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Wallace H. Coulter Department of Biomedical Engineering

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Now showing 1 - 10 of 14
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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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    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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    Biophysical and biochemical control of three-dimensional embryonic stem cell differentiation and morphogenesis
    (Georgia Institute of Technology, 2014-04-08) Kinney, Melissa
    Stem cell differentiation is regulated by the complex interplay of multiple parameters, including adhesive intercellular interactions, cytoskeletal and extracellular matrix remodeling, and gradients of agonists and antagonists that individually and collectively vary as a function of spatial locale and temporal stages of development. Directed differentiation approaches have traditionally focused on the delivery of soluble morphogens and/or the manipulation of culture substrates in two-dimensional, monolayer cultures, with the objective of achieving large yields of homogeneously differentiated cells. However, a more complete understanding of stem cell niche complexity motivates tissue engineering approaches to inform the development of physiologically relevant, biomimetic models of stem cell differentiation. The capacity of pluripotent stem cells to simultaneously differentiate toward multiple tissue-specific cell lineages has prompted the development of new strategies to guide complex, three-dimensional morphogenesis of functional tissue structures. The objective of this project was to characterize the spatiotemporal dynamics of stem cell biophysical characteristics and morphogenesis, to inform the development of ESC culture technologies to present defined and tunable cues within the three-dimensional spheroid microenvironment. The hypothesis was that the biophysical and biochemical cues present within the 3D microenvironment are altered in conjunction with morphogenesis as a function of stem cell differentiation stage. Understanding biochemical and physical tissue morphogenesis, including the relationships between remodeling of cytoskeletal elements and intercellular adhesions, associated developmentally relevant signaling pathways, and the physical properties of the EB structure together elucidate fundamental cellular interactions governing embryonic morphogenesis and cell specification. Together, this project has established a foundation for controlling, characterizing, and systematically perturbing aspects of stem cell microenvironments in order to guide the development of complex, functional tissue structures for regenerative therapies.
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    Osteoinductive material derived from differentiating embryonic stem cells
    (Georgia Institute of Technology, 2012-04-15) Sutha, Ken
    The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.
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    Biomaterial integration within 3D stem cell aggregates for directed differentiation
    (Georgia Institute of Technology, 2011-11-14) Bratt-Leal, Andrés Miguel
    The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation. The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs. Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.