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.
Highlights
Stem cells are unique cells that have the capacity for self renewal, exhibit pluripotency and can proliferate indefinitely in culture while maintaining epigenetic and karyotypic stability [1]
In our differentiated human NPs (hNPs) cultures we have detected high levels of these transcription factors concomitant with the expression of terminal differentiated markers. It appears that Pax3 and Pax7 expression at 10 weeks of hNPs differentiation in vitro resembles more to the in vivo mouse model rather than that of the chick. We believe that this observation together with the expression of crest specific markers can explains the commitment of our differentiated hNPs to yield a high proportion of peripherin expressing neurons following a total of 10 weeks in culture
Characterization of the expression pattern of different growth factors receptors in hNPs in culture showed that 8 weeks old hNPs, as opposed to 3 weeks old hNPs express several basic signaling receptors required for responding to different growth factors which are responsible of peripheral nervous system (PNS) differentiation
Summary
Stem cells are unique cells that have the capacity for self renewal, exhibit pluripotency and can proliferate indefinitely in culture while maintaining epigenetic and karyotypic stability [1]. Derivation of Neural Progenitors (NPs) from hESC, in the presence of Noggin as a pre-step for differentiation, yielded high numbers of neurons of various subtypes [3] These human NPs (hNPs) were capable of extensive proliferation in vitro and expressed early neuroectoderm markers [3]. These NPs-derived PNS neurons can serve as a platform for studying PNS-related neuropathies such as Familial Dysautonomia (FD) which affects the normal development and survival of the sensory and autonomic nervous system [13,14] Using this differentiation system, we show for the first time in human PNS neurons the normal pattern of expression of the protein IKAP/ hELP1, in which a splicing mutation of the gene encoding this protein causes FD [15,16,17]. 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
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