Abstract
The dorsal root ganglia (DRG) consist of a multitude of sensory neuronal subtypes that function to relay sensory stimuli, including temperature, pressure, pain and position to the central nervous system. Our knowledge of DRG sensory neurons have been predominantly driven by animal studies and considerably less is known about the human DRG. Human embryonic stem cells (hESC) are valuable resource to help close this gap. Our previous studies reported an efficient system for deriving neural crest and DRG sensory neurons from hESC. Here we show that this differentiation system gives rise to heterogeneous populations of sensory neuronal subtypes as demonstrated by phenotypic and functional analyses. Furthermore, using microelectrode arrays the maturation rate of the hESC-derived sensory neuronal cultures was monitored over 8 weeks in culture, showing their spontaneous firing activities starting at about 12 days post-differentiation and reaching maximum firing at about 6 weeks. These studies are highly valuable for developing an in vitro platform to study the diversity of sensory neuronal subtypes found within the human DRG.
Highlights
Innervate muscle tissue to provide sensory feedback information on muscle pressure and tension and have large diameter, myelinated fibres with fast conduction velocities (Aα fibres)[1,3]
We previously published an efficient protocol for deriving neural crest from Human embryonic stem cells (hESC) that involves treatment with small molecule inhibitors of activin/nodal (SB431542) and GSK3β (CHIR99021) pathways followed by bone morphogenic protein 2 (BMP2)[6]
Further differentiation of crest progenitors to dorsal root ganglia (DRG) sensory neurons is achieved by culturing in media supplemented with nerve growth factor (NGF), neurotrophin 3 (NT3) and brain-derived neurotrophic factor (BDNF) that support neural differentiation to all DRG sensory neuronal lineages
Summary
Innervate muscle tissue to provide sensory feedback information on muscle pressure and tension and have large diameter, myelinated fibres with fast conduction velocities (Aα fibres)[1,3]. New studies emerging using single cell gene expression profiling of adult rodent DRGs demonstrates further complexity in defining sensory neuronal subtypes and identifying characteristic gene expression profiles and functionalities[4,5]. Using microelectrode arrays (MEA), we observe that hESC-derived DRG neurons are functionally mature, showing phenotypic specification and active firing activities within 8 weeks in culture and are responsive to noxious chemical, heat and osmotic swelling stimuli. These studies are highly significant for understanding the development, molecular and functional characteristics of human DRG sensory neurons, which can be applied for developing therapies to treat peripheral sensory neuropathies
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