Quinton Smith

Design and development of microbioreactors for long-term cell culture in controlled oxygen microenvironments.

The ability to control the oxygen level to which cells are exposed in tissue culture experiments is crucial for many applications. Here, we design, develop and test a microbioreactor (MBR) for long-term cell culture studies with the capability to accurately control and continuously monitor the dissolved oxygen (DO) level in the cell microenvironment. In addition, the DO level can be controlled independently from other cues, such as the viscous shear-stress acting on the cells. We first analyze the transport of oxygen in the proposed device and determine the materials and dimensions that are compatible with uniform oxygen tension and low shear-stress at the cell level. The device is also designed to culture a statistically significant number of cells. We use fully transparent materials and the overall design of the device is compatible with live-cell imaging. The proposed system includes real-time read-out of actual DO levels, is simple to fabricate at low cost, and can be easily expanded to control the concentration of other microenvironmental solutes. We performed control experiments in the absence of cells to demonstrate that the MBR can be used to accurately modulate DO levels ranging from atmospheric level to 1%, both under no flow and perfusion conditions. We also demonstrate cancer cell attachment and viability within the MBR. The proposed MBR offers the unprecedented capability to perform on-line measurement and analysis of DO levels in the microenvironment of adherent cultures and to correlate them with various cellular responses.

Stochasticity and Spatial Interaction Govern Stem Cell Differentiation Dynamics.

Stem cell differentiation underlies many fundamental processes such as development, tissue growth and regeneration, as well as disease progression. Understanding how stem cell differentiation is controlled in mixed cell populations is an important step in developing quantitative models of cell population dynamics. Here we focus on quantifying the role of cell-cell interactions in determining stem cell fate. Toward this, we monitor stem cell differentiation in adherent cultures on micropatterns and collect statistical cell fate data. Results show high cell fate variability and a bimodal probability distribution of stem cell fraction on small (80–140 μm diameter) micropatterns. On larger (225–500 μm diameter) micropatterns, the variability is also high but the distribution of the stem cell fraction becomes unimodal. Using a stochastic model, we analyze the differentiation dynamics and quantitatively determine the differentiation probability as a function of stem cell fraction. Results indicate that stem cells can interact and sense cellular composition in their immediate neighborhood and adjust their differentiation probability accordingly. Blocking epithelial cadherin (E-cadherin) can diminish this cell-cell contact mediated sensing. For larger micropatterns, cell motility adds a spatial dimension to the picture. Taken together, we find stochasticity and cell-cell interactions are important factors in determining cell fate in mixed cell populations.

Micropattern size-dependent endothelial differentiation from a human induced pluripotent stem cell line

The multifaceted extracellular milieu presents biochemical and biophysical stimuli that influence stem cell differentiation. Two‐dimensional (2D) micropatterned substrates allow the presentation of these cues in spatially defined geometries that have been demonstrated to guide stem cell fate decisions. Leveraging stem cells to reconstruct microvasculature, made up of an inner lining of endothelial cells (ECs) supported by pericytes, is critical to tissue‐engineering advances; thus, methods to improve endothelial differentiation efficiency are vital to these efforts. In this study, we examine the hypothesis that the diameter of micropatterned islands influences endothelial differentiation from human induced pluripotent stem cells (hiPSCs). Comparing island diameters of 80, 140, 225 and 500 µm, we found that co‐cultures of control ECs and pericytes did not yield variable ratios of cell types; however, when hiPSCs were differentiated toward a bicellular population of ECs and pericytes on these varying micropattern feature sizes, we found that smaller islands promoted EC differentiation efficiency, yielding a derived population composed of 70% ECs, which exhibited a greater sprouting propensity. Differentiation on the largest feature size exhibited a smaller EC yield, similar to that on non‐patterned substrates. Taken together, these data demonstrate that micropatterned islands of varying diameters can be used to modulate EC differentiation efficiency. Copyright

Hypoxia Affects the Structure of Breast Cancer Cell-Derived Matrix to Support Angiogenic Responses of Endothelial Cells

Hypoxia, a common feature of the tumor environment and participant in tumor progression, is known to alter gene and protein expression of several Extracellular Matrix (ECM) proteins, many of which have roles in angiogenesis. Previously, we reported that ECM deposited from co-cultures of Neonatal Fibroblasts (NuFF) with breast cancer cells, supported 3-dimensional vascular morphogenesis. Here, we sought to characterize the hypoxic ECM and to identify whether the deposited ECM induce angiogenic responses in Endothelial Cells (ECs). NuFF and MDA-MB-231 breast cancer cells were co-cultured, subjected to alternating cycles of 24 hours of 1% (hypoxia) and 21% (atmospheric) oxygen and de-cellularized for analyses of deposited ECM. We report differences in mRNA expression profiles of matrix proteins and crosslinking enzymes relevant to angiogenesis in hypoxia-exposed co-cultures. Interestingly, overt differences in the expression of ECM proteins were not detected in the de-cellularized ECM; however, up-regulation of the cell-binding fragment of fibronecin was observed in the conditioned media of hypoxic co-cultures. Ultrastructure analyses of the de-cellularized ECM revealed differences in fiber morphology with hypoxic fibers more compact and aligned, occupying a greater percent area and having larger diameter fibers than atmospheric ECM. Examining the effect of hypoxic ECM on angiogenic responses of ECs, morphological differences in Capillary-Like Structures (CLS) formed atop de-cellularized hypoxic and atmospheric ECM were not evident. Interestingly, we found that hypoxic ECM regulated the expression of angiogenic factors and matrix metalloproteinases in CLS. Overall, we report that in vitro, hypoxia does not alter the composition of the ECM deposited by co-cultures of NuFF/MDA-MB-231, but rather alters fiber morphology, and induces vascular expression of angiogenic growth factors and metalloproteinases. Taken together, these results have important implications for understanding how the hypoxic matrix may regulate angiogenesis in tumors.

Compliant substratum guides endothelial commitment from human pluripotent stem cells

Surface stiffness during the early stages of stem cell differentiation affects vascular fate under chemically defined conditions. The role of mechanical regulation in driving human induced pluripotent stem cell (hiPSC) differentiation has been minimally explored. Although endothelial cell (EC) fate from hiPSCs has been demonstrated using small molecules to drive mesoderm induction, the effects of substrate stiffness with regard to EC differentiation efficiency have yet to be elucidated. We hypothesized that substrate compliance can modulate mesoderm differentiation kinetics from hiPSCs and affect downstream EC commitment. To this end, we used polydimethylsiloxane (PDMS)—a transparent, biocompatible elastomeric material—as a substrate to study EC commitment of hiPSCs using a stepwise differentiation scheme. Using physiologically stiff (1.7 MPa) and soft (3 kPa) PDMS substrates, compared to polystyrene plates (3 GPa), we demonstrate that mechanical priming during mesoderm induction activates the Yes-associated protein and drives Wnt/β-catenin signaling. When mesoderm differentiation was induced on compliant PDMS substrates in both serum and serum-free E6 medium, mesodermal genetic signatures (T, KDR, MESP-1, GATA-2, and SNAIL-1) were enhanced. Furthermore, examination of EC fate following stiffness priming revealed that compliant substrates robustly improve EC commitment through VECad, CD31, vWF, and eNOS marker expression. Overall, we show that substrate compliance guides EC fate by enhancing mesoderm induction through Wnt activation without the addition of small molecules. These findings are the first to show that the mechanical context of the differentiation niche can be as potent as chemical cues in driving EC identity from hiPSCs.

Extracellular Matrix Regulation of Stem Cell Fate

Purpose of ReviewThe extracellular matrix (ECM) presents a complex myriad of biochemical and physical cues in the stem cell niche and is able to modulate stem cell fate and function. This review summarizes engineering approaches that have exploited natural and synthetic biomaterials to understand ECM regulation of stem cell fate. Specifically, we demonstrate how these studies have advanced our understanding of vascular maturation and mesenchymal lineage specification.Recent FindingsECM mechanics have emerged as a critical cue in stem cell lineage specification. With the introduction of mechanically dynamic materials, which mirror the non-linear elastic behavior of natural matrices, our understanding of differentiation behavior has evolved.SummaryWhile studies using conventional culture employing rigid, two-dimensional surfaces have greatly advanced our understanding of stem cell differentiation, they overlook the complexity of ECM in the stem cell environment. Implementing defined analogs, through material science and tissue engineering approaches, will allow us to mirror the dynamic nature of ECM and fully elucidate how stem cells differentiate.

Cytoskeletal tension regulates mesodermal spatial organization and subsequent vascular fate

Significance Human-induced pluripotent stem cells (hiPSCs) offer an opportunity to study how the developing embryo twists, folds, and expands into a collection of highly self-organized tissues. Introducing varying degrees of confinement with surface micropatterning may drive self-organization into tissues that mirror the structure and complexity of the embryo in vitro. Using high-throughput image-processing and machine-learning algorithms, we studied the relationships between cytoskeletal tension, density, and micropattern geometry on the ability to predict pattern formation in early and late-stage hiPSC maturation toward vascular lineages. We find that the combination of these approaches unveils the importance of cell density and cytoskeletal tension in how hiPSCs sense their environment and differentiate to the mesodermal lineage toward vascular fates. Morphogenesis during human development relies on the interplay between physiochemical cues that are mediated in part by cellular density and cytoskeletal tension. Here, we interrogated these factors on vascular lineage specification during human-induced pluripotent stem-cell (hiPSC) fate decision. We found that independent of chemical cues, spatially presented physical cues induce the self-organization of Brachyury-positive mesodermal cells, in a RhoA/Rho-associated kinase (ROCK)-dependent manner. Using unbiased support vector machine (SVM) learning, we found that density alone is sufficient to predict mesodermal fate. Furthermore, the long-withstanding presentation of spatial confinement during hiPSC differentiation led to an organized vascular tissue, reminiscent of native blood vessels, a process dependent on cell density as found by SVM analysis. Collectively, these results show how tension and density relate to vascular identity mirroring early morphogenesis. We propose that such a system can be applied to study other aspects of the stem-cell niche and its role in embryonic patterning.