Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

Biofunctionalized polymer interfaces for capture, isolation, and characterization of bacteria

Doctor of PhilosophyDepartment of Chemical EngineeringRyan R. HansenThe goal of this research is to develop bio-functional interfaces, designed using polymeric materials, for improved separation and isolation of bacteria for detection and characterization. Microbes impact many aspects of our society, from health to environment to industrial processes. In most cases, microbes exist in complex environments, where thousands of other organisms may also be present. Thus, detecting and characterizing specific microbial targets often necessitates that they are first isolated. Polymeric materials hold several advantages for this type of separation. They can be modified with biomolecules to capture specific microorganisms and can be designed to release captured organisms on-demand using an environmental stimulus. This thesis will explore each of these concepts, beginning with (1) the design of patterned polymer interfaces to tailor the surface reactivity towards biomolecules, (2) bio-functionalization of surface polymers with lectin molecules for bacteria capture, and (3) bio-functional, photodegradable hydrogels for dissection of microbes from membrane surfaces during early-stage biofouling events. The first portion of this thesis aims at fabricating micro/nano-structured patterns of the novel block co-polymer, poly(glycidyl methacrylate)–block–poly(vinyl dimethyl azlactone) (PGMA₅₆-b-PVDMA₁₇₅) onto silicon slides. These polymers use azlactone-based reactions to covalently couple biomolecules to the surface. Bottom-up and top-down chemical co-patterning methods, including microcontact printing, parylene lift-off, and interface directed assembly are investigated for formation of reproducible, brush-like and crosslinked polymers on the substrates. The second portion of this thesis uses these polymer interfaces to capture microbial contaminants from solution using lectin-based binding. Lectin-functionalized interfaces are promising for affinity-based microorganism capture and isolation of bacteria from samples such as blood, urine, and wastewater. However, the equilibrium dissociation constants (K[subscript]D) of lectin-carbohydrate interactions, 2-3 orders of magnitude higher than antibody-antigen binding constants, results in poor cell capture efficiency. To address this limitation, surfaces are designed to combine reactive polymer coatings that generate high lectin surface densities with nanoscale surface structures, ultimately improving cell capture. Both detection sensitivity and bactericidal impact of these optimized surfaces are characterized. Finally, the competing effects on capture due to lectin surface density and due to exopolysaccharide expression levels on the bacteria cell surface is compared. The final portion of this thesis focuses on the use of lectin-functionalized, photodegradable hydrogels to separate and isolate microbes that attach to membrane surfaces during early-stage biofouling, an approach termed polymer surface dissection (PSD). Photo-responsive, biofunctional polyethylene glycol (PEG)-based hydrogels are developed to detach targeted biofilm flocs or cells adhered onto PVDF membranes. A patterned illumination tool then delivers light to the hydrogel in a spatiotemporally controlled manner to release an extracted floc without damage. Microbes can then be sequenced to identify the composition of biofilm flocs at different stages of aggregation. The PSD approach can be used to characterize biofouling in many membrane-based bioseparation processes, here it has been developed to investigate membrane biofouling in anaerobic membrane bioreactors. Understanding the initial stages of biofouling from a mechanistic standpoint could help understand the critical microorganisms in wastewater communities that initiate the biofouling process, information that can inform novel techniques to mitigate biofilm formation

Laser nano-neurosurgery from gentle manipulation to nano-incision of neuronal cells and scaffolds: an advanced neurotechnology tool

Current optical approaches are progressing far beyond the scope of monitoring the structure and function of living matter, and they are becoming widely recognized as extremely precise, minimally-invasive, contact-free handling tools. Laser manipulation of living tissues, single cells, or even single-molecules is becoming a well-established methodology, thus founding the onset of new experimental paradigms and research fields. Indeed, a tightly focused pulsed laser source permits complex tasks such as developing engineered bioscaffolds, applying calibrated forces, transfecting, stimulating, or even ablating single cells with subcellular precision, and operating intracellular surgical protocols at the level of single organelles. In the present review, we report the state of the art of laser manipulation in neuroscience, to inspire future applications of light-assisted tools in nano-neurosurgery

3D Nanoprinting Technologies for Tissue Engineering Applications

Tissue engineering recovers an original function of tissue by replacing the damaged part with a new tissue or organ regenerated using various engineering technologies. This technology uses a scaffold to support three-dimensional (3D) tissue formation. Conventional scaffold fabrication methods do not control the architecture, pore shape, porosity, or interconnectivity of the scaffold, so it has limited ability to stimulate cell growth and to generate new tissue. 3D printing technologies may overcome these disadvantages of traditional fabrication methods. These technologies use computers to assist in design and fabrication, so the 3D scaffolds can be fabricated as designed and standardized. Particularly, because nanofabrication technology based on two-photon absorption (2PA) and on controlled electrospinning can generate structures with submicron resolution, these methods have been evaluated in various areas of tissue engineering. Recent combinations of 3D nanoprinting technologies with methods from molecular biology and cell dynamics have suggested new possibilities for improved tissue regeneration. If the interaction between cells and scaffold system with biomolecules can be understood and controlled and if an optimal 3D environment for tissue regeneration can be realized, 3D nanoprinting will become an important tool in tissue engineering

Two-photon sensitive biomaterials for dynamic control of cellular microenvironments

Two-photon (2P) activable photocleavable protecting groups (PPGs) can be introduced in polymer networks as photodegradation sites or as blocking groups for active sites, which enable the alternation of mechanical properties and biochemical signals and allow to study consequent cell response in a spatiotemporal controlled manner. So far, the design of high efficient 2P activable hydrogels is challenging. This Thesis presents novel designs of photodegradable hydrogels that contain the 4’-methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl (PMNB) PPG. PMNB-gels formed under physiological conditions and showed tuneable hydrolytic stability and adequate rate for cell encapsulation. Moreover, PMNB-gels can be photodegraded efficiently upon 2P excitation (λ = 740 nm). Preliminary experiments of PMNB-gels as 4D matrices for the investigation of cell response are presented. In a second part, a 2P-activatable PPGs endowed with an extended π conjugation was demonstrated and introduced to yield the RGD cell adhesive peptide. The targeted peptide is obtained but only in low yield due to its low stability. The results of this Thesis provide new tools for instructing cells in 3D cultures using 2P-activated processes and demonstrate the potential of photochemistry for the realization of 4D biomaterials.Zwei-Photonen-(2P)-aktivierbare photolytisch spaltbare Schutzgruppen (PPGs) können in Polymernetzwerke als photokysestellen oder als Schutzgruppen für aktive Stellen eingeführt werden, das Alternieren von mechanischen Eigenschaften und biochemischen Signalen ermöglichen und es erlauben, die daraus resultierende Zellreaktion in einer räumlich-zeitlich kontrollierten Weise zu untersuchen. Bisher ist das Design von hocheffizienten 2P-aktivierbaren Hydrogelen eine Herausforderung. In dieser Arbeit werden neuartige Designs von photodegradierbaren Hydrogelen vorgestellt, die 4'-Methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl (PMNB) PPG enthalten. PMNB-Gele bildeten sich unter physiologischen Bedingungen und zeigten eine einstellbare hydrolytische Stabilität und eine angemessene Geschwindigkeit für die Immobilisierung von Zellen. Darüber hinaus können PMNB-Gele bei 2P-Anregung (λ = 740 nm) effizient photolytisch abgebaut werden. Es werden erste Experimente mit PMNB-Gelen als 4D-Matrizen für die Untersuchung der Zellreaktion vorgestellt. In einem zweiten Teil wurde ein eine 2P-aktivierbares PPGs mit einer verlängerten π-Konjugation demonstriert und eingeführt, um das zelladhäsive RGD-Peptid zu erhalten. Das angestrebte Peptid wurde gewonnen, allerdings aufgrund seiner geringen Stabilität nur in geringer Ausbeute. Die Ergebnisse dieser Arbeit liefern neue Werkzeuge für die Steuerung von Zellen in 3D-Kulturen mit Hilfe von 2P-aktivierbaren Prozessen und zeigen das Potenzial der Photochemie für die Realisierung von 4D-Biomaterialien

InVERT molding for scalable control of tissue microarchitecture

Complex tissues contain multiple cell types that are hierarchically organized within morphologically and functionally distinct compartments. Construction of engineered tissues with optimized tissue architecture has been limited by tissue fabrication techniques, which do not enable versatile microscale organization of multiple cell types in tissues of size adequate for physiological studies and tissue therapies. Here we present an ‘Intaglio-Void/Embed-Relief Topographic molding’ method for microscale organization of many cell types, including induced pluripotent stem cell-derived progeny, within a variety of synthetic and natural extracellular matrices and across tissues of sizes appropriate for in vitro, pre-clinical, and clinical studies. We demonstrate that compartmental placement of non-parenchymal cells relative to primary or induced pluripotent stem cell-derived hepatocytes, compartment microstructure, and cellular composition modulate hepatic functions. Configurations found to sustain physiological function in vitro also result in survival and function in mice for at least 4 weeks, demonstrating the importance of architectural optimization before implantation.National Institutes of Health (U.S.) (EB008396)National Institutes of Health (U.S.) (DK56966)National Cancer Institute (U.S.) (Cancer Center Support Core Grant P30-CA14051)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (1F32DK091007)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (1F32DK095529)National Science Foundation (U.S.). Graduate Research Fellowship Program (1122374

Competition and Conjugation Between Agrobacterial Cooperators and Cheaters

Doctor of PhilosophyDepartment of BiologyThomas G PlattNatural selection favors selfish behaviors that undermine the stability of cooperative systems. Despite this, cooperation is widespread throughout nature, including in the microbial world. This dissertation examines the interactions between bacterial cheaters and cooperators, and how these interactions influence the evolution of cooperation and virulence. Bacterial cooperators often pay a fitness cost to provide a benefit, a public good, that benefits the cooperator and other nearby individuals. These public goods drive the emergence of cheaters, individuals that do not pay the costs of cooperation but do benefit from the public goods produced by cooperators. Because cheaters have an inherent competitive advantage over cooperators, they threaten the stability of cooperative systems. Cooperative individuals may employ strategies that antagonize cheaters, thereby preventing cheaters from spreading through the population. In Agrobacterium tumefaciens, a plant pathogen and the causative agent of crown gall disease, cooperation is a key feature of its infection of host plants. Cooperative agrobacteria carry the tumor inducing (Ti) plasmid that encodes the virulence genes required for the genetic transformation of plants. The act of infecting the plants is metabolically costly, involving the expression of the vir genes required to form a type IV secretion system and the effectors that mediate delivery of the T-DNA (transferred DNA) into the plant’s genome. This genetic transformation of the plant by the T-DNA results in the misregulation of plant hormones and the production of opines, small compounds that serve as a nutrient source for agrobacteria. Cheater agrobacteria, individuals that do not infect plants, but do catabolize opines, replicate faster than their cooperative counterparts. Yet, despite this expected competitive advantage, natural cheaters have not been observed dominating the agrobacterial populations associated with natural galls. My research focuses on three main areas related to the interaction between agrobacterial cooperators and cheaters: the population ecology of cheaters, conjugation of the Ti plasmid into cheaters, and screening platforms for the study of competitive interactions between cheaters and cooperators. Broadly, I demonstrated that ΔvirA mutants are cheaters that have a large fitness advantage over virulent agrobacteria. Further, I showed that in gall-like environments the expression of virulence is very costly for agrobacterial cooperators. As a result, agrobacterial cheaters readily arise de novo in these environments. I explored the antagonistic interactions between cheaters and cooperators, focusing on whether pathogenic agrobacteria used horizontal gene transfer as a policing mechanism to prevent cheaters from taking over. In A. tumefaciens, conjugation is regulated in response to bacterial cell density by quorum sensing (QS). To understand the effects of conjugation on cheater policing, I carried out competitions involving mutants lacking TraR. I found that TraR is necessary for conjugation and that traR+ cooperators compete more favorably against ΔvirA cheaters than do traR- cooperators. However, conjugation alone will not antagonize cheater spread when they are already present in a population. In contrast, when cheaters arise via de novo mutations, conjugation can serve as a policing mechanism against freeloaders. Finally, driven by the limitations of current platforms for the screening of microbial interactions, I collaborated on the development of a new tool for the screening of microbial interactions. I used a photodegradable hydrogel that allows for high-throughput screening of bacterial populations in just one experimental trial. As a proof-of-principle, I studied a known interaction between an agrobacterial cooperator and a cheater and demonstrated that our approach allows the screening of entire transposon mutant libraries in a single experiment. In a first screen of this kind, I was able to identify, extract, and characterize rare cells (9/28,000) using this high-throughput approach. Thus, photodegradable hydrogels offer a powerful, straightforward, and adaptable approach that can be used not only for the screening of cheater-cooperator competitive interactions, but also more broadly for the study of other microbial interactions

Hydrogel interfaces for applications in microbial biotechnology

Doctor of PhilosophyDepartment of Chemical EngineeringRyan R. HansenHydrogels are three-dimensional, water-swollen, highly crosslinked polymers that can be designed to provide biocompatible and biofunctional interfaces for cells and biomolecules. With facile fabrication and precise control over chemistry, pore size, and mechanical properties, hydrogels have been studied extensively in various areas of biomedical and bioengineering, particularly in drug delivery and tissue engineering applications. However, hydrogels have not been well-studied or well-applied to many emerging applications in microbiology. This thesis explores two new applications involving hydrogel interfaces: (1) photodegradable hydrogels for high-throughput screening and isolation of rare bacteria and (2) hydrogels for protection of electroactive biofilms from environmental shocks in microbial electrolysis cell systems. The initial portion of this thesis focuses on the use of photodegradable hydrogels for microbial cell screening and rare cell isolation. The photodegradable hydrogel used here was formed with polyethylene glycol (PEG) o-nitrobenzyl acrylate and PEG-tetrathiol macromers, which form three-dimensional hydrogels through thiol-acrylate addition reactions to encapsulate heterogenous populations of bacterial cells. The individual entrapped cells can be cultured into clonal microcolonies due to the suitable hydrogel mesh size for nutrient transport to the cells. Cells are monitored en masse and rare cells showing unique growth phenotypes are identified and extracted from the hydrogel interface using a high-resolution light patterning tool. The optimum experimental setup for achieving high throughput observation and clean extraction was developed. Release kinetics with light dose, the effect of light pattern on cell morphology, and the DNA quality of the extracted cells after exposure to 365 nm light patterns was also investigated. We demonstrated the use of this approach as a screening interface by rapidly screening a mutant library of the Gram-negative bacteria Agrobacterium tumefaciens to identify, isolate, and genetically characterize strains with rare growth profiles. The reported method offers an inexpensive and practical approach to cell screening and cell sorting and can be applied to a wide range of applications where isolating phenotypically pure cells from complex, heterogenous mixtures is essential. This includes applications in microbiology, microbial therapeutics, and biomedical diagnostics. The next section of this thesis focuses on developing PEG-based hydrogels that are designed to protect electroactive biofilms from harsh environmental stressors. The coating was fabricated using PEG-tetrathiol and PEG-divinyl sulfone macromers that form hydrogels with crosslinks resistant to degradation from acid or base hydrolysis, while still promoting nutrient diffusion and electron transport. Methods of fabricating anodes containing electroactive biofilms with the hydrogels are first reported, followed by investigation of the hydrolytic stability of the coatings. Transport of a carbon source (acetate) through the coating is then modeled, and the long-term stability and compatibility of the coating over the biofilm is investigated. Lastly, the effect of the coating on the biofilm recovery from an environmental shock (ammonium exposure) is demonstrated to emphasize the potential benefit of the coating. Finally, the future directions of hydrogels in these applications are recommended, which include discussion on developing a hydrogel chemistry that is degradable on exposure to a near-infrared (NIR) light source as well as discussion on chemical and biological hydrogel additives that will improve its performance

Screening and discovery of symbiotic and antagonistic microbial networks using microwell recovery arrays

Doctor of PhilosophyDepartment of Chemical EngineeringRyan R. HansenThomas G. PlattUnderstanding the dynamic interactions among soil and plant rhizosphere microbiomes is critical for predicting community function and developing improved probiotic and biocontrol agents for plant growth, biofuel production, and human health. However, uncovering these interactions is a grand challenge in microbiology due to the lack of experimental tools suitable for discovery and characterization. This research develops high throughput microwell recovery arrays (MRA) combined with advanced bioinformatics techniques to screen and detect microbial interactions across soil/root microbial communities to uncover bacteria species with an important function. The first part of this thesis describes developing a novel, light-responsive, step-polymerized poly(ethylene glycol) hydrogel membrane to retrieve cells from MRAs with a high degree of spatial control. The utility of microwell arrays, particularly in screening applications, could be significantly expanded if cells of interest could be removed from individual wells for subsequent genetic and phenotypic characterizations. The photodegradability of the membrane permits exchange of nutrients and waste products and seals motile bacteria within microwells and enables individual wells of interest to be opened using a patterned UV light for selective release and retrieval. The second part of the thesis demonstrates the unique application of the MRA platform to discover multi-membered consortia that generate emergent outcomes. The platform was initially developed to discover dual-species co-culture and interactions between two well-characterized interaction pairs, Agrobacterium tumefaciens and Pseudomonas aeruginosa. After investigating the on-chip co-culture using this pair, Populus trichocarpa rhizosphere microbiome was screened for strains affecting the growth of Pantoea sp. YR343, an indole-3-acetic acid (IAA) producing, plant growth-promoting bacteria isolated from Populus deltoides rhizosphere. The third chapter of the thesis uses this approach for enhancing the survival and colonization of commercial nitrogen-fixing, plant growth-promoting bacteria, Azospirillum brasilense, into maize roots to improve crop yield. Diazotrophs such as Azospirillum brasilense function as biofertilizers by colonizing plant roots and enhancing plant productivity through symbiotic interactions within the rhizosphere. Using the MRAs, new isolates showing that promote A. brasilense growth were extracted and identified by 16S sequencing as Serratia mercescens, Serratia nematodiphila, Serratia urelytica, Pantoea agglomerans, Enterobacter tabaci, and Acinetobacter bereziniae, and the interactions were validated off-chip in 96 well plate reader. Also, the growth enhancement and the improvement of the survival and colonization of A. brasilense in Zea mays roots were validated in plant growth chamber experiments, demonstrating the potential to apply the interactions found in vitro towards in vivo systems of agricultural relevance. In the final chapter of the thesis, the screening capabilities using MRAs were further extended towards screening non-pathogenic Agrobacterium isolates for the growth inhibition of pathogenic A. tumefaciens, which is a key plant biotechnology tool and also the causative agent of Crown Gall disease. MRAs were used to combine fluorescently labeled A. tumefaciens sp.15955 with non-pathogenic Agrobacterium isolates collected from native plant roots at the Konza Prairie Biological Station (Manhattan, KS) to uncover several candidates for inhibiting A.tumefaciens sp. 15955 growth. The discovery of such growth-inhibiting isolates will help improve plant productivity by using them as reliable biocontrol agents that prevent Crown Gall disease, and further demonstrates the unique capability of the MRA platform to screen natural isolate collections to discover bacteria capable of inhibiting pathogens