Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

Abstract

BackgroundThe absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons.Methods/FindingsOur method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia.Conclusions/SignificanceWe conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.

Human Sensory Neurons Derived from Induced Pluripotent Stem Cells Support Varicella-Zoster Virus Infection

“…We previously generated human iPS cells from fibroblasts by the introduction of four reprogramming genes using non-integrating adenovirus [24]. There have only been a few studies directed at generating sensory neurons from either ES or iPS cells, with the majority of them focusing on peripheral nervous system diseases, such as familial dysautonomia [25]–[28]. Most of these studies used murine stromal cell lines, such as PA6 or MS5, that promote neural differentiation of stem cells via their “stromal cell-derived inducing activity” [29], [30], followed by the formation of either neural rosettes or neurospheres, a spherical cluster of neural stem cells [31], [32].…”

Transplantation of Mammalian Embryonic Stem Cells and Their Derivatives to Avian Embryos

“…Most recently, GFP-expressing hESC differentiated neurally with noggin (as in the previous study) to make neurospheres, were dissociated and microinjected into the damaged chick dorsal neural tube [39]. In sections made through the injection site after 5-7 additional days of incubation, most of these cells appeared to integrate into the spinal cord of the host.…”

Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells

“…Electrophysiological recordings were performed using an EPC-10 patch-clamp amplifier; results were recorded and analyzed using Patch master V2X69 software (HEKA Electronics Gmbh, Lambrecht, Germany) [26]. The extracellular solution consisted of 128 mM NaCl, 3 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 15 mM HEPES (SigmaeAldrich) and15 mM glucose (SigmaeAldrich), adjusted to a pH of 7.2e7.4 and an osmolarity of 340 mOsm.…”