Limits...
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.

Show MeSH

Related in: MedlinePlus

Electrophysiological analysis of NPs derived PNS neurons.The current-voltage (IV) relationship acquired from cell in voltage clamp mode, holding potential was −60 mV (A, upper panel). The inward current deflections are the sodium currents evoked by 200 ms-long step depolarization from −60 to +40 mV with a 20 mV increment (A, lower panel). Representative traces of single action potential (B, left panel) and repetitive action potential firing (B, right panel) acquired from the cell in current clamp mode by 2 ms and 200 ms-long suprathreshold current injection (B, left and right lower panels, respectively). Increase in intracellular calcium concentration evoked by high KCl application (C), Representative trace from fura-2 AM-loaded cell in response to local 70 mM KCl application is indicated by arrows. These experiments were repeated 4 times, using cells from different cultures each time, obtaining the same results.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2823780&req=5

pone-0009290-g003: Electrophysiological analysis of NPs derived PNS neurons.The current-voltage (IV) relationship acquired from cell in voltage clamp mode, holding potential was −60 mV (A, upper panel). The inward current deflections are the sodium currents evoked by 200 ms-long step depolarization from −60 to +40 mV with a 20 mV increment (A, lower panel). Representative traces of single action potential (B, left panel) and repetitive action potential firing (B, right panel) acquired from the cell in current clamp mode by 2 ms and 200 ms-long suprathreshold current injection (B, left and right lower panels, respectively). Increase in intracellular calcium concentration evoked by high KCl application (C), Representative trace from fura-2 AM-loaded cell in response to local 70 mM KCl application is indicated by arrows. These experiments were repeated 4 times, using cells from different cultures each time, obtaining the same results.

Mentions: Besides expressing neuronal markers including SV2 and VAChT, as shown above, these cells show clear mature neuronal morphology that may also indicate neuron functionality. To examine the functional maturity of our hNPs derived neurons we performed electrophysiological recordings using the whole-cell patch clamp technique and intracellular calcium measurements using the FURA-2AM assay. For these analyses 8 weeks old NPs were seeded onto poly-D-Lysine and laminin coated cover slips followed by incubation for 14 days, until they reached 10 weeks in culture, as described above. Human NPs-derived neurons were chosen for recording based on their morphology, exhibiting well developed neuronal shapes. We then performed a series of electrophysiological recording from these cells. Cells were held in the “voltage-clamp” mode and 100 ms depolarization pulses to increasing voltages were applied. As can be seen from Figure 3A, when the cells were depolarized to above −20 mV, they responded with both fast sodium and delayed potassium currents. Moreover, when the cells were hold in the current clamp mode, a short depolarization pulse elicits an action potential (Figure 3B). These results demonstrate that the NPs derived cells differentiated into electrophysiologically mature neurons. We next examined if these cells exhibit calcium influx following depolarization, a process that is crucial for neurotransmitter release. Intracellular calcium measurements were performed by loading the cells with the fluorescent indicator, Fura-2AM, concomitantly with the application of high a KCl solution. This experiment revealed transient changes in calcium influx within the tested cells (Figure 3C). The KCl application caused membrane depolarization, and as a consequence, opening of voltage-dependent calcium channels. As a result, the intracellular calcium concentration increased; hence, indicating functionality of voltage-dependent calcium channels which is essential for neurotransmitter release. Altogether, these results indicate that the NPs-derived PNS neurons developed essential physiological machineries of functional mature 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)

Electrophysiological analysis of NPs derived PNS neurons.The current-voltage (IV) relationship acquired from cell in voltage clamp mode, holding potential was −60 mV (A, upper panel). The inward current deflections are the sodium currents evoked by 200 ms-long step depolarization from −60 to +40 mV with a 20 mV increment (A, lower panel). Representative traces of single action potential (B, left panel) and repetitive action potential firing (B, right panel) acquired from the cell in current clamp mode by 2 ms and 200 ms-long suprathreshold current injection (B, left and right lower panels, respectively). Increase in intracellular calcium concentration evoked by high KCl application (C), Representative trace from fura-2 AM-loaded cell in response to local 70 mM KCl application is indicated by arrows. These experiments were repeated 4 times, using cells from different cultures each time, obtaining the same results.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009290-g003: Electrophysiological analysis of NPs derived PNS neurons.The current-voltage (IV) relationship acquired from cell in voltage clamp mode, holding potential was −60 mV (A, upper panel). The inward current deflections are the sodium currents evoked by 200 ms-long step depolarization from −60 to +40 mV with a 20 mV increment (A, lower panel). Representative traces of single action potential (B, left panel) and repetitive action potential firing (B, right panel) acquired from the cell in current clamp mode by 2 ms and 200 ms-long suprathreshold current injection (B, left and right lower panels, respectively). Increase in intracellular calcium concentration evoked by high KCl application (C), Representative trace from fura-2 AM-loaded cell in response to local 70 mM KCl application is indicated by arrows. These experiments were repeated 4 times, using cells from different cultures each time, obtaining the same results.
Mentions: Besides expressing neuronal markers including SV2 and VAChT, as shown above, these cells show clear mature neuronal morphology that may also indicate neuron functionality. To examine the functional maturity of our hNPs derived neurons we performed electrophysiological recordings using the whole-cell patch clamp technique and intracellular calcium measurements using the FURA-2AM assay. For these analyses 8 weeks old NPs were seeded onto poly-D-Lysine and laminin coated cover slips followed by incubation for 14 days, until they reached 10 weeks in culture, as described above. Human NPs-derived neurons were chosen for recording based on their morphology, exhibiting well developed neuronal shapes. We then performed a series of electrophysiological recording from these cells. Cells were held in the “voltage-clamp” mode and 100 ms depolarization pulses to increasing voltages were applied. As can be seen from Figure 3A, when the cells were depolarized to above −20 mV, they responded with both fast sodium and delayed potassium currents. Moreover, when the cells were hold in the current clamp mode, a short depolarization pulse elicits an action potential (Figure 3B). These results demonstrate that the NPs derived cells differentiated into electrophysiologically mature neurons. We next examined if these cells exhibit calcium influx following depolarization, a process that is crucial for neurotransmitter release. Intracellular calcium measurements were performed by loading the cells with the fluorescent indicator, Fura-2AM, concomitantly with the application of high a KCl solution. This experiment revealed transient changes in calcium influx within the tested cells (Figure 3C). The KCl application caused membrane depolarization, and as a consequence, opening of voltage-dependent calcium channels. As a result, the intracellular calcium concentration increased; hence, indicating functionality of voltage-dependent calcium channels which is essential for neurotransmitter release. Altogether, these results indicate that the NPs-derived PNS neurons developed essential physiological machineries of functional mature 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