Protocol to derive postganglionic parasympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessment

Abstract

Accessing untransformed human primary parasympathetic neurons is extremely challenging due to their inaccessibility in patients. Therefore, a human pluripotent stem cell-based approach to generate pure parasympathetic neurons is valuable for studying their development, maturation, and pathology. Here, we present a protocol to derive parasympathetic neurons from human pluripotent stem cells. We describe steps for human pluripotent stem cell replating, neural crest replating, cryobanking, and parasympathetic neuron differentiation. We then detail procedures for functional assays via multielectrode array (MEA) analysis. For complete details on the use and execution of this protocol, please refer to Wu et al.¹.

Keywords: Developmental biology; Neuroscience; Stem Cells.

Graphical abstract

Figure 1

Checkpoints for the differentiation of parasympathetic neurons (A) Schematic of parasympathetic neuron differentiation protocol. (B) Table of checkpoints that cultures should go through for proper differentiation: hPSC (d0, white arrows point to bright borders), NC (d10, white arrows point to ridges), SCP (d16), and parasympathetic neurons (d30). (C) Phase contrast images of healthy hPSCs (left, white arrows point to bright borders) and unhappy hPSCs with differentiation (right, white arrows point to differentiating cells). (D) Phase contrast images of d10 NC ridges with not enough BMP4 (left), enough BMP4 (middle, white arrows point to ridges), and too much BMP4 (right, white arrows point to blisters). (E) Phase contrast image of seeding density for d1 NC. (F) Immunofluorescent image of d10 NC: DAPI (blue) and SOX10 (red). (G) NC can be directly replated or frozen to produce parasympathetic neurons. (H) Schematic of sympathetic neuron differentiation protocol. (I) Gene expression of SOX10, S100B, and PHOX2B by RT-qPCR for d14 sympathoblasts compared to d16 SCPs. n=4–7 biological replicate. Unpaired two-tailed t test.

Figure 2

Options for Schwann cell precursor replating on D16 (A) Phase contrast image of d11 SCP spheroids (left) and d16 SCP spheroids (right). (B) Phase contrast images of d30 parasympathetic neurons derived through spheroid plating (top) or dissociated plating (bottom). (C) Phase contrast image of d30 parasympathetic neurons with poor efficiency due to frequent feeding.

Figure 3

Markers for human pluripotent stem cell-derived parasympathetic neurons (A) Expression of ChAT, VAChT, CHRNB4, and NPY2R by immunofluorescence for d30 parasympathetic neurons derived from either spheroid plating (top) or dissociated plating (bottom). (B) Gene expression of d30 parasympathetic neuron markers (ASCL1, PHOX2B, PRPH, CHRNA3, ChAT, VAChT, NPY2R) by RT-qPCR derived either through spheroid plating (left) or dissociated plating (right). n= 5 biological replicates.

Figure 4

Electrophysiological measurement of human pluripotent stem cell-derived parasympathetic neurons (A) Schematic of plating for MEA analysis: d16 SCP spheroids are dissociated and replated onto MEA plates. (B) Mean firing rate, wMFR, and number of bursts for d30 parasympathetic neurons. n= 6–7 biological replicates. Unpaired two-tailed t test.