-07-05) Zimmermann, Joshua A.
    Mesenchymal stem/stromal cells (MSCs) are potent modulators of inflammatory and immune responses due to their ability to secrete soluble paracrine factors that regulate both innate and adaptive immunity and repolarize cells from pro-inflammatory to anti-inflammatory or pro-resolving phenotypes. The ability of MSCs to modulate multiple components that contribute to the complexity of an immune response further motivates the use of MSCs to treat diseases such as graft-versus host disease, inflammatory bowel disease, and autoimmune disorders. Multiple paracrine and immunomodulatory factors are expressed by MSCs that mediate suppression of immune cells and the coordinated action of the immunomodulatory secretome of MSCs is necessary to regulate complex immune responses. Importantly, many of these immunomodulatory factors are not constitutively expressed by resting MSCs and their expression is strongly induced by exposure of MSCs to inflammatory cytokines. Thus, MSC immunomodulation is highly dependent on the local inflammatory milieu to activate immunomodulatory factor expression and the efficacy of MSC-based cellular therapies is therefore highly dependent on the in vivo environment they are exposed to after injection. This environment may be highly variable based on the individual and disease being treated, the stage of inflammation, and the site of MSC transplantation. Therefore, the objective of this dissertation was to develop strategies to enhance intrinsic MSC immunomodulatory activity to improve cellular therapies for the treatment of inflammatory and immune diseases. Three-dimensional MSC constructs offer a promising approach to control the microenvironment and thereby the immunomodulatory activity of MSCs while also enhancing acute cell survival and persistence after transplantation in vivo. Furthermore, engineering the physical and chemical elements of the MSC construct microenvironment through biomaterial-based approaches serves as a novel route to regulate the temporal presentation of inflammatory factors in order to sustain immunomodulatory activity in vivo. Altogether, this strategy offers a novel translatable means of controlling MSC paracrine activity post-transplantation and therefore, improve the efficacy of MSC-based treatment strategies for inflammatory and immune diseases.
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    Engineering a Platform to Harness Pluripotent Stem Cell-Derived Paracrine Factors
    (Georgia Institute of Technology, 2015-11-10) Wilson, Jenna L
    The results of initial stem cell transplantation studies indicate that many of the observed functional improvements are due to transient paracrine actions of the transplanted stem cells, rather than the stem cells permanently engrafting and replacing the damaged cellular material. Thus, research on the identity and potency of paracrine factors secreted by stem cells has become an increased area of focus in the regenerative medicine field. Due to the mitogenic and morphogenic roles of embryonic stem cells (ESCs) during the early stages of development, they are an underexplored cell population which likely possess a unique and potent secretome. A potential application for the milieu of mitogens and morphogens produced by pluripotent stem cells is the restoration of the proliferative and regenerative capacity of adult stem cell populations, as these multipotent cells have a limited ability for expansion outside the body and are also negatively regulated by dysfunctional signals in vivo which are implicated in the reduced capacity for regeneration with injury or aging. To take advantage of the stimulatory potential of pluripotent cell-derived signals, the goal of this project was to develop a controlled means of harnessing and delivering soluble factors derived from pluripotent stem cells. This objective was accomplished through the (1) development of a microencapsulation-based culture system for ESC aggregates, (2) design of a novel upstream bioreactor for encapsulated ESC culture which enabled the concentration and delivery of stem cell secreted products, (3) characterization of the global expression profile of ESC-secreted factors, and (4) investigation of the influence of ESC-derived factors on adult stem and progenitor populations. Ultimately, this project established pluripotent stem cells as a unique source of potent growth factors and cytokines which can be regulated and concentrated using engineering design parameters to enable multiple applications in the field of regenerative medicine.
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    Incorporation of bio-inspired microparticles within embryonnic stem cell aggregates for directed differentiation
    (Georgia Institute of Technology, 2015-08-04) Sullivan, Denise D.
    Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.
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    Delivery of prolyl hydroxylase inhibitors to MSC spheroids for enhanced angiogenic factor secretion
    (Georgia Institute of Technology, 2015-07-24) Lassahn, Katy Ann
    Mesenchymal stromal cells (MSCs) are of clinical interest due to their ability to differentiate towards musculoskeletal lineages, modulate inflammatory responses, and promote new blood vessel formation. Angiogenesis is an important aspect of both tissue engineering and wound healing because it is critical to deliver essential nutrients and oxygen in order to facilitate tissue repair and regeneration. In hypoxic environments, the pro-angiogenic effects of MSCs are enhanced through stimulation of the HIF-1α pathway. A class of small molecules termed prolyl hydroxylase inhibitors (PHDi), stabilize HIF-1α through inhibition of the enzyme that degrades HIF-1α in the presence of oxygen. Thus, a chemically induced hypoxic cell response could be engineered to enable greater control over the pro-angiogenic secretory response of transplanted cells through varying the duration and dosage of exposure to PHDi. Treatment of MSCs with PHDi has been shown to enhance cell survival, improve bone regeneration, and increase new vessel formation in vivo. In addition to treatment with PHDi, the culture format of MSC growth can affect the angiogenic properties of MSCs. The culture of MSCs as spheroids has been shown to promote secretion of angiogenic growth factors such as VEGF. Thus, delivery of PHDi to MSC spheroids may have a greater effect on the angiogenic properties of MSCs than monolayer treatment. Sustained delivery of PHDi may be achieved within spheroids via biomaterial based microparticle incorporation. The microparticle delivery of PHDi within spheroids may allow for localized delivery of PHDi in order to reduce potential off target effects if delivered in vivo. The objective of this project is to determine the effect of sustained delivery of PHDi on the angiogenic properties of MSC spheroids. It is hypothesized that sustained delivery of a PHDi in MSC spheroids via MP incorporation will enhance the angiogenic factor secretion of the MSC spheroids compared to spheroid culture alone. To address this hypothesis, three candidate PHDi were screened to determine appropriate dosage based on VEGF secretion and efficiency of encapsulation within MPs. IOX2 was chosen to be encapsulated in poly lactic-co-glycolic acid (PLGA) microparticles (MPs) as a vehicle for sustained delivery within the spheroids. The effect of PHDi delivery on pro-angiogenic factor secretion was assessed by measuring expression of HIF-1α, secretion of angiogenic growth factors such as VEGF, and HUVEC migration assays. While soluble PHDi treatment of MSC spheroids had a significant effect on pro-angiogenic factor secretion, delivery of PHDi via PLGA MPs was unsuccessful. The ability to modulate the hypoxia response of MSC spheroids through PHDi delivery may prolong and enhance the pro-angiogenic effects of hypoxic environments on MSCs, thus alternative biomaterials should be investigated in the future for efficient PHDi delivery.
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    Analyzing multicellular interactions: A hybrid computational and biological pattern recognition approach
    (Georgia Institute of Technology, 2015-04-07) White, Douglas
    Pluripotent embryonic stem cells (ESCs) can differentiate into all somatic cell types, making them a useful platform for studying a variety of cellular phenomenon. Furthermore, ESCs can be induced to form aggregates called embryoid bodies (EBs) which recapitulate the dynamics of development and morphogenesis. However, many different factors such as gradients of soluble morphogens, direct cell-to-cell signaling, and cell-matrix interactions have all been implicated in directing ESC differentiation. Though the effects of individual factors have often been investigated independently, the inherent difficulty in assaying combinatorial effects has made it difficult to ascertain the concerted effects of different environmental parameters, particularly due to the spatial and temporal dynamics associated with such cues. Dynamic computational models of ESC differentiation can provide powerful insight into how different cues function in combination both spatially and temporally. By combining particle based diffusion models, cellular agent based approaches, and physical models of morphogenesis, a multi-scale, rules-based modeling framework can provide insight into how each component contributes to differentiation. I propose to investigate the complex regulatory cues which govern complex morphogenic behavior in 3D ESC systems via a computational rules based modeling approach. The objective of this study is to examine how spatial patterns of differentiation by ESCs arise as a function of the microenvironment. The central hypothesis is that spatial control of soluble morphogens and cell-cell signaling will allow enhanced control over the patterns and efficiency of stem cell differentiation in embryoid bodies.
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    Proteolytically degradable microparticles for engineering the extracellular microenvironment of pluripotent stem cell aggregates
    (Georgia Institute of Technology, 2014-12-12) Nguyen, Anh H.
    During embryo development, extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) and promotes downstream cell specifications. Pluripotent stem cell (PSC) aggregates can recapitulate various aspects of embryogenesis in vitro, and incorporation of biomaterial microparticles also provides an ideal platform to study cell-biomaterial interactions. Stem cell interactions with ECM-based biomaterials can impact tissue remodeling and differentiation propensity via modulation of MMP activity. This work investigated the MMP activity and subsequent mesenchymal differentiation of embryonic stem cell (ESC) aggregates with incorporated gelatin methacrylate (GMA) MPs with either low (20%) or high (90%) cross-linking densities, corresponding to faster or slower degradation rate, respectively. GMA MP incorporation increased total MMP and MMP-2 levels within 3D ESC aggregates in a substrate-dependent manner. GMA MP-incorporated aggregates also expressed higher levels of epithelial-to-mesenchymal transition markers and displayed enhanced mesenchymal morphogenesis than aggregates without MPs, and the MP-mediated effects were completely abrogated with MMP inhibitor treatment. This work predicts that control of proteolytic responses via introducing ECM-based MPs may offer a novel avenue to engineer the ECM microenvironment to modulate stem cell differentiation.
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    Chondroitin sulfate microparticles modulate TGF-B1-induced chondrogenesis in human mesenchymal stem cell spheroids
    (Georgia Institute of Technology, 2014-04-28) Goude, Melissa Chou
    Due to the limited intrinsic healing ability of mature cartilage tissue, stem cell therapies offer the potential to restore cartilage lost due to trauma or arthritis. Mesenchymal stem cells (MSCs) are a promising cell source due to their ability to differentiate into various adult tissues under specific biochemical and physical cues. Current MSC chondrogenic differentiation strategies employ large pellets, however, we have previously developed a high-throughput technique to form small MSC aggregates (500-1,000 cells) that may reduce diffusion barriers while maintaining a multicellular structure that is analogous to cartilaginous condensations. The objective of this study was to examine the effects on chondrogenesis of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within these small MSC spheroids when cultured in the presence of transforming growth factor-β1 (TGF-β1) over 21 days. Spheroids +MP induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids -MP, although no large differences in immunostaining for these matrix molecules were observed by day 21. Collagen I and X was also detected in the ECM of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. Because chondrogenesis was not hindered by the presence of CSMA MPs, this study demonstrates the utility of this culture system to further examine the effects of matrix molecules on MSC phenotype, as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific target tissues.