A human neuron injury model for molecular studies of axonal regeneration
ABSTRACT The enhancement of regeneration of damaged axons in both the peripheral and central nervous systems is a widely pursued goal in clinical medicine. Although some of the molecular mechanisms involved in the intrinsic neurite regeneration program have been elucidated, much additional study is required for development of new therapeutics. The majority of studies in the field of axonal regeneration have utilized animal models due to obvious limitations of the accessibility of human neural tissues. Here we describe the use of human embryonic stem cell (hESC)-derived neurons as a novel model for studying neuronal responses to axonal injury. Neurons were generated using PA6 induction and neurites injured in vitro using trituration or laser microdissection. Lesioned neurons re-extended neurites with distinct growth cones. Expression of proteins associated with regeneration were observed in this human in vitro system, including appearance of importin beta1 in processes after neuritomy. Laser-transected hESC-derived neuronal cultures were analyzed for their transcriptional response to injury using Affymetrix expression microarrays. Profound changes in gene expression were observed over a time course of 2 to 24 hours after lesion. The expression of several genes reported to be involved in axonal injury responses in animal models changed following injury of hESC-derived neurons. Thus, hESC-derived neurons may be a useful in vitro model system for mechanistic studies on human axonal injury and regeneration.
SourceAvailable from: Ching-Hui Lin[Show abstract] [Hide abstract]
ABSTRACT: Obtaining single dissociated cells from neurospheres is difficult using non-enzymatic methods. In this paper we report the development of a microfluidic-chip-based approach that utilizes flow and microstructures to dissociate neurospheres. We show that this microfluidic-chip-based neurosphere dissociation method can generate high yields of single cells from dissociated neurospheres of mouse KT98 and DC115 cell models (passage number: 3 - 8, diameter range: 40 - 250 m): 90% and 95%, respectively. The microfluidic-chip-dissociated cells had high viabilities (80-85%) and the ability to re-grow into neurospheres, demonstrating the applicability of this device to neurosphere-assay applications. In addition, the dissociated cells retained their normal differentiation potentials, as shown by their capabilities to differentiate into three neural lineages (neurons, astroglia, and oligodendrocytes) when cultured in differentiation culture conditions. Since this microfluidic chip-based method does not require the use of enzymatic reagents, the risk of contamination from exogenous substances could be reduced, making it an attractive tool for a wide range of applications where neurosphere dissociation is needed.Analytical Chemistry 11/2013; 85(24). DOI:10.1021/ac402724b · 5.83 Impact Factor
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ABSTRACT: The extensive lengths of neuronal processes necessitate efficient mechanisms for communication with the cell body. Neuronal regeneration after nerve injury requires new transcription; thus, long-distance retrograde signalling from axonal lesion sites to the soma and nucleus is required. In recent years, considerable progress has been made in elucidating the mechanistic basis of this system. This has included the discovery of a priming role for early calcium waves; confirmation of central roles for mitogen-activated protein kinase signalling effectors, the importin family of nucleocytoplasmic transport factors and molecular motors such as dynein; and demonstration of the importance of local translation as a key regulatory mechanism. These recent findings provide a coherent mechanistic framework for axon-soma communication in the injured nerve and shed light on the integration of cytoplasmic and nuclear transport in all eukaryotic cells.Nature Reviews Neuroscience 12/2013; DOI:10.1038/nrn3609 · 31.38 Impact Factor
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ABSTRACT: Human embryonic stem cells (hESCs) are potentially an unlimited source of neurons for study and therapy for human disease. Directed differentiation of hESCs has been performed using many different methods, often via neural precursor intermediates generated from aggregates of hESC. We describe here a protocol based on commercially available reusable silicone micromolds and two small molecule growth factor inhibitors to simply and reproducibly generate human neurons from hESC. Hundreds of neurospheres were generated with a single pipettation of hESC into agarose multiwell plates made with the micromolds. This was followed by suspension culture with two medium changes, and plating of clumps cut from the neurospheres on laminin-coated coverslips. After two weeks of terminal differentiation, 90%+ of cells expressed neuronal proteins, and many of the neurons expressed markers of peripheral sensory neurons. The neurons made with this method underwent productive infection with the human-specific pathogenic virus varicella zoster, demonstrating the utility of the neurons for addressing clinically relevant research questions. This simple method should allow laboratories experienced in growing human pluripotent cells to easily generate neurons for studies of nerve cell biology and pathology.Journal of neuroscience methods 01/2013; 214(1). DOI:10.1016/j.jneumeth.2012.12.026 · 1.96 Impact Factor