A long-term goal of the research in our laboratory is to re-create the components and compartments of mammalian systems, and in particular human systems, to engineer functional hybrid models. The initial period for this Bioengineering Research Grant (BRG) was to investigate the motoneuron to muscle segment of the reflex arc to understand in vitro what are possible outcomes when a human stem cell is implanted in a rat model and then apply it to improve the outcomes in an in vivo system. We have made a number of breakthroughs with this model system that move us closer to the goal of creating a functional in vitro model of the reflex arc: 1) We have cultivated embryonic and adult rat motoneurons as well as motoneurons derived from human fetal stem cells (hFSC). 2) We have demonstrated neuromuscular junction formation between these rat and human derived motoneurons with myotubes derived from both rat and human adult satellite cells. However, we have also created mature myotubes that exhibit sarcomere formation, express myosin heavy chain, have established the EC coupling apparatus as well as Type I and Type II muscle isoforms. We have also differentiated the myotubes into intrafusal fibers and integrated then with the MEMS constructs and demonstrated that these are functional mechanoreceptor synapses. 3) Glial cells have been incorporated into our model system and we have demonstrated Schwann cell myelination of motoneurons, with complete Node of Ranvier formation, and derived pure populations of MBP (myelin basic protein) positive oligodendrocytes from human mesenchymal stem cells. 4) We have successfully demonstrated in vivo integration of the differentiated hFSC derived motoneurons into an adult rat spinal cord injury model and shown significant locomotor recovery as accessed via the BBB test scoring system. This renewal BRG application seeks to complete the development of the entire reflex arc in vitro using primary rat cells and then with cells derived from human stem cells. We will also initiate experiments to increase the throughput of first the motoneurons to muscle segment of the stretch reflex are and then for the entire reflex arc. The Hypothesis is that we can recreate a functional in vitro model of the reflex arc with primary rat cells and achieve the same result with cells derived from human stem cells that will be a prototype for the next generation screens for drug discovery and toxicology. The continuation of the grant will enable advances for 1) the creation of a human cell based reflex arc in a defined, serum-free environment and 2) the development of new technology for utilization in the next level of Systems Biology and high-information content screens. Our expertise in surface chemistry, Micro-ElectroMechanical Systems (MEMS) fabrication and developmental/cellular/molecular biology has now allowed the construction of complex functional neuronal systems in vitro. Our team possesses the necessary expertise in all aspects of this project, as well as the facilities to effectively and successfully perform the research.

Public Health Relevance

The goal of this project is to engineer a system to model one of the most fundamental motor circuits in the human body, the spinal reflex arc. We will use nanotechnology and microelectronics in combination with biomedical engineering techniques to build this hybrid biological/non-biological system. Potential benefits include prevention, diagnosis, and treatment of developmental abnormalities in the spinal cord, rehabilitation of chronic neurological/muscle disorders and accompanying pain, and new strategies for prosthetic and orthotic design and evaluation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS050452-07
Application #
8217190
Study Section
Special Emphasis Panel (ZRG1-ETTN-F (02))
Program Officer
Owens, David F
Project Start
2005-09-23
Project End
2016-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
7
Fiscal Year
2012
Total Cost
$401,518
Indirect Cost
$120,309
Name
University of Central Florida
Department
None
Type
Organized Research Units
DUNS #
150805653
City
Orlando
State
FL
Country
United States
Zip Code
32826
Guo, Xiufang; Greene, Keshel; Akanda, Nesar et al. (2014) In vitro Differentiation of Functional Human Skeletal Myotubes in a Defined System. Biomater Sci 2:131-138
Davis, Hedvika; Gonzalez, Mercedes; Stancescu, Maria et al. (2014) A phenotypic culture system for the molecular analysis of CNS myelination in the spinal cord. Biomaterials 35:8840-5
Esch, Mandy B; Smith, Alec S T; Prot, Jean-Matthieu et al. (2014) How multi-organ microdevices can help foster drug development. Adv Drug Deliv Rev 69-70:158-69
Smith, A S T; Long, C J; Pirozzi, K et al. (2014) A multiplexed chip-based assay system for investigating the functional development of human skeletal myotubes in vitro. J Biotechnol 185:15-8
Smith, Alec S T; Long, Christopher J; McAleer, Christopher et al. (2014) Utilization of microscale silicon cantilevers to assess cellular contractile function in vitro. J Vis Exp :e51866
Sung, Jong Hwan; Srinivasan, Balaji; Esch, Mandy Brigitte et al. (2014) Using physiologically-based pharmacokinetic-guided "body-on-a-chip" systems to predict mammalian response to drug and chemical exposure. Exp Biol Med (Maywood) 239:1225-39
Guo, Xiufang; Spradling, Severo; Stancescu, Maria et al. (2013) Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials 34:4418-27
Rumsey, John W; McAleer, Christopher; Das, Mainak et al. (2013) Myelination and node of Ranvier formation on sensory neurons in a defined in vitro system. In Vitro Cell Dev Biol Anim 49:608-18
Sung, Jong H; Esch, Mandy B; Prot, Jean-Matthieu et al. (2013) Microfabricated mammalian organ systems and their integration into models of whole animals and humans. Lab Chip 13:1201-12
Edwards, Darin; Stancescu, Maria; Molnar, Peter et al. (2013) Two cell circuits of oriented adult hippocampal neurons on self-assembled monolayers for use in the study of neuronal communication in a defined system. ACS Chem Neurosci 4:1174-82

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