Motor neurons and endothelial cells additively promote development and fusion of human iPSC-derived skeletal myocytes.

BACKGROUND: Neurovascular cells have wide-ranging implications on skeletal muscle biology regulating myogenesis, maturation, and regeneration. Although several in vitro studies have investigated how motor neurons and endothelial cells interact with skeletal myocytes independently, there is limited knowledge about the combined effect of neural and vascular cells on muscle maturation and development. METHODS: Here, we report a triculture system comprising human-induced pluripotent stem cell (iPSC)-derived skeletal myocytes, human iPSC-derived motor neurons, and primary human endothelial cells maintained under controlled media conditions. Briefly, iPSCs were differentiated to generate skeletal muscle progenitor cells (SMPCs). These SMPCs were seeded at a density of 5 × 104 cells/well in 12-well plates and allowed to differentiate for 7 days before adding iPSC-derived motor neurons at a concentration of 0.5 × 104 cells/well. The neuromuscular coculture was maintained for another 7 days in coculture media before addition of primary human umbilical vein endothelial cells (HUVEC) also at 0.5 × 104 cells/well. The triculture was maintained for another 7 days in triculture media comprising equal portions of muscle differentiation media, coculture media, and vascular media. Extensive morphological, genetic, and molecular characterization was performed to understand the combined and individual effects of neural and vascular cells on skeletal muscle maturation. RESULTS: We observed that motor neurons independently promoted myofiber fusion, upregulated neuromuscular junction genes, and maintained a molecular niche supportive of muscle maturation. Endothelial cells independently did not support myofiber fusion and downregulated expression of LRP4 but did promote expression of type II specific myosin isoforms. However, neurovascular cells in combination exhibited additive increases in myofiber fusion and length, enhanced production of Agrin, along with upregulation of several key genes like MUSK, RAPSYN, DOK-7, and SLC2A4. Interestingly, more divergent effects were observed in expression of genes like MYH8, MYH1, MYH2, MYH4, and LRP4 and secretion of key molecular factors like amphiregulin and IGFBP-4. CONCLUSIONS: Neurovascular cells when cultured in combination with skeletal myocytes promoted myocyte fusion with concomitant increase in expression of various neuromuscular genes. This triculture system may be used to gain a deeper understanding of the effects of the neurovascular niche on skeletal muscle biology and pathophysiology.

Bedside to bench: the outlook for psychedelic research.

There has recently been a resurgence of interest in psychedelic compounds based on studies demonstrating their potential therapeutic applications in treating post-traumatic stress disorder, substance abuse disorders, and treatment-resistant depression. Despite promising efficacy observed in some clinical trials, the full range of biological effects and mechanism(s) of action of these compounds have yet to be fully established. Indeed, most studies to date have focused on assessing the psychological mechanisms of psychedelics, often neglecting the non-psychological modes of action. However, it is important to understand that psychedelics may mediate their therapeutic effects through multi-faceted mechanisms, such as the modulation of brain network activity, neuronal plasticity, neuroendocrine function, glial cell regulation, epigenetic processes, and the gut-brain axis. This review provides a framework supporting the implementation of a multi-faceted approach, incorporating in silico, in vitro and in vivo modeling, to aid in the comprehensive understanding of the physiological effects of psychedelics and their potential for clinical application beyond the treatment of psychiatric disorders. We also provide an overview of the literature supporting the potential utility of psychedelics for the treatment of brain injury (e.g., stroke and traumatic brain injury), neurodegenerative diseases (e.g., Parkinson's and Alzheimer's diseases), and gut-brain axis dysfunction associated with psychiatric disorders (e.g., generalized anxiety disorder and major depressive disorder). To move the field forward, we outline advantageous experimental frameworks to explore these and other novel applications for psychedelics.

Emergent structural and functional properties of hippocampal multi-cellular aggregates.

Hippocampal neural networks are distinctly capable of integrating multi-modal sensory inputs to drive memory formation. Neuroscientific investigations using simplified in vitro models have greatly relied on planar (2D) neuronal cultures made from dissociated tissue. While these models have served as simple, cost-effective, and high-throughput tools for examining various morphological and electrophysiological characteristics of hippocampal networks, 2D cultures fail to reconstitute critical elements of the brain microenvironment that may be necessary for the emergence of sophisticated integrative network properties. To address this, we utilized a forced aggregation technique to generate high-density (>100,000 cells/mm3) multi-cellular three-dimensional aggregates using rodent embryonic hippocampal tissue. We contrasted the emergent structural and functional properties of aggregated (3D) and dissociated (2D) cultures over 28 days in vitro (DIV). Hippocampal aggregates displayed robust axonal fasciculation across large distances and significant neuronal polarization, i.e., spatial segregation of dendrites and axons, at earlier time points compared to dissociated cultures. Moreover, we found that astrocytes in aggregate cultures self-organized into non-overlapping quasi-domains and developed highly stellate morphologies resembling astrocyte structures in vivo. We maintained cultures on multi-electrode arrays (MEAs) to assess spontaneous electrophysiological activity for up to 28 DIV. We found that 3D networks of aggregated cultures developed highly synchronized networks and with high burstiness by 28 DIV. We also demonstrated that dual-aggregate networks became active by 7 DIV, in contrast to single-aggregate networks which became active and developed synchronous bursting activity with repeating motifs by 14 DIV. Taken together, our findings demonstrate that the high-density, multi-cellular, 3D microenvironment of hippocampal aggregates supports the recapitulation of emergent biofidelic morphological and functional properties. Our findings suggest that neural aggregates may be used as segregated, modular building blocks for the development of complex, multi-nodal neural network topologies.

Papaya latex mediated synthesis of prism shaped proteolytic gold nanozymes.

Beyond natural enzymes, the artificially synthesized nanozymes have attracted a significant interest as it can overcome the limitations of the former. Here, we report synthesis of shape controlled nanozymes showing proteolytic activity using Carica papaya L. (papaya) latex. The nanozymes synthesized under optimized reaction conditions exhibited sharp SPR peak around 550 nm with high abundance (45.85%) of prism shaped particles. FTIR analysis and coagulation test indicated the presence of papaya latex enzymes as capping agents over the gold nanoprisms. The milk clot assay and the inhibition test with egg white confirmed the proteolytic activity of the nanozymes and the presence of cysteine protease on it, respectively. The nanozymes were found to be biocompatible and did not elicit any toxic response in both in-vitro and in-vivo study. Based on our findings, we envisage that these biocompatible, shape-specific nanozymes can have potential theragnostic applications.

Engineered neuronal microtissue provides exogenous axons for delayed nerve fusion and rapid neuromuscular recovery in rats.

Nerve injury requiring surgical repair often results in poor functional recovery due to the inability of host axons to re-grow long distances and reform meaningful connections with the target muscle. While surgeons can re-route local axon fascicles to the target muscle, there are no technologies to provide an exogenous source of axons without sacrificing healthy nerves. Accordingly, we have developed tissue engineered neuromuscular interfaces (TE-NMIs) as the first injectable microtissue containing motor and sensory neurons in an anatomically-inspired architecture. TE-NMIs provide axon tracts that are intended to integrate with denervated distal structures and preserve regenerative capacity during prolonged periods without host innervation. Following implant, we found that TE-NMI axons promoted Schwann cell maintenance, integrated with distal muscle, and preserved an evoked muscle response out to 20-weeks post nerve transection in absence of innervation from host axons. By repopulating the distal sheath with exogenous axons, TE-NMIs also enabled putative delayed fusion with proximal host axons, a phenomenon previously not achievable in delayed repair scenarios due to distal axon degeneration. Here, we found immediate electrophysiological recovery after fusion with proximal host axons and improved axon maturation and muscle reinnervation at 24-weeks post-transection (4-weeks following delayed nerve fusion). These findings show that TE-NMIs provide the potential to improve functional recovery following delayed nerve repair.

Innervation: the missing link for biofabricated tissues and organs.

Innervation plays a pivotal role as a driver of tissue and organ development as well as a means for their functional control and modulation. Therefore, innervation should be carefully considered throughout the process of biofabrication of engineered tissues and organs. Unfortunately, innervation has generally been overlooked in most non-neural tissue engineering applications, in part due to the intrinsic complexity of building organs containing heterogeneous native cell types and structures. To achieve proper innervation of engineered tissues and organs, specific host axon populations typically need to be precisely driven to appropriate location(s) within the construct, often over long distances. As such, neural tissue engineering and/or axon guidance strategies should be a necessary adjunct to most organogenesis endeavors across multiple tissue and organ systems. To address this challenge, our team is actively building axon-based "living scaffolds" that may physically wire in during organ development in bioreactors and/or serve as a substrate to effectively drive targeted long-distance growth and integration of host axons after implantation. This article reviews the neuroanatomy and the role of innervation in the functional regulation of cardiac, skeletal, and smooth muscle tissue and highlights potential strategies to promote innervation of biofabricated engineered muscles, as well as the use of "living scaffolds" in this endeavor for both in vitro and in vivo applications. We assert that innervation should be included as a necessary component for tissue and organ biofabrication, and that strategies to orchestrate host axonal integration are advantageous to ensure proper function, tolerance, assimilation, and bio-regulation with the recipient post-implant.

Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss.

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.

A Porcine Model of Peripheral Nerve Injury Enabling Ultra-Long Regenerative Distances: Surgical Approach, Recovery Kinetics, and Clinical Relevance.

BACKGROUND: Millions of Americans experience residual deficits from traumatic peripheral nerve injury (PNI). Despite advancements in surgical technique, repair typically results in poor functional outcomes due to prolonged periods of denervation resulting from long regenerative distances coupled with slow rates of axonal regeneration. Novel surgical solutions require valid preclinical models that adequately replicate the key challenges of clinical PNI. OBJECTIVE: To develop a preclinical model of PNI in swine that addresses 2 challenging, clinically relevant PNI scenarios: long segmental defects (≥5 cm) and ultra-long regenerative distances (20-27 cm). Thus, we aim to demonstrate that a porcine model of major PNI is suitable as a potential framework to evaluate novel regenerative strategies prior to clinical deployment. METHODS: A 5-cm-long common peroneal nerve or deep peroneal nerve injury was repaired using a saphenous nerve or sural nerve autograft, respectively. Histological and electrophysiological assessments were performed at 9 to 12 mo post repair to evaluate nerve regeneration and functional recovery. Relevant anatomy, surgical approach, and functional/histological outcomes were characterized for both repair techniques. RESULTS: Axons regenerated across the repair zone and were identified in the distal stump. Electrophysiological recordings confirmed these findings and suggested regenerating axons reinnervated target muscles. CONCLUSION: The models presented herein provide opportunities to investigate peripheral nerve regeneration using different nerves tailored for specific mechanisms of interest, such as nerve modality (motor, sensory, and mixed fiber composition), injury length (short/long gap), and total regenerative distance (proximal/distal injury).

Stretch growth of motor axons in custom mechanobioreactors to generate long-projecting axonal constructs.

The central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to distant target muscle. However, current in vitro models have not replicated this feature owing to challenges in generating motor axon tracts beyond a few millimeters in length. To address this, we have developed a novel combination of microtissue engineering and mechanically assisted growth techniques to create long-projecting centimeter-scale motor axon tracts. Here, primary motor neurons were isolated from rat spinal cords and induced to form engineered microspheres via forced aggregation in custom microwells. This technique yielded healthy motor neurons projecting dense, fasciculated axonal tracts. Within our custom-built mechanobioreactors, motor neuron culture conditions, neuronal/axonal architecture, and mechanical growth conditions were optimized to generate parameters for robust and efficient stretch growth of motor axons. We found that axons projecting from motor neuron aggregates were able to tolerate displacement rates at least 10 times greater than those by axons projecting from dissociated motor neurons. The growth and structural characteristics of these stretch-grown motor axons were compared with that of benchmark stretch-grown sensory axons, revealing increased motor axon fasciculation. Finally, motor axons were integrated with myocytes and stretch grown to create novel long-projecting axonal-myocyte constructs that recreate characteristic dimensions of native nerve-muscle anatomy. This is the first demonstration of mechanical elongation of spinal motor axons and may have applications as anatomically inspired in vitro testbeds or as tissue-engineered living scaffolds for targeted axon tract reconstruction following nervous system injury or disease.

Microfabricated intracortical extracellular matrix-microelectrodes for improving neural interfaces.

Intracortical neural microelectrodes, which can directly interface with local neural microcircuits with high spatial and temporal resolution, are critical for neuroscience research, emerging clinical applications, and brain computer interfaces (BCI). However, clinical applications of these devices remain limited mostly by their inability to mitigate inflammatory reactions and support dense neuronal survival at their interfaces. Herein we report the development of microelectrodes primarily composed of extracellular matrix (ECM) proteins, which act as a bio-compatible and an electrochemical interface between the microelectrodes and physiological solution. These ECM-microelectrodes are batch fabricated using a novel combination of micro-transfer-molding and excimer laser micromachining to exhibit final dimensions comparable to those of commercial silicon-based microelectrodes. These are further integrated with a removable insertion stent which aids in intracortical implantation. Results from electrochemical models and in vivo recordings from the rat's cortex indicate that ECM encapsulations have no significant effect on the electrochemical impedance characteristics of ECM-microelectrodes at neurologically relevant frequencies. ECM-microelectrodes are found to support a dense layer of neuronal somata and neurites on the electrode surface with high neuronal viability and exhibited markedly diminished neuroinflammation and glial scarring in early chronic experiments in rats.

Electrospun silk-polyaniline conduits for functional nerve regeneration in rat sciatic nerve injury model.

The present study describes the fabrication of polyaniline-silk fibroin (PASF) nanocomposite-based nerve conduits and their subsequent implantation in a rat sciatic nerve injury model for peripheral nerve regeneration. This is the first in vivo study of polyaniline-based nerve conduits describing the safety and efficacy of the conduits in treating peripheral nerve injuries. The nanocomposite was synthesized by electrospinning a mixture of silk fibroin protein and polyaniline wherein the silk nanofibers were observed to be uniformly coated with polyaniline nanoparticles. Tubular shaped nerve conduits were subsequently formed by multiple rolling of the electrospun sheet over a stainless steel mandrel. The conduits were characterized in vitro for their physico-chemical properties as well as their compatibility with rat Schwann cells. Upon implantation in a 10 mm sciatic nerve injury model, the conduits were evaluated for their neuro-regenerative potential through extensive electrophysiological studies and monitoring of gait pattern over a course of 12 months. Gross examination, histological and ultra-structure analyses of the conduits and the regenerated nerve were also performed to evaluate morphological regeneration of transected nerve. PASF nanocomposite conduits seeded with Schwann cell (cell seeded PASF) exhibited excellent nerve conduction velocity (NCV) (50 m s-1), compound muscle action potential (CMAP) (12.8 mV), motor unit potential (MUP) (124 µV), growth of healthy tissue along the nerve gap and thick myelination of axons 12 months after implantation indicating enhanced neuro-regeneration. The excellent functional recovery achieved by animals implanted with cell seeded PASF conduits (86.2% NCV; 80.00% CMAP; 76.07% MUP) are superior to outcomes achieved previously with similar electrically conductive conduits. We believe that the present study would encourage further research in developing electrically active neural implants using synthetic conducting polymers and the in vivo applications of the same.

De novo transcriptome of the muga silkworm, Antheraea assamensis (Helfer).

Antheraea assamensis (Lepidoptera: Saturniidae), is a semi-domesticated silkworm known to be endemic to Assam and the adjoining hilly areas of Northeast India. It is the only producer of a unique, commercially important variety of golden silk called "muga silk". Herein, we report the de novo transcriptome of A. assamensis reared on Machilus bombycina leaves for the first time. Short reads generated by high throughput sequencing of cDNA libraries from multiple tissues, viz. alimentary canal, silk gland and residual body of the 5th instar of muga silkworm were assembled into transcripts via a de novo assembly pipeline followed by functional annotation and classification. A total of 1,21,433 transcripts were generated from ~231 million raw reads of which ~74% (89,583) were either allocated a functional annotation or categorized under Pfam/COG/KEGG categories. Identification of differentially expressed transcripts and their comparative sequence analysis revealed candidate genes related to silk synthesis, viz. silk gland factor-1 and 3, sericin-like transcript, etc. with conserved forkhead, homeo- and POU domains. Several candidate anti-microbial peptides which may have potential anti-bacterial, anti-fungal or anti-parasitic activity in A. assamensis were also identified. T/A and AT/TA were predicted to be the most abundant mono- and di-nucleotide simple sequence repeat markers in the transcriptome. Transcriptome validation was carried out by quantitative real-time PCR (qPCR) amplification of eight transcripts. The resources generated by this study will expand the periphery of existing genomic data on A. assamensis facilitating future in-depth studies on its unknown aspects.

Bio-inspired nano tools for neuroscience.

Research and treatment in the nervous system is challenged by many physiological barriers posing a major hurdle for neurologists. The CNS is protected by a formidable blood brain barrier (BBB) which limits surgical, therapeutic and diagnostic interventions. The hostile environment created by reactive astrocytes in the CNS along with the limited regeneration capacity of the PNS makes functional recovery after tissue damage difficult and inefficient. Nanomaterials have the unique ability to interface with neural tissue in the nano-scale and are capable of influencing the function of a single neuron. The ability of nanoparticles to transcend the BBB through surface modifications has been exploited in various neuro-imaging techniques and for targeted drug delivery. The tunable topography of nanofibers provides accurate spatio-temporal guidance to regenerating axons. This review is an attempt to comprehend the progress in understanding the obstacles posed by the complex physiology of the nervous system and the innovations in design and fabrication of advanced nanomaterials drawing inspiration from natural phenomenon. We also discuss the development of nanomaterials for use in Neuro-diagnostics, Neuro-therapy and the fabrication of advanced nano-devices for use in opto-electronic and ultrasensitive electrophysiological applications. The energy efficient and parallel computing ability of the human brain has inspired the design of advanced nanotechnology based computational systems. However, extensive use of nanomaterials in neuroscience also raises serious toxicity issues as well as ethical concerns regarding nano implants in the brain. In conclusion we summarize these challenges and provide an insight into the huge potential of nanotechnology platforms in neuroscience.

Data in support of in vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration.

In the present data article we report the in vitro and in vivo biocompatibility of fabricated nerve conduits described in Das et al. [1]. Green synthesised gold nanoparticles (GNPs) were evaluated for their cytotoxicity in rat Schwann cells (SCTM41). We also describe herein the adhesion and proliferation of Schwann cells over the nanofibrous scaffolds. Methods describing surgical implantation of conduits in a rat sciatic nerve injury model, confirming its accurate implantation as well as the porosity and swelling tendency of the nerve conduits are illustrated in the various figures and graphs.

In vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration.

We report a novel silk-gold nanocomposite based nerve conduit successfully tested in a neurotmesis grade sciatic nerve injury model in rats over a period of eighteen months. The conduit was fabricated by adsorbing gold nanoparticles onto silk fibres and transforming them into a nanocomposite sheet by electrospinning which is finally given a tubular structure by rolling on a stainless steel mandrel of chosen diameter. The conduits were found to promote adhesion and proliferation of Schwann cells in vitro and did not elicit any toxic or immunogenic responses in vivo. We also report for the first time, the monitoring of muscular regeneration post nerve conduit implantation by recording motor unit potentials (MUPs) through needle electromyogram. Pre-seeding the conduits with Schwann cells enhanced myelination of the regenerated tissue. Histo-morphometric and electrophysiological studies proved that the nanocomposite based conduits pre-seeded with Schwann cells performed best in terms of structural and functional regeneration of severed sciatic nerves. The near normal values of nerve conduction velocity (50 m/sec), compound muscle action potential (29.7 mV) and motor unit potential (133 µV) exhibited by the animals implanted with Schwann cell loaded nerve conduits in the present study are superior to those observed in previous reports with synthetic materials as well as collagen based nerve conduits. Animals in this group were also able to perform complex locomotory activities like stretching and jumping with excellent sciatic function index (SFI) and led a normal life.

Functional nucleic-acid-based sensors for environmental monitoring.

Efforts to replace conventional chromatographic methods for environmental monitoring with cheaper and easy to use biosensors for precise detection and estimation of hazardous environmental toxicants, water or air borne pathogens as well as various other chemicals and biologics are gaining momentum. Out of the various types of biosensors classified according to their bio-recognition principle, nucleic-acid-based sensors have shown high potential in terms of cost, sensitivity, and specificity. The discovery of catalytic activities of RNA (ribozymes) and DNA (DNAzymes) which could be triggered by divalent metallic ions paved the way for their extensive use in detection of heavy metal contaminants in environment. This was followed with the invention of small oligonucleotide sequences called aptamers which can fold into specific 3D conformation under suitable conditions after binding to target molecules. Due to their high affinity, specificity, reusability, stability, and non-immunogenicity to vast array of targets like small and macromolecules from organic, inorganic, and biological origin, they can often be exploited as sensors in industrial waste management, pollution control, and environmental toxicology. Further, rational combination of the catalytic activity of DNAzymes and RNAzymes along with the sequence-specific binding ability of aptamers have given rise to the most advanced form of functional nucleic-acid-based sensors called aptazymes. Functional nucleic-acid-based sensors (FNASs) can be conjugated with fluorescent molecules, metallic nanoparticles, or quantum dots to aid in rapid detection of a variety of target molecules by target-induced structure switch (TISS) mode. Although intensive research is being carried out for further improvements of FNAs as sensors, challenges remain in integrating such bio-recognition element with advanced transduction platform to enable its use as a networked analytical system for tailor made analysis of environmental monitoring.