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

Valensi-Kurtz M, Lefler S, Cohen MA, Aharonowiz M, Cohen-Kupiec R, Sheinin A, Ashery U, Reubinoff B, Weil M - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT

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.

Methods/findings: 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/significance: 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.

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Characterization of IKAP/hELP1 expression in hNPs derived PNS neurons in vitro and in vivo.(A–B) comparative analysis of the expression of IKAP/hELP1 in hNPs derived PNS cultures (A) and in human fibroblasts (B) under the same confocal microscopy settings, showing higher levels of IKAP/hELP1 in the hNPs derived neurons. (C–F) Confocal micrographs showing the expression of IKAP/hELP1 in hNPs derived PNS neurons in vitro as judged by Peripherin positive staining. Peripherin+ cells show high levels of IKAP/ELP1 (D and, indicated by arrowheads) in contrast to non-peripherin expressing cells in the culture (fig. 5D and E, indicated by asterisks). (G) Magnified area of the same confocal plane (C–F) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of PNS neurons. Confocal micrographs showing the expression of IKAP/hELP1 in GFP+ hNPs derived PNS neurons in vivo, which express Brn3a, at the implanted chick spinal cord (H–K). (L) Magnified area of the same confocal plane (H–K) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of human PNS neurons in vivo. Scale bars are indicated in representative images.
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pone-0009290-g005: Characterization of IKAP/hELP1 expression in hNPs derived PNS neurons in vitro and in vivo.(A–B) comparative analysis of the expression of IKAP/hELP1 in hNPs derived PNS cultures (A) and in human fibroblasts (B) under the same confocal microscopy settings, showing higher levels of IKAP/hELP1 in the hNPs derived neurons. (C–F) Confocal micrographs showing the expression of IKAP/hELP1 in hNPs derived PNS neurons in vitro as judged by Peripherin positive staining. Peripherin+ cells show high levels of IKAP/ELP1 (D and, indicated by arrowheads) in contrast to non-peripherin expressing cells in the culture (fig. 5D and E, indicated by asterisks). (G) Magnified area of the same confocal plane (C–F) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of PNS neurons. Confocal micrographs showing the expression of IKAP/hELP1 in GFP+ hNPs derived PNS neurons in vivo, which express Brn3a, at the implanted chick spinal cord (H–K). (L) Magnified area of the same confocal plane (H–K) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of human PNS neurons in vivo. Scale bars are indicated in representative images.

Mentions: We detected IKAP/hELP1 expression in PNS neurons as shown in Figure 5. IKAP/hELP1 was expressed in the cytoplasm of peripherin expressing neurons. In comparison to non-neuronal cells, like human fibroblasts, IKAP/hELP1 expression in PNS neurons seems to be much higher as judged by the intensity of the monoclonal antibody staining under the same confocal scanning conditions (Figure 5A and B). It should be noted that, IKAP/hELP1 expression in human PNS neurons (peripherin positives) seems to be more granular and localized along the axons and dendrite terminals (Figure 5C–E, indicated by arrowheads) in contrast to non-peripherin expressing cells in the culture (Figure 5C–E, indicated by asterisks). According to previous studies IKAP/hELP1 was found to be part of the Elongator complex involved in RNA transcription process in yeast and mammalian cell lines [26], [27] therefore we performed in more detail a confocal localization analysis of IKAP/hELP1 in PNS neurons. We did not observe any significant expression of IKAP/hELP1 in the nuclei of these PNS neurons (shown as orthogonal section in Figure 5G which is a field magnification from 5D). Similar results of IKAP/hELP1 expression and intracellular localization were obtained with implanted GFP-hNPs derived PNS neurons in vivo as judged by coexpression of IKAP/ELP1 with Brn3a positive nuclear staining, respectively (Figure 5H–K and magnified orthogonal section 5L). Moreover, we have also observed similar cytoplasmic localization of IKAP/hELP1 in hTAU positive hNPs derived neurons in vivo (Figure S3A–F). Altogether, these observations may indicate that IKAP/hELP1 is differently regulated in PNS neurons and other cell types. This may support the assumption that IKAP/hELP1 plays a special role in PNS neurons. This assumption is in accord with the fact that the FD mutation of the IKAP gene selectively affects PNS development and postnatal PNS neurons.


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

Valensi-Kurtz M, Lefler S, Cohen MA, Aharonowiz M, Cohen-Kupiec R, Sheinin A, Ashery U, Reubinoff B, Weil M - PLoS ONE (2010)

Characterization of IKAP/hELP1 expression in hNPs derived PNS neurons in vitro and in vivo.(A–B) comparative analysis of the expression of IKAP/hELP1 in hNPs derived PNS cultures (A) and in human fibroblasts (B) under the same confocal microscopy settings, showing higher levels of IKAP/hELP1 in the hNPs derived neurons. (C–F) Confocal micrographs showing the expression of IKAP/hELP1 in hNPs derived PNS neurons in vitro as judged by Peripherin positive staining. Peripherin+ cells show high levels of IKAP/ELP1 (D and, indicated by arrowheads) in contrast to non-peripherin expressing cells in the culture (fig. 5D and E, indicated by asterisks). (G) Magnified area of the same confocal plane (C–F) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of PNS neurons. Confocal micrographs showing the expression of IKAP/hELP1 in GFP+ hNPs derived PNS neurons in vivo, which express Brn3a, at the implanted chick spinal cord (H–K). (L) Magnified area of the same confocal plane (H–K) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of human PNS neurons in vivo. Scale bars are indicated in representative images.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823780&req=5

pone-0009290-g005: Characterization of IKAP/hELP1 expression in hNPs derived PNS neurons in vitro and in vivo.(A–B) comparative analysis of the expression of IKAP/hELP1 in hNPs derived PNS cultures (A) and in human fibroblasts (B) under the same confocal microscopy settings, showing higher levels of IKAP/hELP1 in the hNPs derived neurons. (C–F) Confocal micrographs showing the expression of IKAP/hELP1 in hNPs derived PNS neurons in vitro as judged by Peripherin positive staining. Peripherin+ cells show high levels of IKAP/ELP1 (D and, indicated by arrowheads) in contrast to non-peripherin expressing cells in the culture (fig. 5D and E, indicated by asterisks). (G) Magnified area of the same confocal plane (C–F) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of PNS neurons. Confocal micrographs showing the expression of IKAP/hELP1 in GFP+ hNPs derived PNS neurons in vivo, which express Brn3a, at the implanted chick spinal cord (H–K). (L) Magnified area of the same confocal plane (H–K) showing orthogonal analysis of IKAP/hELP1 localization mainly in the cytosol of human PNS neurons in vivo. Scale bars are indicated in representative images.
Mentions: We detected IKAP/hELP1 expression in PNS neurons as shown in Figure 5. IKAP/hELP1 was expressed in the cytoplasm of peripherin expressing neurons. In comparison to non-neuronal cells, like human fibroblasts, IKAP/hELP1 expression in PNS neurons seems to be much higher as judged by the intensity of the monoclonal antibody staining under the same confocal scanning conditions (Figure 5A and B). It should be noted that, IKAP/hELP1 expression in human PNS neurons (peripherin positives) seems to be more granular and localized along the axons and dendrite terminals (Figure 5C–E, indicated by arrowheads) in contrast to non-peripherin expressing cells in the culture (Figure 5C–E, indicated by asterisks). According to previous studies IKAP/hELP1 was found to be part of the Elongator complex involved in RNA transcription process in yeast and mammalian cell lines [26], [27] therefore we performed in more detail a confocal localization analysis of IKAP/hELP1 in PNS neurons. We did not observe any significant expression of IKAP/hELP1 in the nuclei of these PNS neurons (shown as orthogonal section in Figure 5G which is a field magnification from 5D). Similar results of IKAP/hELP1 expression and intracellular localization were obtained with implanted GFP-hNPs derived PNS neurons in vivo as judged by coexpression of IKAP/ELP1 with Brn3a positive nuclear staining, respectively (Figure 5H–K and magnified orthogonal section 5L). Moreover, we have also observed similar cytoplasmic localization of IKAP/hELP1 in hTAU positive hNPs derived neurons in vivo (Figure S3A–F). Altogether, these observations may indicate that IKAP/hELP1 is differently regulated in PNS neurons and other cell types. This may support the assumption that IKAP/hELP1 plays a special role in PNS neurons. This assumption is in accord with the fact that the FD mutation of the IKAP gene selectively affects PNS development and postnatal PNS neurons.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT

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.

Methods/findings: 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/significance: 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.

Show MeSH
Related in: MedlinePlus