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

Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models.

To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states

Sensing the difference: the influence of anisotropic cues on cell behavior

From tissue morphogenesis to homeostasis, cells continuously experience and respond to physical, chemical and biological cues commonly presented in gradients. In this article we focus our discussion on the importance of nano/micro topographic cues on cell activity, and the role of anisotropic milieus play on cell behavior, mostly adhesion and migration. We present the need to study physiological gradients in vitro. To do this, we review different cell migration mechanisms and how adherent cells react to the presence of complex tissue-like environments and cell-surface stimulation in 2D and 3D (e.g. ventral/dorsal anisotropy)

Modeling the growth of multicellular cancer spheroids in a\ud bioengineered 3D microenvironment and their treatment with an\ud anti-cancer drug

A critical step in the dissemination of ovarian cancer cells is the formation of multicellular spheroids from cells shed from the primary tumor. The objectives of this study were to establish and validate bioengineered three-dimensional (3D) microenvironments for culturing ovarian cancer cells in vitro and simultaneously to develop computational models describing the growth of multicellular spheroids in these bioengineered matrices. Cancer cells derived from human epithelial ovarian carcinoma were embedded within biomimetic hydrogels of varying stiffness and cultured for up to 4 weeks. Immunohistochemistry was used to quantify the dependence of cell proliferation and apoptosis on matrix stiffness, long-term culture and treatment with the anti-cancer drug paclitaxel.\ud \ud Two computational models were developed. In the first model, each spheroid was treated as an incompressible porous medium, whereas in the second model the concept of morphoelasticity was used to incorporate details about internal stresses and strains. Each model was formulated as a free boundary problem. Functional forms for cell proliferation and apoptosis motivated by the experimental work were applied and the predictions of both models compared with the output from the experiments. Both models simulated how the growth of cancer spheroids was influenced by mechanical and biochemical stimuli including matrix stiffness, culture time and treatment with paclitaxel. Our mathematical models provide new perspectives on previous experimental results and have informed the design of new 3D studies of multicellular cancer spheroids

Mechanobiology of Epithelial Clusters in ECMs of Diverse Mechanical Properties

Cell clusters reside in complex extracellular matrices (ECMs) of varying mechanical properties. Epithelial cells sense and translate the mechanical cues presented by the surrounding ECM into biochemical signals through a process called ‘mechanotransduction’, which controls fundamental aspects of disease and development. During the course of metastasis, mechanical changes in the tumor microenvironment can lead to declustering of epithelial cells through a process called epithelial-to-mesenchymal transition (EMT). Throughout different steps of metastasis, escaped epithelial clusters encounter heterogeneous tissues of varying mechanical properties that ultimately influence their behavior in distant locations within the body. This dissertation investigates the mechanobiology of epithelial clusters inside mechanically heterogeneous tissue microenvironments. Chapter 1 provides an introduction for the mechanobiology of epithelial clusters and describes how mechanical properties of the microenvironment mediates behavior of epithelial cells. Chapter 2 addresses the mechano-regulated epithelial to mesenchymal transition inside matrices of varying stiffness and confinement. Growing evidence suggests that high extracellular matrix (ECM) stiffness induces EMT. Yet, very little is known about how various geometrical parameters of the ECM might influence EMT. To this end, we develop polyacrylamide (PA)-microchannels based matrix platform to culture mammary epithelial cell clusters in ECMs of tunable stiffness and confinement. Our results demonstrate that ECM confinement alone is able to induce EMT in epithelial clusters surrounded by a soft matrix, which otherwise protects against EMT in unconfined environments. Also, we demonstrate that stiffness- and confinement-induced EMT work through cell-matrix adhesions and cytoskeletal polarization, respectively. In chapter 3, we examined the combinatorial effect of phenotypic heterogeneity and matrix heterogeneity in determining the overall migration of the migrating clusters and the motion of individual cells in the cluster. These findings may provide insights into the effect of cellular heterogeneity on the epithelial dynamics during the early stage of cancer progression. In chapter 4, we examined the collective migration of epithelial cells across physically diverse matrices. Although the influence of matrix stiffness on cell migration is well-recognized, it remains unknown whether these matrix-dependent cellular features persist even after cells move to a new microenvironment. We have discovered that epithelial cells primed on a stiff matrix migrate faster, generate higher actomyosin expression, and retain nuclear YAP even after arriving on a soft matrix, as compared to their control behavior on a homogeneously soft matrix. Our results have uncovered a mechanical memory of past matrix stiffness in collective migration of normal and cancer epithelial cells. The depletion of YAP dramatically reduces this memory-dependent migration. These revelations imply that, during metastasis, changes in tumor microenvironment stiffness may influence the future invasion of escaping tumor cells

Quantitative signature for architectural organization of regulatory factors using intranuclear informatics

Regulatory machinery for replication and gene expression is punctately organized in supramolecular complexes that are compartmentalized in nuclear microenvironments. Quantitative approaches are required to understand the assembly of regulatory machinery within the context of nuclear architecture and to provide a mechanistic link with biological control. We have developed \u27intranuclear informatics\u27 to quantify functionally relevant parameters of spatially organized nuclear domains. Using this informatics strategy we have characterized post-mitotic reestablishment of focal subnuclear organization of Runx (AML/Cbfa) transcription factors in progeny cells. By analyzing point mutations that abrogate fidelity of Runx intranuclear targeting, we establish molecular determinants for the spatial order of Runx domains. Our novel approach provides evidence that architectural organization of Runx factors may be fundamental to their tissue-specific regulatory function

Growth of confined cancer spheroids: a combined experimental and mathematical modelling approach

We have integrated a bioengineered three-dimensional platform by generating multicellular cancer spheroids in a controlled microenvironment with a mathematical model to investigate\ud confined tumour growth and to model its impact on cellular processes

Spatially and Temporally Controlled Mechanical Signals to Direct Human Mesenchymal Stem Cell Behavior

In order to effectively incorporate stem cells into tissue engineering solutions, a deeper understanding of the microenvironment factors that influence their behaviors is necessary. Specifically, the inherent mechanics of the extracellular matrix (ECM) have been shown to profoundly effect multiple stem cell behaviors such as their morphology, proliferation, differentiation, and secretion of factors. The effect of matrix mechanics on stem cells has been investigated using a wide range of material systems; however, many of these systems lack the mechanical complexity that native tissues possess in terms of their spatial and temporal properties, as well as context (2D vs. 3D). In order to determine the effect of heterogeneous and dynamic mechanical signals on stem cells, a sequential crosslinking technique was developed that allowed for formation of hydrogels with a wide range in mechanical properties in terms of magnitude, context, and spatiotemporal presentation. Hydrogels with tunable mechanics were synthesized using methacrylate hyaluronic acid (MeHA) in a sequential process: 1) Michael-type `addition\u27 crosslinking using dithiothreitol to consume a fraction of the methacrylate groups, and 2) UV-initiated `radical\u27 crosslinking using controlled UV light exposure in the presence of a photoinitiator to consume unreacted methacrylates. Using this approach, we demonstrated local control of stem cell morphology, proliferation, and differentiation (adipogenesis and osteogenesis) in both patterned and gradient systems on 2D hydrogels. We further investigated the effects of mechanics in a 3D context using non-porous and porous presentations of controlled mechanics. In the non-porous system, cell behavior was shown to be dependent on mechanics as threshold responses were observed related to the ability of hMSCs to adopt a spread or rounded morphology within the hydrogel. In the 3D macroporous system, mechanics were spatially and temporally modulated and hMSC morphology, proliferation, differentiation, and secretion of angiogenic and cytokine factors were shown to be dependent on the local and temporal presentation of mechanical signals. This dissertation work emphasizes the importance of the magnitude, context, and presentation of mechanical signals and highlights this sequential crosslinking process as a model system for future investigations into heterogeneous, dynamic microenvironments, as well as a novel platform for developing future tissue engineering strategies

Combining Smart Material Platforms and New Computational Tools to Investigate Cell Motility Behavior and Control

Cell-extracellular matrix (ECM) interactions play a critical role in regulating important biological phenomena, including morphogenesis, tissue repair, and disease states. In vivo, cells are subjected to various mechanical, chemical, and electrical cues to collectively guide their functionality within a specific microenvironment. To better understand the mechanisms regulating cell adhesive, differentiation, and motility dynamics, researchers have developed in vitro platforms to synthetically mimic native tissue responses. While important information about cell-ECM interactions have been revealed using these systems, a knowledge gap currently exists regarding how cell responses in static environments relate to the dynamic cell-ECM interaction behaviors observed in vivo. Advances at the intersection of materials science, biophysics, and cell biology have recently enabled the production of dynamic ECM mimics where cells can be exposed to controlled mechanical, electrical or chemical cues to directly decouple cell-ECM related behaviors from cell-cell or cell-environmental factors. Utilization of these dynamic synthetic biomaterials will enable discovery of novel mechanisms fundamental in tissue development, homeostasis, repair, and disease. In this dissertation, the primary goal was to evaluate how mechanical changes in the ECM regulate cell motility and polarization responses. This was accomplished through two major aims: 1) by developing a modular image processing tool that could be applied in complex synthetic in vitro microenvironments to asses cell motility dynamics, and 2) to utilize that tool to advance understanding of mechanobiology and mechanotransduction processes associated with development, wound healing, and disease progression. Therefore, the first portion of this thesis (Chapters 2 and 3) dealt with proof of concept for our newly developed automated cell tracking system, termed ACTIVE (automated contour-based tracking for in vitro environments), while the second portion of this thesis (Chapter 4-7) addressed applying this system in multiple experimental designs to synthesize new knowledge regarding cell-ECM or cell-cell interactions. In Chapter 1, we introduced why cell-ECM interactions are essential for in vivo processes and highlighted the current state of the literature. In Chapter 2, we demonstrated that ACTIVE could achieve greater than 95% segmentation accuracy at multiple cell densities, while improving two-body cell-cell interaction error by up to 43%. In Chapter 3 we showed that ACTIVE could be applied to reveal subtle differences in fibroblast motility atop static wrinkled or static non-wrinkled surfaces at multiple cell densities. In Chapters 4 and 5, we characterized fibroblast motility and intracellular reorganization atop a dynamic shape memory polymer biomaterial, focusing on the role of the Rho-mediated pathway in the observed responses. We then utilized ACTIVE to identify differences in subpopulation dynamics of monoculture versus co-culture endothelial and smooth muscle cells (Chapter 6). In Chapter 7, we applied ACTIVE to investigate E. coli biofilm formation atop poly(dimethylsiloxane) surfaces with varying stiffness and line patterns. Finally, we presented a summary and future work in Chapter 8. Collectively, this work highlights the capabilities of the newly developed ACTIVE tracking system and demonstrates how to synthesize new information about mechanobiology and mechanotransduction processes using dynamic biomaterial platforms

In vivo imaging reveals transient microglia recruitment and functional recovery of photoreceptor signaling after injury.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo