Photothermal modulation of human stem cells using light-responsive 2D nanomaterials.

Two-dimensional (2D) molybdenum disulfide (MoS2) nanomaterials are an emerging class of biomaterials that are photoresponsive at near-infrared wavelengths (NIR). Here, we demonstrate the ability of 2D MoS2 to modulate cellular functions of human stem cells through photothermal mechanisms. The interaction of MoS2 and NIR stimulation of MoS2 with human stem cells is investigated using whole-transcriptome sequencing (RNA-seq). Global gene expression profile of stem cells reveals significant influence of MoS2 and NIR stimulation of MoS2 on integrins, cellular migration, and wound healing. The combination of MoS2 and NIR light may provide new approaches to regulate and direct these cellular functions for the purposes of regenerative medicine as well as cancer therapy.

Wnt signaling regulates neural plate patterning in distinct temporal phases with dynamic transcriptional outputs.

The process that partitions the nascent vertebrate central nervous system into forebrain, midbrain, hindbrain, and spinal cord after neural induction is of fundamental interest in developmental biology, and is known to be dependent on Wnt/ß-catenin signaling at multiple steps. Neural induction specifies neural ectoderm with forebrain character that is subsequently posteriorized by graded Wnt signaling: embryological and mutant analyses have shown that progressively higher levels of Wnt signaling induce progressively more posterior fates. However, the mechanistic link between Wnt signaling and the molecular subdivision of the neural ectoderm into distinct domains in the anteroposterior (AP) axis is still not clear. To better understand how Wnt mediates neural AP patterning, we performed a temporal dissection of neural patterning in response to manipulations of Wnt signaling in zebrafish. We show that Wnt-mediated neural patterning in zebrafish can be divided into three phases: (I) a primary AP patterning phase, which occurs during gastrulation, (II) a mes/r1 (mesencephalon-rhombomere 1) specification and refinement phase, which occurs immediately after gastrulation, and (III) a midbrain-hindbrain boundary (MHB) morphogenesis phase, which occurs during segmentation stages. A major outcome of these Wnt signaling phases is the specification of the major compartment divisions of the developing brain: first the MHB, then the diencephalic-mesencephalic boundary (DMB). The specification of these lineage divisions depends upon the dynamic changes of gene transcription in response to Wnt signaling, which we show primarily involves transcriptional repression or indirect activation. We show that otx2b is directly repressed by Wnt signaling during primary AP patterning, but becomes resistant to Wnt-mediated repression during late gastrulation. Also during late gastrulation, Wnt signaling becomes both necessary and sufficient for expression of wnt8b, en2a, and her5 in mes/r1. We suggest that the change in otx2b response to Wnt regulation enables a transition to the mes/r1 phase of Wnt-mediated patterning, as it ensures that Wnts expressed in the midbrain and MHB do not suppress midbrain identity, and consequently reinforce formation of the DMB. These findings integrate important temporal elements into our spatial understanding of Wnt-mediated neural patterning and may serve as an important basis for a better understanding of neural patterning defects that have implications in human health.

Analysis of the wnt1 regulatory chromosomal landscape.

One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain-hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1 regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.

Enabling tools for engineering collagenous tissues integrating bioreactors, intravital imaging, and biomechanical modeling.

Many investigators have engineered diverse connective tissues having good mechanical properties, yet few tools enable a global understanding of the associated formation of collagen fibers, the primary determinant of connective tissue stiffness. Toward this end, we developed a biomechanical model for collagenous tissues grown on polymer scaffolds that accounts for the kinetics of polymer degradation as well as the synthesis and degradation of multiple families of collagen fibers in response to cyclic strains imparted in a bioreactor. The model predicted well both overall thickness and stress-stretch relationships for tubular engineered vessels cultured for 8 weeks, and suggested that a steady state had not yet been reached. To facilitate future refinements of the model, we also developed bioreactors that enable intravital nonlinear optical microscopic imaging. Using these tools, we found that collagen fiber alignment was driven strongly by nondegraded polymer fibers at early times during culture, with subsequent mechano-stimulated dispersal of fiber orientations as polymer fibers degraded. In summary, mathematical models of growth and remodeling of engineered tissues cultured on polymeric scaffolds can predict evolving tissue morphology and mechanics after long periods of culture, and related empirical observations promise to further our understanding of collagen matrix development in vitro.

Quantifiable Intravital Light Sheet Microscopy.

Live imaging of zebrafish embryos that maintains normal development can be difficult to achieve due to a combination of sample mounting, immobilization, and phototoxicity issues that, once overcome, often still results in image quality sufficiently poor that computer-aided analysis or even manual analysis is not possible. Here, we describe our mounting strategy for imaging the zebrafish midbrain-hindbrain boundary (MHB) with light sheet fluorescence microscopy (LSFM) and pilot experiments to create a study-specific set of parameters for semiautomatically tracking cellular movements in the embryonic midbrain primordium during zebrafish segmentation.

The Sterol Trafficking Pathway in Arabidopsis thaliana.

In plants, the trafficking mechanisms by which sterols move through the plant and into target cells are unknown. Earlier studies identified endosomes as primary candidates for internalization of sterols in plants, but these results have come into question. Here, we show that in elongating root cells, the internalization of sterol occurs primarily by a non-endocytic mechanism. Added fluorescent sterols [dehydroergosterol (DHE) and BODIPY-cholesterol (BCh)] do not initially label endosomes identified by fluorescent protein markers or by internalized FM4-64. Instead, the nuclear envelope, an organelle not associated with the endocytic pathway but part of the endoplasmic reticulum (ER), becomes labeled. This result is supported by experiments with the inducible overexpression of auxilin-2-like protein (AUX2 line), which blocks most endocytosis upon induction. Internalization and nuclear envelope labeling still occur in induced AUX2 cells. Longer-term incubation labels the oil body, a site involved in sterol storage. Although the first site of localization, the nuclear envelope, is part of the ER, other domains of the ER do not accumulate the label. The trafficking pathway differs from vesicular endocytosis and points toward a different pathway of sterol transport possibly involving other mechanisms, such as ER-plasma membrane contact sites and cytoplasmic transport.

Building a three-dimensional model of early-stage zebrafish embryo brain.

We introduce a computational approach to build three-dimensional (3D) surface mesh models of the early-stage zebrafish brain primordia from time-series microscopy images. The complexity of the early-stage brain primordia and lack of recognizable landmarks pose a distinct challenge for feature segmentation and 3D modeling. Additional difficulty arises because of noise and variations in pixel intensity. We overcome these by using a hierarchical approach in which simple geometric elements, such as "beads" and "bonds," are assigned to represent local features and their connectivity is used to smoothen the surface while retaining high-curvature regions. We apply our method to build models of two zebrafish embryo phenotypes at discrete time points between 19 and 28 h post-fertilization and collect measurements to quantify development. Our approach is fast and applicable to building models of other biological systems, as demonstrated by models from magnetic resonance images of the human fetal brain. The source code, input scripts, sample image files, and generated outputs are publicly available on GitHub.

Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis.

From the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like.

Structural inhomogeneity and fiber orientation in the inner arterial media.

The microstructural orientation of vascular wall constituents is of interest to scientists and clinicians because alterations in their native states are associated with various cardiovascular diseases. In the arterial media, the orientation of these constituents is often described as circumferential. However, it has been noted that, just below the endothelial surface, the vascular wall constituents are oriented axially. To further study this reported change in orientation, and to resolve previous observations (which were made under conditions of no load), we used nonlinear optical microscopy to examine the orientation of collagen and elastin fibers in the inner medial region of bovine common carotid arteries. Images were obtained from this part of the arterial wall under varying degrees of mechanical strain: 0%, 10% axial, 10% circumferential, and 10% biaxial. We observed that close to the endothelium these components are aligned in the axial direction but abruptly change to a circumferential alignment at a depth of approximately 20 mum from the endothelial surface. The application of mechanical strain resulted in a significantly greater degree of fiber alignment, both collagen and elastin, in the strain direction, regardless of their initial unloaded orientation. Furthermore, variations in strain conditions resulted in an increase or a decrease in the overall degree of fiber alignment in the subendothelial layer depending on the direction of the applied strain. This high-resolution investigation adds more detail to existing descriptions of complex structure-function relationships in vascular tissue, which is essential for a better understanding of the pathophysiological processes resulting from injury, disease progression, and interventional therapies.

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices.

The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.

Revisiting optical clearing with dimethyl sulfoxide (DMSO).

Functional optical characterization of disease progression and response to therapy suffers from loss of spatial resolution and imaging depth due to scattering. Here we report on the ability of dimethyl sulfoxide (DMSO) alone to reduce the optical scattering of skin. We observed a threefold reduction in the scattering of skin with topical DMSO application. With an in vivo window chamber model, we observed a threefold increase in light transmittance through the preparation and enhanced visualization of subsurface microvasculature. Collectively, our data demonstrate the potential of DMSO alone to mitigate effects of scattering, which we expect will improve molecular imaging studies.

Delivery of sub-10-fs pulses for nonlinear optical microscopy by polarization-maintaining single mode optical fiber.

Broadband, sub-10-fs pulses, can be propagated through polarization-maintaining single mode fiber (PMF) for use in nonlinear optical microscopy (NLOM). We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al(2)O(3) (75 MHz repetition rate) oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. The minimized time-bandwidth product of the laser pulses at the NLOM focus allowed the nonlinear excitation of multiple fluorophores simultaneously without central wavelength tuning. Polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

Rabbit cornea microstructure response to changes in intraocular pressure visualized by using nonlinear optical microscopy.

PURPOSE: To characterize the microstructural response of the rabbit cornea to changes in intraocular pressure (IOP) by using nonlinear optical microscopy (NLOM). METHODS: Isolated rabbit corneas were mounted on an artificial anterior chamber in series with a manometer and were hydrostatically pressurized by a reservoir. The chamber was mounted on an upright microscope stage of a custom-built NLOM system for corneal imaging without using exogenous stains or dyes. Second harmonic generation in collagen was used to image through the full thickness of the central corneal stroma at IOPs between 5 and 20 mm Hg. Microstructural morphology changes as a function of IOP were used to characterize the depth-dependent response of the central cornea. RESULTS: Regional collagen lamellae architecture through the full thickness of the stroma was specifically imaged as a function of IOP. Hypotensive corneas showed gaps between lamellar structures that decreased in size with increasing IOP. These morphologic features appear to result from interwoven lamellae oriented along the anterior-posterior axis and parallel to the cornea surface. They appear throughout the full thickness and disappear with tension in the anterior but persist in the posterior central cornea, even at hypertensive IOP. CONCLUSIONS: NLOM reveals interwoven collagen lamellae sheets through the full thickness of the rabbit central cornea oriented along the anterior-posterior axis and parallel to the surface. The nondestructive nature of NLOM allows 3-dimensional imaging of stromal architecture as a function of IOP in situ. Collagen morphologic features were used as an indirect measure of depth-dependent mechanical response to changes in IOP.

Vascular Smooth Muscle Contractile Function Declines With Age in Skeletal Muscle Feed Arteries.

Aging induces a progressive decline in vasoconstrictor responses in central and peripheral arteries. This study investigated the hypothesis that vascular smooth muscle (VSM) contractile function declines with age in soleus muscle feed arteries (SFA). Contractile function of cannulated SFA isolated from young (4 months) and old (24 months) Fischer 344 rats was assessed by measuring constrictor responses of denuded (endothelium removed) SFA to norepinephrine (NE), phenylephrine (PE), and angiotensin II (Ang II). In addition, we investigated the role of RhoA signaling in modulation of VSM contractile function. Structural and functional characteristics of VSM cells were evaluated by fluorescence imaging and atomic force microscopy (AFM). Results indicated that constrictor responses to PE and Ang II were significantly impaired in old SFA, whereas constrictor responses to NE were preserved. In the presence of a Rho-kinase inhibitor (Y27632), constrictor responses to NE, Ang II, and PE were significantly reduced in young and old SFA. In addition, the age-group difference in constrictor responses to Ang II was eliminated. ROCK1 and ROCK2 content was similar in young and old VSM cells, whereas pROCK1 and pROCK2 were significantly elevated in old VSM cells. Aging was associated with a reduction in smooth muscle α-actin stress fibers and recruitment of proteins to cell-matrix adhesions. Old VSM cells presented an increase in integrin adhesion to the matrix and smooth muscle γ-actin fibers that was associated with increased cell stiffness. In conclusion, our results indicate that VSM contractile function declined with age in SFA. The decrement in contractile function was mediated in part by RhoA/ROCK signaling. Upregulation of pROCK in old VSM cells was not able to rescue contractility in old SFA. Collectively, these results indicate that changes at the VSM cell level play a central role in the reduced contractile function of aged SFA.

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing "cellularization" at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more "physiological" method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

Midbrain-Hindbrain Boundary Morphogenesis: At the Intersection of Wnt and Fgf Signaling.

A constriction in the neural tube at the junction of the midbrain and hindbrain is a conserved feature of vertebrate embryos. The constriction is a defining feature of the midbrain-hindbrain boundary (MHB), a signaling center that patterns the adjacent midbrain and rostral hindbrain and forms at the junction of two gene expression domains in the early neural plate: an anterior otx2/wnt1 positive domain and a posterior gbx/fgf8 positive domain. otx2 and gbx genes encode mutually repressive transcription factors that create a lineage restriction boundary at their expression interface. Wnt and Fgf genes form a mutually dependent feedback system that maintains their expression domains on the otx2 or gbx side of the boundary, respectively. Constriction morphogenesis occurs after these conserved gene expression domains are established and while their mutual interactions maintain their expression pattern; consequently, mutant studies in zebrafish have led to the suggestion that constriction morphogenesis should be considered a unique phase of MHB development. We analyzed MHB morphogenesis in fgf8 loss of function zebrafish embryos using a reporter driven by the conserved wnt1 enhancer to visualize anterior boundary cells. We found that fgf8 loss of function results in a re-activation of wnt1 reporter expression posterior to the boundary simultaneous with an inactivation of the wnt1 reporter in the anterior boundary cells, and that these events correlate with relaxation of the boundary constriction. In consideration of other results that correlate the boundary constriction with Wnt and Fgf expression, we propose that the maintenance of an active Wnt-Fgf feedback loop is a key factor in driving the morphogenesis of the MHB constriction.

Inverted orientation improves decellularization of whole porcine hearts.

In structurally heterogeneous organs, such as heart, it is challenging to retain extracellular matrix integrity in the thinnest regions (eg, valves) during perfusion decellularization and completely remove cellular debris from thicker areas. The high inflow rates necessary to maintain physiologic pressure can distend or damage thin tissues, but lower pressures prolong the process and increase the likelihood of contamination. We examined two novel retrograde decellularization methods for porcine hearts: inverting the heart or venting the apex to decrease inflow rate. We measured flow dynamics through the aorta (Ao) and pulmonary artery (PA) at different Ao pressures and assessed the heart's appearance, turbidity of the outflow solutions, and coronary perfusion efficiency. We used rectangle image fitting of decellularized heart images to obtain a heart shape index. Using nonlinear optical microscopy, we determined the microstructure of collagen and elastin fibers of the aortic valve cusps. DNA, glycosaminoglycan, and residual detergent levels were compared. The inverted method was superior to the vented method, as shown by a higher coronary perfusion efficiency, more cell debris outflow, higher collagen and elastin content inside the aortic valve, lower DNA content, and better retention of the heart shape after decellularization. To our knowledge, this is the first study to use flow dynamics in a whole heart throughout the decellularization procedure to provide real-time information about the success of the process and the integrity of the vulnerable regions of the matrix. Heart orientation was important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. STATEMENT OF SIGNIFICANCE: The use of decellularized tissue as a suitable scaffold for engineered tissue has emerged over the past decade as one of the most promising biofabrication platforms. The decellularization process removes all native cells, leaving the natural biopolymers, extracellular matrix materials and native architecture intact. This manuscript describes heart orientation as important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. To our knowledge, this is the first study to assess flow dynamics in a whole heart throughout the decellularization procedure. Our findings compared to currently published methods demonstrate that continuous complex real-time measurements and analyses are required to produce an optimal scaffold for cardiac regeneration.

Nck deficiency is associated with delayed breast carcinoma progression and reduced metastasis.

Although it is known that noncatalytic region of tyrosine kinase (Nck) regulates cell adhesion and migration by bridging tyrosine phosphorylation with cytoskeletal remodeling, the role of Nck in tumorigenesis and metastasis has remained undetermined. Here we report that Nck is required for the growth and vascularization of primary tumors and lung metastases in a breast cancer xenograft model as well as extravasation following injection of carcinoma cells into the tail vein. We provide evidence that Nck directs the polarization of cell-matrix interactions for efficient migration in three-dimensional microenvironments. We show that Nck advances breast carcinoma cell invasion by regulating actin dynamics at invadopodia and enhancing focalized extracellular matrix proteolysis by directing the delivery and accumulation of MMP14 at the cell surface. We find that Nck-dependent cytoskeletal changes are mechanistically linked to enhanced RhoA but restricted spatiotemporal activation of Cdc42. Using a combination of protein silencing and forced expression of wild-type/constitutively active variants, we provide evidence that Nck is an upstream regulator of RhoA-dependent, MMP14-mediated breast carcinoma cell invasion. By identifying Nck as an important driver of breast carcinoma progression and metastasis, these results lay the groundwork for future studies assessing the therapeutic potential of targeting Nck in aggressive cancers.

Molecular interactions of exogenous chemical agents with collagen--implications for tissue optical clearing.

Reduction of optical scattering in turbid biological tissues using nonreactive chemical agents has potential applications for light-based diagnostics and therapeutics. Optical clearing effects by exogenous chemical agents, in particular sugars and sugar alcohols, have been found to be temporary with tissue rehydration. Applications with dermatologic laser therapies are now being investigated, but suffer from the inability of studied agents to penetrate the superficial layers of human skin. Selection, design, and refinement of topically effective chemical agents are hindered by a lack of fundamental understanding of tissue clearing mechanisms. We present recent work, particularly from the biochemistry community, detailing molecular interactions between chemical agents and collagen. This body of work demonstrates the perturbative effects of sugars and sugar alcohols on collagen high-order structures at micro- and nanometer length scales by screening noncovalent bonding forces. In addition, these studies emphasize the nonreactive nature of agent-collagen interactions and the ability of noncovalent bonding forces to recover with agent removal and drive reassembly of destabilized collagen structures. A mechanism of tissue optical clearing is proposed based on agent destabilization of high-order collagen structures.

Collagen solubility correlates with skin optical clearing.

Biomedical optics and photomedicine applications are challenged by the turbidity of most biological tissue systems. Nonreactive, biocompatible chemical agents can induce a reversible reduction in optical scattering of collagenous tissues such as human skin. Herein we show that a chemical agent's tissue optical clearing potential is directly related to its collagen solubility, providing a rational design basis for effective, percutaneous formulations.