Real Time Imaging of Human Progenitor Neurogenesis

Human neural progenitors are increasingly being employed in drug screens and emerging cell therapies targeted towards neurological disorders where neurogenesis is thought to play a key role including developmental disorders, Alzheimer’s disease, and depression. Key to the success of these applications is understanding the mechanisms by which neurons arise. Our understanding of development can provide some guidance but since little is known about the specifics of human neural development and the requirement that cultures be expanded in vitro prior to use, it is unclear whether neural progenitors obey the same developmental mechanisms that exist in vivo. In previous studies we have shown that progenitors derived from fetal cortex can be cultured for many weeks in vitro as undifferentiated neurospheres and then induced to undergo neurogenesis by removing mitogens and exposing them to supportive substrates. Here we use live time lapse imaging and immunocytochemical analysis to show that neural progenitors use developmental mechanisms to generate neurons. Cells with morphologies and marker profiles consistent with radial glia and recently described outer radial glia divide asymmetrically and symmetrically to generate multipolar intermediate progenitors, a portion of which express ASCL1. These multipolar intermediate progenitors subsequently divide symmetrically to produce CTIP2+ neurons. This 3-cell neurogenic scheme echoes observations in rodents in vivo and in human fetal slice cultures in vitro, providing evidence that hNPCs represent a renewable and robust in vitro assay system to explore mechanisms of human neurogenesis without the continual need for fresh primary human fetal tissue. Knowledge provided by this and future explorations of human neural progenitor neurogenesis will help maximize the safety and efficacy of new stem cell therapies by providing an understanding of how to generate physiologically-relevant cell types that maintain their identities when placed in diagnostic or transplantation environments

High-speed in vitro intensity diffraction tomography

We demonstrate a label-free, scan-free intensity diffraction tomography technique utilizing annular illumination (aIDT) to rapidly characterize large-volume three-dimensional (3-D) refractive index distributions in vitro. By optimally matching the illumination geometry to the microscope pupil, our technique reduces the data requirement by 60 times to achieve high-speed 10-Hz volume rates. Using eight intensity images, we recover volumes of ∼350 μm  ×  100 μm  ×  20  μm, with near diffraction-limited lateral resolution of   ∼  487  nm and axial resolution of   ∼  3.4  μm. The attained large volume rate and high-resolution enable 3-D quantitative phase imaging of complex living biological samples across multiple length scales. We demonstrate aIDT’s capabilities on unicellular diatom microalgae, epithelial buccal cell clusters with native bacteria, and live Caenorhabditis elegans specimens. Within these samples, we recover macroscale cellular structures, subcellular organelles, and dynamic micro-organism tissues with minimal motion artifacts. Quantifying such features has significant utility in oncology, immunology, and cellular pathophysiology, where these morphological features are evaluated for changes in the presence of disease, parasites, and new drug treatments. Finally, we simulate the aIDT system to highlight the accuracy and sensitivity of the proposed technique. aIDT shows promise as a powerful high-speed, label-free computational microscopy approach for applications where natural imaging is required to evaluate environmental effects on a sample in real time.https://arxiv.org/abs/1904.06004Accepted manuscrip

Collective and single cell behavior in epithelial contact inhibition

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition". The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured MDCK cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition

Morphology and Nanomechanics of Sensory Neurons Growth Cones following Peripheral Nerve Injury

A prior peripheral nerve injury in vivo, promotes a rapid elongated mode of sensory neurons neurite regrowth in vitro. This in vitro model of conditioned axotomy allows analysis of the cellular and molecular mechanisms leading to an improved neurite re-growth. Our differential interference contrast microscopy and immunocytochemistry results show that conditioned axotomy, induced by sciatic nerve injury, did not increase somatic size of adult lumbar sensory neurons from mice dorsal root ganglia sensory neurons but promoted the appearance of larger neurites and growth cones. Using atomic force microscopy on live neurons, we investigated whether membrane mechanical properties of growth cones of axotomized neurons were modified following sciatic nerve injury. Our data revealed that neurons having a regenerative growth were characterized by softer growth cones, compared to control neurons. The increase of the growth cone membrane elasticity suggests a modification in the ratio and the inner framework of the main structural proteins

Characterization of Embryonic Stem (ES) Neuronal Differentiation Combining Atomic Force, Confocal and DIC Microscopy Imaging

The ultimate clinical implementation of embryonic stem cells will require methods and protocols to turn these unspecialized cells into the fully functioning cell types found in a wide variety of tissues and organs. In order to achieve this, it is necessary to clearly understand the signals and cues that direct embryonic stem cell differentiation. This book provides a snapshot of current research on the differentiation of embryonic stem cells to a wide variety of cell types, including neural, cardiac, endothelial, osteogenic, and hepatic cells. In addition, induced pluripotent stem cells and other pluripotent stem cell sources are described. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines

Computational illumination for high-speed in vitro Fourier ptychographic microscopy

We demonstrate a new computational illumination technique that achieves large space-bandwidth-time product, for quantitative phase imaging of unstained live samples in vitro. Microscope lenses can have either large field of view (FOV) or high resolution, not both. Fourier ptychographic microscopy (FPM) is a new computational imaging technique that circumvents this limit by fusing information from multiple images taken with different illumination angles. The result is a gigapixel-scale image having both wide FOV and high resolution, i.e. large space-bandwidth product (SBP). FPM has enormous potential for revolutionizing microscopy and has already found application in digital pathology. However, it suffers from long acquisition times (on the order of minutes), limiting throughput. Faster capture times would not only improve imaging speed, but also allow studies of live samples, where motion artifacts degrade results. In contrast to fixed (e.g. pathology) slides, live samples are continuously evolving at various spatial and temporal scales. Here, we present a new source coding scheme, along with real-time hardware control, to achieve 0.8 NA resolution across a 4x FOV with sub-second capture times. We propose an improved algorithm and new initialization scheme, which allow robust phase reconstruction over long time-lapse experiments. We present the first FPM results for both growing and confluent in vitro cell cultures, capturing videos of subcellular dynamical phenomena in popular cell lines undergoing division and migration. Our method opens up FPM to applications with live samples, for observing rare events in both space and time

A Cell-Based Small Molecule Screening Method for Identifying Inhibitors of Epithelial-Mesenchymal Transition in Carcinoma

Epithelial Mesenchymal Transition (EMT) is a crucial mechanism for carcinoma progression, as it provides routes for in situ carcinoma cells to dissociate and become motile, leading to localized invasion and metastatic spread. Targeting EMT therefore represents an important therapeutic strategy for cancer treatment. The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics. Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target. We have developed a novel screening assay that can lead to the identification of compounds that can inhibit EMT initiated by growth factor signaling. This assay is designed as a high-content screening assay where both cell growth and cell migration can be analyzed simultaneously via time-course imaging in multi-well plates. Using this assay, we have validated several compounds as viable EMT inhibitors. In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling. Overall, this EMT screening method provides a foundation for improving the therapeutic value of recently developed compounds in advanced stage carcinoma

In vitro neutrophil migration is associated with inhaled corticosteroid treatment and serum cytokines in pediatric asthma.

Background: Different asthma phenotypes are driven by molecular endotypes. A Th1-high phenotype is linked to severe, therapy-refractory asthma, subclinical infections and neutrophil inflammation. Previously, we found neutrophil granulocytes (NGs) from asthmatics exhibit decreased chemotaxis towards leukotriene B4 (LTB4), a chemoattractant involved in inflammation response. We hypothesized that this pattern is driven by asthma in general and aggravated in a Th1-high phenotype. Methods: NGs from asthmatic nd healthy children were stimulated with 10 nM LTB4/100 nM N-formylmethionine-leucyl-phenylalanine and neutrophil migration was documented following our prior SiMA (simplified migration assay) workflow, capturing morphologic and dynamic parameters from single-cell tracking in the images. Demographic, clinical and serum cytokine data were determined in the ALLIANCE cohort. Results: A reduced chemotactic response towards LTB4 was confirmed in asthmatic donors regardless of inhaled corticosteroid (ICS) treatment. By contrast, only NGs from ICS-treated asthmatic children migrate similarly to controls with the exception of Th1-high donors, whose NGs presented a reduced and less directed migration towards the chemokines. ICS-treated and Th1-high asthmatic donors present an altered surface receptor profile, which partly correlates with migration. Conclusions: Neutrophil migration in vitro may be affected by ICS-therapy or a Th1-high phenotype. This may be explained by alteration of receptor expression and could be used as a tool to monitor asthma treatment

Tumor Cell Migration and Interaction with ECM and Stroma in 3D Tissue Models

Tumors are often described as ”wounds that do not heal”. Tumor progression and woundhealing both feature sustained proliferative signaling, evasion from immune destruction, cell death resistance, inflammation, angiogenesis, extracellular matrix(ECM) remodeling, and activated cell migration. Unlike normal wound healing which often ends up with restored tissue homeostasis, pathological ECM remodeling is frequently implicated in fibrotic diseases and solid tumors. In addition, the dysregulated ECM signatures are directly associated with poor prognosis and also immunotherapy failure in certain types of cancers. In this dissertation, we used 3D in vitro cocultures to understand how tumor cells co-op with stromal/immune components, how ECM remodeling is hijacked, and how ECM architecture impacts tumor progression. We first investigated the impact of fibroblasts. Fibroblasts are the most abundant cell types in the tumor stroma. The density of cancer-associated fibroblasts(CAFs) has been shown directly correlated with poor prognosis in some types of solid tumors. To uncover potential mechanisms behind the quantitative relationship between CAFs and tumor dissemination, we developed our coculture model by varying the density ratios between normal human lung fibroblasts and breast cancer cells(MDA-MB-231s). We found that fibroblasts increase tumor cell motility and facilitate the transition from confined to diffusive tumor cell motions, indicative of an uncaging effect. Furthermore, the ECM is globally and locally remodeled substantially with the presence of fibroblasts. Moreover, these fibroblast-mediated phenomena are in part dependent on matrix metalloproteinases. We then investigated the impact of macrophages. In this study, we developed a 3D collagen co-culture system to mimic the melanoma microenvironment and investigate how interactions between melanoma cancer cells, fibroblasts, and macrophages shape the early stages of macrophage immune activity. In this in vitro model, we captured the macrophage immunosuppressive transition. Macrophages in the model displayed increased motility and acquired a phenotype that was similar to tumor-associated macrophages(TAMs) from melanoma tumors. This model may provide a platform for further studies on TAMs targeted immune therapy in melanoma. In the end, we investigated the impact of ECM architecture in tumor progression. Reconstruction of a biomimetic scaffold is critical in 3D in vitro models. Here we introduce a new type of thick collagen bundles that highly mimic in vivo ECM structure. We fabricated this type of thickened collagen bundles by introducing mechanical agitation during the transient gelation process. Thickened collagen patches are interconnected with a loose collagen network, highly resembling collagen architecture in human skin scars. This type of thickened collagen bundles promotes tumor cell dissemination. The effect is significantly augmented in the presence of fibroblasts. The application of this type of collagen triggers different morphology and migration behaviors of tumor cells and highlights the importance of mesoscale architectures. Overall, this dissertation investigated the roles of stromal and non-stromal components in tumor progression through 3D in vitro models. The coculture models established in this dissertation may be further extended to test novel therapeutics targeted at CAFs, TAMs or ECM architecture