Enriched population of PNS neurons derived from human embryonic stem cells as a platform for studying peripheral neuropathies.
BACKGROUND
The 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.


RESULTS
Our 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
We 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.
Valensi-Kurtz
Moran
M
Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel.
Lefler
Sharon
S
Cohen
Malkiel A
MA
Aharonowiz
Michal
M
Cohen-Kupiec
Rachel
R
Sheinin
Anton
A
Ashery
Uri
U
Reubinoff
Benjamin
B
Weil
Miguel
M
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D004402:Dysautonomia, Familial / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D009474:Neurons / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D017933:Peripheral Nervous System / Q000166:cytology* / Q000378:metabolism; D010523:Peripheral Nervous System Diseases / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D011189:Potassium Chloride / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D011984:Sensory Receptor Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D035602:Transcriptional Elongation Factors
D002352:Carrier Proteins; C114720:Elp1 protein, human; C503514:FOXD3 protein, human; D051858:Forkhead Transcription Factors; D055758:SOX9 Transcription Factor; D035602:Transcriptional Elongation Factors; D011189:Potassium Chloride