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

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.

Figure 1. hESC derived NPs express neural crest PNS markers and PNS related growth factor receptors.

(A) Schematic flow diagram of the differentiation protocol of hESC derived NPs along 10 weeks culture. (B) RT-PCR analysis of mRNA extracts from hNPs at 5 and 8 weeks in suspension cultures express various early neural crest specific markers prior to final differentiation. Expression of the housekeeping gene GAPDH served as reaction control for each sample. (C–H) Cross section of 5 weeks old NPs in suspension cultures expressing the migratory-crest marker HNK-1 throughout the sphere section (D), while at the edge of the sphere section colocalization of the PNS neuronal marker peripherin and the pan neuronal marker β-Tubulin III is observed (C, F and G respectively). Scale bars are indicated in A and C. (I) Comparative RT-PCR analysis of mRNA extracts from hNPs at 3 and 8 weeks in suspension cultures express different pattern of neuronal differentiation-related growth factor receptors. Expression of the housekeeping gene RPL-27 served as reaction control for each sample.

Figure 2. In vitro PNS differentiation capacity of hESC derived NPs.

(A–S) Eight weeks old Human NPs cultured on laminin for 12 days showing extensive neurite outgrowth expressing the pan neuronal marker β-tubulin III (A–D). These cells also express the PNS marker Peripherin (H,H,K,N and Q) together with the neural crest marker HNK-1 (F), the synaptic vesicle marker SV2 (I) and vesicular acetylcholine transporter VAChT (L), indicative of their maturity, the PNS markers Brn3a (O) and Istet-1 (R). (T) Quantitative analysis from these experiments (N–R) of the proportion of peripherin positive cells from total cells in the culture and the relative numbers of double stained Peripherin+/Brn3a+ and Peripherin+/Islet-1+ from total Peripherin+ cells. (Scale bar: in A-B = 0.5 mm; in C-S = 50 µm).

Figure 3. 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.

Figure 4. In vivo PNS differentiation of 8 weeks old hNPs implanted into the chick developing neural tube.

(A) Eight weeks old hESC expressing GFP derived NPs in suspension before cell dissociation. (B) Illustration of the cell transplantation procedure in the dorsal neural tube after removal of one neural fold. (C) Micrograph showing microinjection of GFP+ cells into the implantation site. (D–T) hNPs GFP+ derived cells 7 days after implantation located at the dorsal spinal cord showing extensive migration and neurite outgrowth (D and in high magnification in E). (F–H) implanted human NPs derived cells are specifically identified with anti-GFP antibodies (F) and with anti human specific nuclear antigen (G). Implanted hNPs express the human specific neuronal microtubule-associated protein Tau (I–K, and in high magnification in L–N). These cells also express the PNS markers, Peripherin (O–Q) and Brn3a (Q–S), Scale bars are indicated in representative images.

Figure 5. 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.