Soluble Gradients Research Articles

Impacts of atmospheric deposition on the heavy metal mobilization and bioavailability in soils amended by lime

Atmospheric deposition is an important source of heavy metal in agricultural soils, but there is limited research on the mobility of these metals in soil and their impact on soil amendment. Here, we performed a dust incubation experiment in soils in the laboratory and a factorial transplant experiment at three field sites with a gradient of atmospheric deposition to examine the impacts of atmospherically deposited heavy metals (Cu, Cd, and Pb) on the mobility and bioavailability in soils with and without lime applications. Results showed that the atmospherically deposited heavy metals showed high mobility and were primarily presented in the soluble ionic fractions in the wet part and acid-exchangeable and reducible fractions in the dry part of atmospheric deposition. Atmospheric dust addition caused the 2p3/2 and 2p1/2 electrons of Cu atoms in uncontaminated soils to transition the 3d vacant states, resulting in similar copper absorption peaks as atmospheric particles by the observation of X-ray absorption near-edge spectroscopy (XANES). In the field, atmospheric deposition can only increase the mobile fractions in the surface soils, but not in the deeper layers. However, the deposition can increase the soluble and diffusive gradients in thin films (DGT)-measured bioavailable fractions in profile along with the soil depth. Lime applications cannot significantly reduce the mobile fractions of heavy metals in the surface soils exposed to atmospheric deposition, but significantly reduce the heavy metal concentrations in soil solutions and the DGT-measured bioavailable concentrations, particularly in the deeper layer (6–10 cm). The major implication is that atmospherically deposited heavy metals can significantly increase their bioavailable concentrations in the plough horizon of soil and constrain the effects of soil amendments on heavy metal immobilization, thereby increasing the risks of crop uptake.

Defined Microenvironments Trigger In Vitro Gastrulation in Human Pluripotent Stem Cells.

Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by treating pluripotent stem cells with soluble morphogens to trigger differentiation. However, in vivo gastrulation is a multistage process coordinated through feedback between soluble gradients and biophysical forces, with the multipotent epiblast transforming to the primitive streak followed by germ layer segregation. Here, the authors show how constraining pluripotent stem cells to hydrogel islands triggers morphogenesis that mirrors the stages preceding in vivo gastrulation, without the need for exogenous supplements. Within hours of initial seeding, cells display a contractile phenotype at the boundary, which leads to enhanced proliferation, yes-associated protein (YAP) translocation, epithelial to mesenchymal transition, and emergence of SRY-box transcription factor 17 (SOX17)+ T/BRACHYURY+ cells. Molecular profiling and pathway analysis reveals a role for mechanotransduction-coupled wingless-type (WNT) signaling in orchestrating differentiation, which bears similarities to processes observed in whole organism models of development. After two days, the colonies form multilayered aggregates, which can be removed for further growth and differentiation. This approach demonstrates how materials alone can initiate gastrulation, thereby providing in vitro models of development and a tool to support organoid bioengineering efforts.

A Stable Chemokine Gradient Controls Directional Persistence of Migrating Dendritic Cells.

Navigation of dendritic cells (DCs) from the site of infection to lymphoid organs is guided by concentration gradients of CCR7 ligands. How cells interpret chemokine gradients and how they couple directional sensing to polarization and persistent chemotaxis has remained largely elusive. Previous experimental systems were limited in the ability to control fast de novo formation of the final gradient slope, long-lasting stability of the gradient and to expose cells to dynamic stimulation. Here, we used a combination of microfluidics and quantitative in vitro live cell imaging to elucidate the chemotactic sensing strategy of DCs. The microfluidic approach allows us to generate soluble gradients with high spatio-temporal precision and to analyze actin dynamics, cell polarization, and persistent directional migration in both static and dynamic environments. We demonstrate that directional persistence of DC migration requires steady-state characteristics of the soluble gradient instead of temporally rising CCL19 concentration, implying that spatial sensing mechanisms control chemotaxis of DCs. Kymograph analysis of actin dynamics revealed that the presence of the CCL19 gradient is essential to stabilize leading edge protrusions in DCs and to determine directionality, since both cytoskeletal polarization and persistent chemotaxis are abrogated in the range of seconds when steady-state gradients are perturbed. In contrast to Dictyostelium amoeba, DCs are unable to decode oscillatory stimulation of soluble chemokine traveling waves into a directional response toward the wave source. These findings are consistent with the notion that DCs do not employ adaptive temporal sensing strategies that discriminate temporally increasing and decreasing chemoattractant concentrations in our setting. Taken together, in our experimental system DCs do not depend on increasing absolute chemokine concentration over time to induce persistent migration and do not integrate oscillatory stimulation. The observed capability of DCs to migrate with high directional persistence in stable gradients but not when subjected to periodic temporal cues, identifies spatial sensing as a key requirement for persistent chemotaxis of DCs.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Cloning, Expression, Purification, and Characterization of Soluble Bioactive Recombinant Human Anterior Gradient Homolog 2 - DsRed Monomer Protein in Escherichia coli

Cloning, Expression, Purification, and Characterization of Soluble Bioactive Recombinant Human Anterior Gradient Homolog 2 - DsRed Monomer Protein in Escherichia coli

Heparan sulfate is essential for thymus growth

Thymus organogenesis and T cell development are coordinated by various soluble and cell-bound molecules. Heparan sulfate (HS) proteoglycans can interact with and immobilize many soluble mediators, creating fields or gradients of secreted ligands. While the role of HS in the development of many organs has been studied extensively, little is known about its function in the thymus. Here, we examined the distribution of HS in the thymus and the effect of its absence on thymus organogenesis and T cell development. We found that HS was expressed most abundantly on the thymic fibroblasts and at lower levels on endothelial, epithelial, and hematopoietic cells. To study the function of HS in the thymus, we eliminated most of HS in this organ by genetically disrupting the glycosyltransferase Ext1 that is essential for its synthesis. The absence of HS greatly reduced the size of the thymus in fetal thymic organ cultures and in vivo, in mice, and decreased the production of T cells. However, no specific blocks in T cell development were observed. Wild-type thymic fibroblasts were able to physically bind the homeostatic chemokines CCL19, CCL21, and CXCL12 ex vivo. However, this binding was abolished upon HS degradation, disrupting the CCL19/CCL21 chemokine gradients and causing impaired migration of dendritic cells in thymic slices. Thus, our results show that HS plays an essential role in the development and growth of the thymus and in regulating interstitial cell migration.

Immobilisation of CXCL8 gradients in microfluidic devices for migration experiments

The release of inflammatory chemokines leads to the formation of chemokine gradients that result in the directed migration of immune cells to the site of injury. In this process, cells respond to soluble gradients (chemotaxis) as well as to immobilised gradients (haptotaxis). Surface-bound chemokine gradients are mostly presented by endothelial cells and supported by glycosaminoglycans (GAGs), such as heparan sulfate, involving the GAG binding site of chemokines. Microfluidic devices have been used to analyse cell migration along soluble chemokine gradients, as these devices allow the generation of stable gradients with resolutions in the range of microns. To immobilise well-controlled soluble gradients of interleukin-8 (CXCL8), an inflammatory chemokine, we developed a simple procedure using a heparin-coated PDMS-microfluidic device. We used these immobilised gradients for migration experiments with CXCL8-responsive THP-1 cells and confirmed directed cell migration. This setup might be useful for the examination of factors that may alter chemotaxis and haptotaxis as well as synergistic and antagonistic effects of other soluble and immobilised chemokines.

B cell zone reticular cell microenvironments shape CXCL13 gradient formation

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.

The Biophysics of Cell Migration: Biasing Cell Motion with Feynman Ratchets

ABSTRACT The concepts and frameworks of soft matter physics and the laws of thermodynamics can be used to describe relevant developmental, physiologic, and pathologic events in which directed cell migration is involved, such as in cancer. Typically, this directionality has been associated with the presence of soluble long-range gradients of a chemoattractant, synergizing with many other guidance cues to direct the motion of cells. In particular, physical inputs have been shown to strongly influence cell locomotion. However, this type of cue has been less explored despite the importance in biology. In this paper, we describe recent in vitro works at the interface between physics and biology, showing how the motion of cells can be directed by using gradient-free environments with repeated local asymmetries. This rectification of cell migration, from random to directed, is a process reminiscent of the Feynman ratchet; therefore, this framework can be used to explain the mechanism behind directed cell motion.

Axonal Growth of Midbrain Dopamine Neurons is Modulated by the Cell Adhesion Molecule ALCAM Through Trans-Heterophilic Interactions with L1cam, Chl1, and Semaphorins.

The growth of axons corresponding to different neuronal subtypes is governed by unique expression profiles of molecules on the growth cone. These molecules respond to extracellular cues either locally though cell adhesion interactions or over long distances through diffusible gradients. Here, we report that that the cell adhesion molecule ALCAM (CD166) can act as an extracellular substrate to selectively promote the growth of murine midbrain dopamine (mDA) neuron axons through a trans-heterophilic interaction with mDA-bound adhesion molecules. In mixed-sex primary midbrain cultures, the growth-promoting effect of ALCAM was abolished by neutralizing antibodies for components of the Semaphorin receptor complex Nrp1, Chl1, or L1cam. The ALCAM substrate was also found to modulate the response of mDA neurites to soluble semaphorins in a context-specific manner by abolishing the growth-promoting effect of Sema3A but inducing a branching response in the presence of Sema3C. These findings identify a previously unrecognized guidance mechanism whereby cell adhesion molecules act in trans to modulate the response of axonal growth cones to soluble gradients to selectively orchestrate the growth and guidance of mDA neurons.SIGNIFICANCE STATEMENT The mechanisms governing the axonal connectivity of midbrain dopamine (mDA) neurons during neural development have remained rather poorly understood relative to other model systems for axonal growth and guidance. Here, we report a series of novel interactions between proteins previously not identified in the context of mDA neuronal growth. Significantly, the results suggest a previously unrecognized mechanism involving the convergence in signaling between local, adhesion and long-distance, soluble cues. A better understanding of the molecules and mechanisms involved in establishment of the mDA system is important as a part of ongoing efforts to understand the consequence of conditions that may result from aberrant connectivity and also for cell replacement strategies for Parkinson's disease.

Generation of 3D Soluble Signal Gradients in Cell-Laden Hydrogels Using Passive Diffusion.

Soluble signal gradients play an important role in organ patterning, cell migration, and differentiation. Currently, signal gradients in 2D cell culture are realized using microfluidics and here cells are exposed to high and nonphysiological shear stress. Tissue morphogenesis (organogenesis) however occurs in 3D and therefore there is a need for simple and practical systems to impose gradients to cells dispersed in 3D matrix. Herein, a 3D gradient generator based on passive diffusion elements that recapitulates interstitial flow and is capable of imposing predictable gradients over long length scales (6 mm) lasting up to 48 h to cells dispersed in a hydrogel environment is reported. Using recombinant human WNT3A (rhWNT3A), the spatiotemporal activation of the canonical WNT pathway in human epithelial kidney cells and human mesenchymal stems cells expressing a green fluorescence protein reporter on a transcription factor/lymphoid enhancer-binding factor (TCF/LEF) promoter is demonstrated. By refining computation models based on experimental findings, the diffusion coefficient of rhWNT3A in presence of human cells in 3D is determined. Furthermore, the formation of rhBMP4 gradients is visualized using immunohistochemistry by staining for phospho-SMAD1/5, the downstream targets of the bone morphogenetic protein (BMP) pathway. The simplicity of the gradient generator is expected to spur its adoption in studying developmental biology paradigms in vitro.

Revisiting Netrin-1: One Who Guides (Axons).

Proper patterning of the nervous system requires that developing axons find appropriate postsynaptic partners; this entails microns to meters of extension through an extracellular milieu exhibiting a wide range of mechanical and chemical properties. Thus, the elaborate networks of fiber tracts and non-fasciculated axons evident in mature organisms are formed via complex pathfinding. The macroscopic structures of axon projections are highly stereotyped across members of the same species, indicating precise mechanisms guide their formation. The developing axon exhibits directionally biased growth toward or away from external guidance cues. One of the most studied guidance cues is netrin-1, however, its presentation in vivo remains debated. Guidance cues can be secreted to form soluble or chemotactic gradients or presented bound to cells or the extracellular matrix to form haptotactic gradients. The growth cone, a highly specialized dynamic structure at the end of the extending axon, detects these guidance cues via transmembrane receptors, such as the netrin-1 receptors deleted in colorectal cancer (DCC) and UNC5. These receptors orchestrate remodeling of the cytoskeleton and cell membrane through both chemical and mechanotransductive pathways, which result in traction forces generated by the cytoskeleton against the extracellular environment and translocation of the growth cone. Through intracellular signaling responses, netrin-1 can trigger either attraction or repulsion of the axon. Here we review the mechanisms by which the classical guidance cue netrin-1 regulates intracellular effectors to respond to the extracellular environment in the context of axon guidance during development of the central nervous system and discuss recent findings that demonstrate the critical importance of mechanical forces in this process.

Estimating cadmium availability to the hyperaccumulator Sedum plumbizincicola in a wide range of soil types using a piecewise function

Estimating the bioavailability and predicting the uptake of metals to hyperaccumulators is very important in developing the field application of phytoextraction. A pot experiment was conducted using 108 agricultural soils covering a wide range of soil properties by the cadmium (Cd) hyperaccumulator Sedum plumbizincicola. The contributions of a range of soil properties to Cd uptake were quantified. Soil total, soluble, CaCl2-extractable and diffusive gradients in thin films (DGT)-extractable Cd concentrations (Cdtotal, Cdsoln, CdCaCl2 and CdDGT) were used to estimate Cd bioavailability and predict shoot Cd concentration (Cdshoot) using a piecewise function. Cdtotal and pH were the two major contributors to Cd uptake. Cdshoot showed a logarithmic increase with Cdtotal from 0.30 to 10.0 mg kg−1 but no further increase when Cd levels exceeded 10 mg kg−1. Soil pH had a discernible negative effect on Cd bioavailability from pH 5.5 to 7.5 but a weak influence at pH < 5.5 or pH > 7.5. This indicates that the optimum pH for phytoextraction with S. plumbizincicola was ~5.5 and lower pH produced little increase in shoot Cd uptake. DGT gave the best estimation of Cd bioavailability across all the data. When Cdtotal > 10 mg kg−1, none of the four measures was accurate enough to predict Cdshoot but when pH > 7.5 all the four measures were well correlated with Cdshoot. Piecewise equations in different ranges of Cdtotal or pH significantly improved the prediction of Cdshoot compared with the global equations derived from all the data. Compared with the piecewise equations, when pH > 7.5 Cdshoot was greatly overestimated with the global equation of Cdtotal. Our study provides useful information on the soils in which phytoextraction with S. plumbizincicola is feasible in the field. CapsuleCd availability to S. plumbizincicola was estimated by a piecewise function in soils with wide ranges of total Cd concentration and pH.

Glioblastoma Cells Respond Directionally to L1CAM Protein Ectodomain

Glioblastoma Multiforme (GBM) is the most common form of brain cancer and is also the most malignant. Part of what makes this cancer so insidious is the invariable tendency for tumors to regrow and kill patients after tumor removal. Currently, there is no definitive treatment for such cases, nor is there a consensus on the cause. It is our hypothesis that a major reason is the expression and release of L1CAM (L1), a neural cell adhesion protein, by GBM cells. Previously, our lab has used Super Scratch assays to show that L1 increases motility and proliferation in glioblastoma cells. However, until now, there has not been research into a possible attractive chemotactic effect of L1 on glioblastoma cells to guide their motility. In this study, we investigated the possibility of the released L1 ectodomain (L1ecto) guiding glioblastoma cells up a concentration gradient of L1ecto released by neighboring cells, and the ability of patterned immobilized L1ecto to stimulate GBM cell motility. This was done in vitro by modifying existing scratch assay protocols to examine not only the velocity of GBM cells, but also the preferential direction of their movement toward other GBM cells either releasing, or not releasing, L1ecto. The results of these experiments showed a significant chemotactic effect of released L1ecto. Responding cells moved significantly faster and directionally toward cells releasing L1ecto compared to their motility toward cells not releasing L1ecto. This suggests cells were responding to a gradient of L1ecto originating from the L1ecto releasing cells. These experiments suggest a possible leader‐follower relationship between L1 releasing and responding GBM cells during brain tumor invasion into surrounding tissue. To test this, we also studied a possible chemotactic relationship in vivo by injecting a mixture of fluorescently labelled red L1 positive and green L1 negative glioblastoma cells into the optic tectum of chick embryos. The embryos developed for ten days, after which the brain was dissected, sectioned with a vibratome, and then optically sectioned using a confocal microscope. 3D models then were rendered to examine the invasion pathways of the cancerous cells. Results thus far suggest a strong “leader‐follower” relationship between L1ecto releasing cells and non‐L1 producing cells. We conclude that 1) released L1ecto can form a soluble gradient that can be detected by GBM cells and guide their direction and stimulate their velocity; 2) immobilized L1ecto also can stimulate and guide GBM cell motility in vitro; and 3) GBM cells releasing L1ecto in brain appear to be able to influence other GBM cells to follow them.Support or Funding InformationWe thank the University of Delaware's Summer Scholars Program, Undergraduate Research Program, Dept. of Biological Sciences Undergraduate Research Fund, ASBMB, and graduate student Reetika Dutt.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The influence of soluble fragments of extracellular matrix (ECM) on tumor growth and morphology

A major challenge in matrix-metalloproteinase (MMP) target validation and MMP-inhibitor-drug development for anti-cancer clinical trials is to better understand their complex roles (often competing with each other) in tumor progression. While there is extensive research on the growth-promoting effects of MMPs, the growth-inhibiting effects of MMPs has not been investigated thoroughly. So we develop a continuum model of tumor growth and invasion including chemotaxis and haptotaxis in order to examine the complex interaction between the tumor and its host microenvironment and to explore the inhibiting influence of the gradients of soluble fragments of extracellular matrix (ECM) density on tumor growth and morphology. Previously, it was shown both computationally (in one spatial dimension) and experimentally that the chemotactic pull due to soluble ECM gradients is anti-invasive, contrary to the traditional view of the role of chemotaxis in malignant invasion [1]. With two-dimensional numerical simulation and using a level set based tumor-host interface capturing method, we examine the effects of chemotaxis on the progression and morphology of a tumor growing in nutrient-rich and nutrient-poor microenvironments which was not investigated before. In particular we examine how the geometry of the growing tumor is affected when placed in different environments. We also investigate the effects of varying ECM degradation rate, the production rate of matrix degrading enzymes (MDE), and the conversion of ECM into soluble ECM. We find that chemotaxis due to ECM-fragment gradients strongly influences tumor growth and morphology, and that the instabilities caused by tumor cell proliferation and haptotactic movements can be prevented if chemotaxis is sufficiently strong. The influence of chemotaxis and the above factors on tumor growth and morphology are found to be more prominent in nutrient-poor environments than in nutrient-rich environments. So we extend our investigations of these antinvasive chemotactic influences by examining the effects of cell-cell and cell-ECM adhesion and low proliferation rate for tumors growing in low-nutrient environments. We find that as the extent of chemotaxis increases, the effects of adhesion on tumor growth and shape become negligible. Under conditions of low cell mitosis, chemotaxis may cause the tumor to shrink, as the extent of chemotaxis increases. Both stable and unstable tumor shrinkage are predicted by our model. Unexpectedly, in some cases chemotaxis may contribute toward developing instability where haptotaxis alone induces stable growth.

Engineering cell aggregates through incorporated polymeric microparticles

Cell-scaled polymer microparticles (MPs) integrated into cellular aggregates have been shown to be a powerful tool to direct cell response. MPs have supported the development of healthy cartilage, islets, nerves, and vasculature by the maintenance of soluble gradients as well as by the local presentation of tethered cues and diffusing proteins and small molecules. MPs integrated with pluripotent stem cells have directed in vivo expansion and differentiation. Looking forward, MPs are expected to support both the characterization and development of in vitro tissue systems for applications such as drug testing platforms. However, useful co-cultures must be designed keeping in mind the limitations and attributes of each material strategy within the context of the overall tissue biology. The present review integrates prospectives from materials development, drug delivery, and tissue engineering to provide a toolbox for the development and application of MPs useful for long-term co-culture within cell aggregates.

A microfluidic device for measuring cell migration towards substrate-bound and soluble chemokine gradients.

Cellular locomotion is a central hallmark of eukaryotic life. It is governed by cell-extrinsic molecular factors, which can either emerge in the soluble phase or as immobilized, often adhesive ligands. To encode for direction, every cue must be present as a spatial or temporal gradient. Here, we developed a microfluidic chamber that allows measurement of cell migration in combined response to surface immobilized and soluble molecular gradients. As a proof of principle we study the response of dendritic cells to their major guidance cues, chemokines. The majority of data on chemokine gradient sensing is based on in vitro studies employing soluble gradients. Despite evidence suggesting that in vivo chemokines are often immobilized to sugar residues, limited information is available how cells respond to immobilized chemokines. We tracked migration of dendritic cells towards immobilized gradients of the chemokine CCL21 and varying superimposed soluble gradients of CCL19. Differential migratory patterns illustrate the potential of our setup to quantitatively study the competitive response to both types of gradients. Beyond chemokines our approach is broadly applicable to alternative systems of chemo- and haptotaxis such as cells migrating along gradients of adhesion receptor ligands vs. any soluble cue.

In This Issue

In This Issue

Mechanical Forces Reshape Differentiation Cues That Guide Cardiomyogenesis.

Soluble morphogen gradients have long been studied in the context of heart specification and patterning. However, recent data have begun to challenge the notion that long-standing in vivo observations are driven solely by these gradients alone. Evidence from multiple biological models, from stem cells to ex vivo biophysical assays, now supports a role for mechanical forces in not only modulating cell behavior but also inducing it de novo in a process termed mechanotransduction. Structural proteins that connect the cell to its niche, for example, integrins and cadherins, and that couple to other growth factor receptors, either directly or indirectly, seem to mediate these changes, although specific mechanistic details are still being elucidated. In this review, we summarize how the wingless (Wnt), transforming growth factor-β, and bone morphogenetic protein signaling pathways affect cardiomyogenesis and then highlight the interplay between each pathway and mechanical forces. In addition, we will outline the role of integrins and cadherins during cardiac development. For each, we will describe how the interplay could change multiple processes during cardiomyogenesis, including the specification of undifferentiated cells, the establishment of heart patterns to accomplish tube and chamber formation, or the maturation of myocytes in the fully formed heart.

Experimental and computational models of neurite extension at a choice point in response to controlled diffusive gradients

Ojective. Axons are guided toward desired targets through a series of choice points that they navigate by sensing cues in the cellular environment. A better understanding of how microenvironmental factors influence neurite growth during development can inform strategies to address nerve injury. Therefore, there is a need for biomimetic models to systematically investigate the influence of guidance cues at such choice points. Approach. We ran an adapted in silico biased turning axon growth model under the influence of nerve growth factor (NGF) and compared the results to corresponding in vitro experiments. We examined if growth simulations were predictive of neurite population behavior at a choice point. We used a biphasic micropatterned hydrogel system consisting of an outer cell restrictive mold that enclosed a bifurcated cell permissive region and placed a well near a bifurcating end to allow proteins to diffuse and form a gradient. Experimental diffusion profiles in these constructs were used to validate a diffusion computational model that utilized experimentally measured diffusion coefficients in hydrogels. The computational diffusion model was then used to establish defined soluble gradients within the permissive region of the hydrogels and maintain the profiles in physiological ranges for an extended period of time. Computational diffusion profiles informed the neurite growth model, which was compared with neurite growth experiments in the bifurcating hydrogel constructs. Main results. Results indicated that when applied to the constrained choice point geometry, the biased turning model predicted experimental behavior closely. Results for both simulated and in vitro neurite growth studies showed a significant chemoattractive response toward the bifurcated end containing an NGF gradient compared to the control, though some neurites were found in the end with no NGF gradient. Significance. The integrated model of neurite growth we describe will allow comparison of experimental studies against growth cone guidance computational models applied to axon pathfinding at choice points.