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. 2018 Aug 10;7:e35786. doi: 10.7554/eLife.35786

Figure 5. Axial progenitor-derived trunk neural crest is an optimal source of sympathoadrenal cells.

(A) Diagram depicting the culture conditions employed to direct axial progenitors (‘NMPs’) toward trunk NC and subsequently sympathoadrenal progenitors (SAP) and sympathetic neurons. (B) FACS analysis of PHOX2B-GFP expression in SAP cells derived from axial progenitors as shown in A. Below: Immunofluorescence analysis of PHOX2B-GFP and PHOX2B protein expression following antibody staining. Scale bar = 100 µm. (C) Quantification of d18 differentiated cells positive for the indicated markers in relation to PHOX2B-GFP expression following antibody staining. In each case four randomly selected representative fields were used to obtain the average number of cells/marker. Total numbers of cells scored: GATA3 (N = 3003), ASCL1 (N = 2575), ISL1 (N = 2963). (D) Immunofluorescence analysis of PHOX2B-GFP together with the indicated markers in day 18 differentiated SAP/sympathetic neurons derived from axial progenitors as shown in A. Scale bar = 100 µm. (E) qPCR expression analysis of indicated SAP/sympathetic neuron markers in d12 and d18 cultures. Error bars = S.E.M. (n = 3). (F) Voltage responses of hPSC-derived sympathetic neurons (after day 19 of differentiation) to current injection. Type I and Type II cells were current clamped and hyperpolarising (negative) and depolarising (positive) current steps were applied (the current injected is shown next to the traces). The resulting membrane potential responses of the cells to these current injections are shown, the traces are overlaid. (G) Analysis of catecholamine production in hPSC-derived sympathetic neurons (after day 19 of differentiation) using a commercial ELISA kit (n = 2). NE, norepinephrine; DA dopamine.

Figure 5—source data 1. Raw data for Figure 5.
elife-35786-fig5-data1.xlsx (10.7KB, xlsx)
DOI: 10.7554/eLife.35786.020

Figure 5.

Figure 5—figure supplement 1. Characterisation of axial progenitor-derived sympathoadrenal progenitors and sympathetic neurons.

Figure 5—figure supplement 1.

(A) Immunofluorescence analysis of PHOX2B-GFP and ASCL1 expression in d12 SAP cells derived from axial progenitors as shown in Figure 5A. Scale bar = 100 µm. (B) Immunofluorescence analysis of DBH, TH and PHOX2B-GFP expression in d 30 sympathetic neurons derived from axial progenitors. Scale bar = 50 µm. (C) Immunofluorescence analysis of PRPH and HOXC9 expression in d19 sympathetic neurons derived from axial progenitors as shown in Figure 5A. Scale bar = 100 µm. (D) Top panels, voltage clamp membrane currents from a Type I (left) and a Type II (right) hPSC-derived sympathetic neuron, elicited by hyperpolarizing and depolarising voltage steps (10 mV nominal decrements) from –84 mV. Actual test potentials reached are shown by some of the traces. INa indicates the initial rapidly inactivating inward Na+ current and IK shows the delayed-rectifier outward K+ current. The bottom panels show average steady-state (left panel) or peak inward (right panel) current-voltage curves from Type I (black, n = 7) and Type II (red, n = 4) hPSC-derived sympathetic neurons. Steady-state current values were measured towards the end of the traces shown in the upper panels, and inward current values were measured at the peak to isolate the Na+ current. Error bar = s.e.m.