Maria Stancescu

Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system.

Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time-lapse recordings and their subsequent quenching by d-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair.

Patterned Cardiomyocytes on Microelectrode Arrays as a Functional, High Information Content Drug Screening Platform

Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional re-entry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes.

A Defined Long-Term In Vitro Tissue Engineered Model of Neuromuscular Junctions

Neuromuscular junction (NMJ) formation, occurring between motoneurons and skeletal muscle, is a complex multistep process involving a variety of signaling molecules and pathways. In vitro motoneuron-muscle co-cultures are powerful tools to study the role of different growth factors, hormones and cellular structures involved in NMJ formation. In this study, a serum-free culture system utilizing defined temporal growth factor application and a non-biological substrate resulted in the formation of robust NMJs. The system resulted in long-term survival of the co-culture and selective expression of neonatal myosin heavy chain, a marker of myotube maturation. NMJ formation was verified by colocalization of dense clusters of acetylcholine receptors visualized using alpha-bungarotoxin and synaptophysin containing vesicles present in motoneuron axonal terminals. This model will find applications in basic NMJ research and tissue engineering applications such as bio-hybrid device development for limb prosthesis and regenerative medicine as well as for high-throughput drug and toxin screening applications.

Skeletal Muscle Tissue Engineering: A Maturation Model Promoting Long Term Survival of Myotubes, Structural Development of the Excitation - Contraction Coupling Apparatus and Neonatal Myosin Heavy Chain Expression

The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation-contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70-90 days) than myotubes from our previously developed system (20-25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.

Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers

The sensory circuit of the stretch reflex arc, composed of specialized intrafusal muscle fibers and type Ia proprioceptive sensory neurons, converts mechanical information regarding muscle length and stretch to electrical action potentials and relays them to the central nervous system. Utilizing a non-biological substrate, surface patterning photolithography and a serum-free medium formulation a co-culture system was developed that facilitated functional interactions between intrafusal muscle fibers and sensory neurons. The presence of annulospiral wrappings (ASWs) and flower-spray endings (FSEs), both physiologically relevant morphologies in sensory neuron-intrafusal fiber interactions, were demonstrated and quantified using immunocytochemistry. Furthermore, two proposed components of the mammalian mechanosensory transduction system, BNaC1 and PICK1, were both identified at the ASWs and FSEs. To verify functionality of the mechanoreceptor elements the system was integrated with a MEMS cantilever device, and Ca(2+) currents were imaged along the length of an axon innervating an intrafusal fiber when stretched by cantilever deflection. This system provides a platform for examining the role of this mechanosensory complex in the pathology of myotonic and muscular dystrophies, peripheral neuropathy, and spasticity inducing diseases like Parkinsons. These studies will also assist in engineering fine motor control for prosthetic devices by improving our understanding of mechanosensitive feedback.

In vitro differentiation of functional human skeletal myotubes in a defined system

In vitro human skeletal muscle systems are valuable tools for the study of human muscular development, disease and treatment. However, published in vitro human muscle systems have so far only demonstrated limited differentiation capacities. Advanced differentiation features such as cross-striations and contractility have only been observed in co-cultures with motoneurons. Furthermore, it is commonly regarded that cultured human myotubes do not spontaneously contract, and any contraction has been considered to originate from innervation. This study developed a serum-free culture system in which human skeletal myotubes demonstrated advanced differentiation. Characterization by immunocytochemistry, electrophysiology and analysis of contractile function revealed these major features: A) well defined sarcomeric development, as demonstrated by the presence of cross-striations. B) finely developed excitation-contraction coupling apparatus characterized by the close apposition of dihydropyridine receptors on T-tubules and Ryanodine receptors on sarcoplasmic reticulum membranes. C) spontaneous and electrically controlled contractility. This report not only demonstrates an improved level of differentiation of cultured human skeletal myotubes, but also provides the first published evidence that such myotubes are capable of spontaneous contraction. Use of this functional in vitro human skeletal muscle system would advance studies concerning human skeletal muscle development and physiology, as well as muscle-related disease and therapy.

Regeneration and characterization of adult mouse hippocampal neurons in a defined in vitro system.

Although the majority of human illnesses occur during adulthood, most of the available in vitro disease models are based upon cells obtained from embryonic/fetal tissues because of the difficulties involved with culturing adult cells. Development of adult mouse neuronal cultures has a special significance because of the abundance of transgenic disease models that use this species. In this study a novel cell culture method has been developed that supports the long-term survival and physiological regeneration of adult mouse hippocampal cells in a serum-free defined environment. In this well-defined, controlled system, adult mouse hippocampal cells survived for up to 21 days in culture. The cultured cells exhibited typical hippocampal neuronal morphology and electrophysiological properties after recovery from the trauma of dissociation, and stained positive for the expected neuronal markers. This system has great potential as an investigative tool for in vitro studies of adult diseases, the aging brain or transgenic models of age-associated disorders.

Altered calcium dynamics in cardiac cells grown on silane-modified surfaces

Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca(2+) because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca(2+) imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca(2+) concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca(2+) changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca(2+) dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems.

Node of Ranvier formation on motoneurons in vitro

One of the most significant interactions between Schwann cells and neurons is myelin sheath formation. Myelination is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems provide a unique modality to study both factors influencing myelination and diseases associated with myelination. Currently, no in vitro system for motoneuron myelination by Schwann cells has been demonstrated. This work details the myelination of motoneuron axons by Schwann cells, with complete Node of Ranvier formation, in a defined in vitro culture system. This defined system utilizes a novel serum-free medium in combination with the non-biological substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA). The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This defined system provides a highly controlled, reproducible model for studying Schwann cell interactions with motoneurons as well as the myelination process and its effect on neuronal plasticity. Furthermore, an in vitro system that would allow studies of motoneuron myelination would be beneficial for understanding peripheral demyelinating neuropathies such as diabetes induced peripheral neuropathy and could lead to a better understanding of CNS demyelinating diseases like multiple sclerosis, as well as neuromuscular junction maturation and maintenance.

Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1

Although sensory neurons constitute a critical component for the proper function of the nervous system, the in vitro differentiation of functional sensory neurons from human stem cells has not yet been reported. This study presents the differentiation of sensory neurons (SNs) from a human neural progenitor cell line, hNP1, and their functional maturation in a defined, in vitro culture system without murine cell feeder layers. The SNs were characterized by immunocytochemistry and their functional maturation was evaluated by electrophysiology. Neural crest (NC) precursors, as one of the cellular derivatives in the differentiation culture, were isolated, propagated, and tested for their ability to generate sensory neurons. The hSC-derived SNs, as well as the NC precursors provide valuable tools for developing in vitro functional systems that model sensory neuron-related neural circuits and for designing therapeutic models for related diseases.