Collective adhesion and displacement of retinal progenitor cells upon extracellular matrix substrates of transplantable biomaterials

Strategies to replace retinal photoreceptors lost to damage or disease rely upon the migration of replacement cells transplanted into sub-retinal spaces. A significant obstacle to the advancement of cell transplantation for retinal repair is the limited migration of transplanted cells into host retina. In this work, we examine the adhesion and displacement responses of retinal progenitor cells on extracellular matrix substrates found in retina as well as widely used in the design and preparation of transplantable scaffolds. The data illustrate that retinal progenitor cells exhibit unique adhesive and displacement dynamics in response to poly-l-lysine, fibronectin, laminin, hyaluronic acid, and Matrigel. These findings suggest that transplantable biomaterials can be designed to improve cell integration by incorporating extracellular matrix substrates that affect the migratory behaviors of replacement cells

Collective Behavior of Drosophila Melanogaster Neural Progenitor and Imaginal Disc Cells within Controlled Microenvironments

Regenerative therapies for the damaged visual system have introduced stem-derived cells to recapitulate developmental processes and initiate functional regeneration in different components of the eye. The developing visual system in Drosophila Melanogaster offers a model in which to analyze the associated processes in retinogenesis. The optic nerve is critical to vision and is developmentally preceded in Drosophila by a structure called the Optic Stalk (OS). Collective migration of neural and retinal progenitor cells (RPCs) from the developing brain lobes (DBL) to the Imaginal Disc (ID), through the OS, is a fundamental part of regenerative strategies in retina. Developmental signals governing retinal cell fate and migration have been well-studied using Drosophila Melanogaster. While conserved signaling pathways are known to drive retinogenesis across invertebrate and vertebrate species, the role(s) of diffusible signaling molecules in the collective migratory processes critical to eye development remain incompletely understood. Invertebrate models remain largely underutilized for in vitro study of cell response to controlled stimuli. In this thesis, the collective behavior and migration of primary Drosophila-derived neural progenitor cells (NPC) and Drosophila imaginal disc cells were analyzed on different extracellular matrices and coatings, poly-L-lysine (PLL), laminin (LM) and Concanavalin- A (Con-A), when exposed to exogenous gradients of growth factors and within microfluidic systems in order to propose an animal model for retinogenesis. The formation of single, small clusters and large clusters of NPCs were observed on all matrices and within the μLane, a bridged microchannel system. Furthermore, small and large clusters were demonstrated chemotaxis, directed cell migration, to gradients of fibroblast growth factor 8 (FGF8), while single cells demonstrated chemokinesis, non- directed cell migration within the μLane. A microfluidic system called the Micro Optic Stalk (μOS), that recapitulated in vivo geometric constraints seen during Drosophila retinal development, was designed, fabricated and validated. When NPCs were cultured and exposed to FGF8 within the μOS, the formation of single cells, small clusters and large clusters was observed. Furthermore, clusters demonstrated chemotaxis and gradient-dependent migration patterns. Imaginal disc cells were studied in order to look at the behavior of the secondary structure involved in retinogenesis. Dm-D17-c3 (D17) cells were examined as they have been utilized for motility studies in literature. D17 cells demonstrated two cell populations, rounded and elongated cells, on PLL and Con- A, while they did not adhere and grew in suspension on LM. Utilizing Boyden Chamber Assays, D17 cells showed significant migration toward brain-derived neurotrophic factor (BDNF), concentration-dependent migration toward Insulin (In), and no significant migration toward FGF8. Furthermore, D17 cells showed high viability when cultured within the μLane when seeded at higher cell densities (7.5 ́105 cells and 1 ́106 cells). Although D17 cells were shown to not be suitable for examination in a retinogenesis model centering on the role of FGF8, they show promise for use in other developmental models and microfluidic systems. Future work will utilize FGFR receptor knock outs in Drosophila within the μOS, in order to further understand FGF8’s role in retinogenesis

In vitro formation of neuroclusters in microfluidic devices and cell migration as a function of stromal-derived growth factor 1 gradients

Central nervous system (CNS) cells cultured in vitro as neuroclusters are useful models of tissue regeneration and disease progression. However, the role of cluster formation and collective migration of these neuroclusters to external stimuli has been largely unstudied in vitro. Here, 3 distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were examined with respect to cluster formation and migration in response to Stromal-Derived Growth Factor (SDF-1). A microfluidic platform was used to distinguish collective migration of neuroclusters from that of individual cells in response to controlled concentration profiles of SDF-1. Cell lines were also compared with respect to expression of CXCR4, the receptor for SDF-1, and the gap junction protein Connexin 43 (Cx43). All cell types spontaneously formed clusters and expressed both CXCR4 and Cx43. RPC clusters exhibited collective chemotactic migration (i.e. movement as clusters) along SDF-1 concentration gradients. MGPCs clusters did not exhibit adhesion-based migration, and migration of MB clusters was inconsistent. This study demonstrates how controlled microenvironments can be used to examine the formation and collective migration of CNS-derived neuroclusters in varied cell populations

Long-range directional growth of neurites induced by magnetic forces

The ability to control the growth and orientation of neurites over long distances has significant implications for regenerative therapies and the development of physiologically relevant brain tissue models. In this study, the forces generated on magnetic nanoparticles internalised within intracellular endosomes are used to direct the orientation of neuronal outgrowth in cell cultures. Following differentiation, neurite orientation was observed after 3 days application of magnetic forces to human neuroblastoma (SH-SY5Y) cells, and after 4 days application to rat cortical primary neurons. The direction of neurite outgrowth was quantified using a 2D Fourier transform analysis, showing agreement with the derived magnetic force vectors. Orientation control was found to be effective over areas >1cm2 using modest forces of ∼10 fN per endosome, apparently limited only by the local confluence of the cells. A bioinformatics analysis of protein expression in cells exposed to magnetic forces revealed changes to cell signaling and metabolic pathways resulting in enhanced carbohydrate metabolism, as well as the perturbation of processes related to cellular organisation and proliferation. Additionally, in cell culture regions where the measured force vectors converged, large (∼100 µm) SH-SY5Y neuroclusters loaded with nanoparticles were found, connected by unusually thick linear neurite fibres. This could suggest a magnetically driven enhancement of neurocluster growth, with the clusters themselves contributing to the local forces that direct outgrowth. Such structures, which have not been previously observed, could provide new insights into the development and possible enhancement of neural circuitry

Chemotactic Migration of Clustered Central Nervous System Progenitor Cells

Clustering of central nervous system (CNS) cells is often utilized for cell growth and characterization, as well as investigated for tissue regeneration and disease progression. Collective CNS cell migration, however, has been largely unstudied. Cell cluster formation and migration play a critical part of modeling in vivo conditions and in development of therapies. Three distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were investigated for cluster formation, upregulation of CXCR4, the receptor for Stromal-Derived Growth Factor (SDF-1), and Connexin 43 expression, a gap junction hemichannel. A microfluidic platform was used to examine the the migration of clusters and single cells in response to controlled concentration gradients of SDF-1. All cell types illustrated self-clustering, as well as upregulated CXCR4 surface expression and increased Connexin 43 expression upon ligand stimulation. Further, RPC clusters exhibited collective, chemotactic migration along SDF-1 concentration gradients, while MB clusters illustrated inconsistent collective migration, and MGPCs clusters did not exhibit adhesion-based migration

Neurostream: Scalable and Energy Efficient Deep Learning with Smart Memory Cubes

open4siHigh-performance computing systems are moving towards 2.5D and 3D memory hierarchies, based on High Bandwidth Memory (HBM) and Hybrid Memory Cube (HMC) to mitigate the main memory bottlenecks. This trend is also creating new opportunities to revisit near-memory computation. In this paper, we propose a flexible processor-in-memory (PIM) solution for scalable and energy-efficient execution of deep convolutional networks (ConvNets), one of the fastest-growing workloads for servers and high-end embedded systems. Our co-design approach consists of a network of Smart Memory Cubes (modular extensions to the standard HMC) each augmented with a many-core PIM platform called NeuroCluster. NeuroClusters have a modular design based on NeuroStream coprocessors (for Convolution-intensive computations) and general-purpose RISC-V cores. In addition, a DRAM-friendly tiling mechanism and a scalable computation paradigm are presented to efficiently harness this computational capability with a very low programming effort. NeuroCluster occupies only 8 percent of the total logic-base (LoB) die area in a standard HMC and achieves an average performance of 240 GFLOPS for complete execution of full-featured state-of-the-art (SoA) ConvNets within a power budget of 2.5 W. Overall 11 W is consumed in a single SMC device, with 22.5 GFLOPS/W energy-efficiency which is 3.5X better than the best GPU implementations in similar technologies. The minor increase in system-level power and the negligible area increase make our PIM system a cost-effective and energy efficient solution, easily scalable to 955 GFLOPS with a small network of just four SMCs.openAzarkhish, Erfan*; Rossi, Davide; Loi, Igor; Benini, LucaAzarkhish, Erfan*; Rossi, Davide; Loi, Igor; Benini, Luc

Invertebrate Retinal Progenitors as Regenerative Models in a Microfluidic System

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by the misdirected and/or insuffcient migration of transplanted cells. RPCs must achieve appropriate spatial and functional positioning in host retina, collectively, to restore vision, whereas movement of clustered cells differs substantially from the single cell migration studied in classical chemotaxis models. Defining how RPCs interact with each other, neighboring cell types and surrounding extracellular matrixes are critical to our understanding of retinogenesis and the development of effective, cell-based approaches to retinal replacement. The current article describes a new bio-engineering approach to investigate the migratory responses of innate collections of RPCs upon extracellular substrates by combining microfluidics with the well-established invertebrate model of Drosophila melanogaster. Experiments utilized microfluidics to investigate how the composition, size, and adhesion of RPC clusters on defined extracellular substrates affected migration to exogenous chemotactic signaling. Results demonstrated that retinal cluster size and composition influenced RPC clustering upon extracellular substrates of concanavalin (Con-A), Laminin (LM), and poly-L-lysine (PLL), and that RPC cluster size greatly altered collective migratory responses to signaling from Fibroblast Growth Factor (FGF), a primary chemotactic agent in Drosophila. These results highlight the significance of examining collective cell-biomaterial interactions on bio-substrates of emerging biomaterials to aid directional migration of transplanted cells. Our approach further introduces the benefits of pairing genetically controlled models with experimentally controlled microenvironments to advance cell replacement therapies

An emerging paradigm of CXCL12 involvement in the metastatic cascade

The chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF1), has emerged as a pivotal regulator in the intricate molecular networks driving cancer progression. As an influential factor in the tumor microenvironment, CXCL12 plays a multifaceted role that spans beyond its traditional role as a chemokine inducing invasion and metastasis. Indeed, CXCL12 has been assigned functions related to epithelial-to-mesenchymal transition, cancer cell stemness, angiogenesis, and immunosuppression, all of which are currently viewed as specialized biological programs contributing to the “metastatic cascade” among other cancer hallmarks. Its interaction with its cognate receptor, CXCR4, initiates a cascade of events that not only shapes the metastatic potential of tumor cells but also defines the niches within the secondary organs that support metastatic colonization. Given the profound implications of CXCL12 in the metastatic cascade, understanding its mechanistic underpinnings is of paramount importance for the targeted elimination of rate-limiting steps in the metastatic process. This review aims to provide a comprehensive overview of the current knowledge surrounding the role of CXCL12 in cancer metastasis, especially its molecular interactions rationalizing its potential as a therapeutic target.</p

A Gal-MS Device to Evaluate Cell Migratory Response to Combined Galvano-Chemotactic Fields

Electric fields have been studied extensively in biomedical engineering (BME) for numerous regenerative therapies. Recent studies have begun to examine the biological effects of electric fields in combination with other environmental cues, such as tissue-engineered extracellular matrices (ECM), chemical gradient profiles, and time-dependent temperature gradients. In the nervous system, cell migration driven by electrical fields, or galvanotaxis, has been most recently studied in transcranial direct stimulation (TCDS), spinal cord repair and tumor treating fields (TTF). The cell migratory response to galvano-combinatory fields, such as magnetic fields, chemical gradients, or heat shock, has only recently been explored. In the visual system, restoration of vision via cellular replacement therapies has been limited by low numbers of motile cells post-transplantation. Here, the combinatory application of electrical fields with other stimuli to direct cells within transplantable biomaterials and/or host tissues has been understudied. In this work, we developed the Gal-MS device, a novel microfluidics device capable of examining cell migratory behavior in response to single and combinatory stimuli of electrical and chemical fields. The formation of steady-state, chemical concentration gradients and electrical fields within the Gal-MS were modeled computationally and verified experimentally within devices fabricated via soft lithography. Further, we utilized real-time imaging within the device to capture cell trajectories in response to electric fields and chemical gradients, individually, as well as in combinatory fields of both. Our data demonstrated that neural cells migrated longer distances and with higher velocities in response to combined galvanic and chemical stimuli than to either field individually, implicating cooperative behavior. These results reveal a biological response to galvano-chemotactic fields that is only partially understood, as well as point towards novel migration-targeted treatments to improve cell-based regenerative therapies

Development of Methodologies for the Solution of the Forward Problem in Magnetic-Field Tomography (MFT) Based on Magnetoencephalography (MEG)

The prime topic of research presented in this report is the development and validation of methodologies for the solution of the forward problem in Magnetic field Tomography based on Magnetoencephalography. Throughout the report full aspects of the accurate solution are discussed, including the development of algorithms and methods for realistic brain model, development of realistic neuronal source, computational approaches, and validation techniques. Every delivered methodology is tested and analyzed in terms of mathematical and computational errors. Optimizations required for error minimization are performed and discussed. Presented techniques are successfully integrated together for different test problems. Results were compared to experimental data where possible for the most of calculated cases. Designed human brain model reconstruction algorithms and techniques, which are based on MRI (Magnetic Resonance Imaging) modality, are proved to be the most accurate among existing in terms of geometrical and material properties. Error estimations and algorithm structure delivers the resolution of the model to be the same as practical imaging resolution of the MRI equipment (for presented case was less than 1mm). Novel neuronal source modelling approach was also presented with partial experimental validation showing improved results in comparison to all existing methods. At the same time developed mathematical basis for practical realization of discussed approach allows computer simulations of any known neuronal formation. Also it is the most suitable method for Finite Element Method (FEM) which was proved to be the best computer solver for complex bio-electrical problems. The mathematical structure for Inverse problem solution which is based on integrated human brain modelling technique and neuronal source modelling approach is delivered and briefly discussed. In the concluding part of the report the practical application case of developed techniques is performed and discussed