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. 2016 Jul 7;19(1):95-106.
doi: 10.1016/j.stem.2016.05.002. Epub 2016 Jun 16.

Functional Coupling With Cardiac Muscle Promotes Maturation of hPSC-Derived Sympathetic Neurons

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Functional Coupling With Cardiac Muscle Promotes Maturation of hPSC-Derived Sympathetic Neurons

Yohan Oh et al. Cell Stem Cell. .
Free PMC article

Abstract

Neurons derived from human pluripotent stem cells (hPSCs) are powerful tools for studying human neural development and diseases. Robust functional coupling of hPSC-derived neurons with target tissues in vitro is essential for modeling intercellular physiology in a dish and to further translational studies, but it has proven difficult to achieve. Here, we derive sympathetic neurons from hPSCs and show that they can form physical and functional connections with cardiac muscle cells. Using multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro and successfully isolated PHOX2B::eGFP+ neurons that exhibit sympathetic marker expression and electrophysiological properties and norepinephrine secretion. Upon pharmacologic and optogenetic manipulation, PHOX2B::eGFP+ neurons controlled beating rates of cardiomyocytes, and the physical interactions between these cells increased neuronal maturation. This study provides a foundation for human sympathetic neuron specification and for hPSC-based neuronal control of organs in a dish.

Figures

Figure 1
Figure 1. Activation of WNT Followed by SHH Signaling Leads Both ASCL1 and PHOX2B Inductions in hPSC-Derived Autonomic Specification
(A and D) Schematics for ASCL1 and PHOX2B gene targeting using homologous recombination enhanced by CRISPR/Cas9 system. (B and E) Immunofluorescence analyses were performed using OCT3/4 (green) and NANOG (red) antibodies. Representative images of OCT3/4+/NANOG+ colonies from the undifferentiated ASCL1::eGFP (B) and PHOX2B::eGFP (E) reporter lines. (C and F) Representative FACS plots of differentiated reporter hESC lines for either (C) ASCL1 or (F) PHOX2B. (G–J) Top, schematics for differentiation; bottom, after addition of (G) the recombinant sonic hedgehog C25II (Shh) protein and SHH agonist (purmorphamine, PMP) or SHH antagonist (cyclopamine, CyP) to the LSB protocol, (H) BMP4 (B4) to the LSB-SP protocol, (I) CHIR99021 (CHIR) and ‘modified-3i’ (m3i; CHIR, DAPT, and PD173074) to the LSB-SP protocol, or (J) m3i and B4 to the LSB protocol, the percentages of ASCL1::eGFP− and PHOX2B::eGFP-expressing cells was measured by using FACS analysis (* P < 0.05; **P < 0.01; ***P < 0.001; unpaired Student’s t-test; n = 3). LSB, LDN193189 and SB431542. SP, Shh plus PMP. (K) Immunofluorescence analyses were performed at day 10 following LSB (day 0–3/4) – m3i (day 2–7) – SP (day 3–10) treatment, using GFP (green) and either ASCL1 (red) or PHOX2B (red) antibodies. (L) qRT-PCR data showing enrichments of each transcript after cell sorting (**P < 0.01; ***P < 0.001; compared to eGFP− population; unpaired Student’s t-test; n = 3). All error bars represent mean + S.E.M. Scale bars, 50 μm. See also Figure S1.
Figure 2
Figure 2. Genetic Reporter hPSC Lines for Four Different Genes Reveal Distinct Stages of Sympathetic Neuronal Differentiation
(A–H) Changes of each reporter gene expression after differentiation using different lines and protocols: for OCT4::eGFP and SOX10::eGFP lines: LSB (day 0–3/4) – CHIR (day 2–10); for ASCL1::eGFP line: LSB (day 0–3/4) – CHIR (day 2–7) – SP (day 3–10); for PHOX2B::eGFP line: LSB (day 0–3/4) – m3i (day 2–7) – SP (day 3–10). The eGFP positive samples were collected by cell sorting at the specific time points as indicated. LSB, LDN193189 and SB431542; m3i, CHIR (CHIR99021), DAPT, and PD173074; SP, Shh plus PMP (purmorphamine). (B) Clustered heat map of global gene expression after microarray analyses (n = 2). (C–F) List of the increased (purple) and decreased (white) genes comparing two groups, as assessed by microarray analysis (**P < 0.01; ***P < 0.001; ANOVA test using Partek Genomics Suite (Partek Inc.); n = 3 for OCT4+, SOX10+, or ASCL1+ cells; n = 2 for PHOX2B+ cells). (G–H) Gene ontology (GO) analysis with upregulated gene list (G, ASCL1+ cells over PHOX2B+ cells; H, PHOX2B+ cells over ASCL1+ cells). All error bars represent mean ± S.E.M. See also Figure S2.
Figure 3
Figure 3. Characterization of FACS-Purified PHOX2B::eGFP+ Sympathetic Neurons from hPSCs
(A) Schematic for differentiation of PHOX2B::eGFP positive cells. LSB, LDN193189 and SB431542; m3i, CHIR99021, DAPT, and PD173074; SP, Shh plus PMP (purmorphamine). B4, BMP4. (B) FACS-purified PHOX2B::eGFP-expressing putative sympathetic neuronal precursors gave rise to peripherin (PRPH, pseudo-green), TH (red), DBH (red), GATA3 (red)-expressing sympathetic neurons. (C) Quantification of PRPH, TH, DBH, and GATA3 positive cells (n = 3). (D) qRT-PCR data showing significant increases of each transcript after sympathetic neuronal differentiation (**P < 0.01; ***P < 0.001; compared to each transcript level of undifferentiated hESCs; unpaired Student’s t-test; n = 3). (E–H) Electrophysiological characterization of PHOX2B::eGFP-expressing sympathetic neurons, including (E) I–V curve (n = 7), representative action potentials upon either (F) administration of 50 mM KCl, or (G) injecting 100 pA current. In (E), 10 of 10 neurons express a considerable amount of sodium channels and potassium channel to conduct both inward and outward current. In (F), 3 of 4 neurons responded to 50 mM KCl. In (G), 4 of 9 neurons fired a single of action potential (Type 1) and 5 of 9 neurons fired a train of action potentials (Type 2); 8 of 9 neurons responded to currents of 100 pA or greater, and 1 (Type 1) of 9 neurons responded to currents of 300 pA or greater. (H) An action potential (top) evoked during a brief depolarizing current step (bottom), with an afterhyperpolarization (AHP); 3 of 4 neurons had medium AHP (lasts 221–428 ms); RMP, resting membrane potential. (I) Release of catecholamines after 50 mM KCl administration were analyzed by using commercial ELISA kit (*P < 0.05; unpaired Student’s t-test; n = 3). DA, dopamine. NE, norepinephrine. All error bars represent mean ± S.E.M. or mean + S.E.M. Scale bars, 100 μm. See also Figure S3.
Figure 4
Figure 4. hPSC-Derived Sympathetic Neurons Can Pharmacologically Control Spontaneous Beating of Neonatal Mouse Ventricular Myocytes
(A) Schematic representation of the in vitro co-culture system, with FACS-purified human ESC-derived PHOX2B::eGFP+ sympathetic neurons (hSNs) and neonatal mouse ventricular myocytes (NMVM), used to test sympathetic-cardiac connections. (B) Transmission electron microscopy (TEM) image of the junctional region between hSN and NMVM following co-culture. Green-colored region indicates the nerve terminal of a hSN, and pink-colored region indicates the NMVM. White arrowheads indicate putative synaptic vesicles. (C) Immunofluorescence analysis was performed using PRPH (pseudo-green, hSNs) and cTnT (red, NMVM) antibodies. Bottom, maximum intensity projection of confocal microscopy z-stacks. Top, ortho-images of boxed region in the bottom image (see Movie S1 for 3-D reconstructed image). (D) Top, schematic diagrams for the control of beating rate of NMVM; bottom, increased beating rates were observed upon the administration of 1 μM nicotine (see Experimental Procedures; *P < 0.05; **P < 0.01; unpaired Student’s t-test; n = 25 for i, 12 for ii, 31 for iii, 12 for iv). Red lines indicate each mean value. (E–F) High speed video image acquisitions and multi-electrode array (MEA) recordings were synchronized (see Supplemental Experimental Procedures for more details). (E) Using motion vector analysis, increased beating rates were observed 6 min after administration of 1 μM nicotine. First peaks of each beating indicate contractions, and second peaks indicate relaxations. (F) Using MEA recordings, increased beating rates were detected 6 min after treatment of 1 μM nicotine. Scale bars in (B) TEM images, 0.5 μm; in (C) confocal images, 5 μm. See also Figure S4 and Movie S1–S3.
Figure 5
Figure 5. Optogenetic Control on hPSC-derived Sympathetic Neurons Lead to Changes of Spontaneous Beating of Neonatal Mouse Ventricular Myocytes
(A) Schematic representation of the in vitro co-culture system, with FACS-purified human ESC-derived hChR2-expressing PHOX2B::eGFP+ sympathetic neurons and neonatal mouse ventricular myocytes (NMVM), for testing sympathetic-cardiac connections. hESC-derived sympathetic neurons were infected with (C–I) hChR2 or (B) LacZ lentivirus. (B–C) Immunofluorescence analyses were performed using TH (red) antibody. (D) Quantification of the proportion of co-localized cells (n = 6). (E) Cultured sympathetic neurons-expressing hChR2-eYFP were targeted in IR-DIC for whole-cell patch clamp recordings. Responses of a neuron to trains of wide-field blue light pulses at a constant frequency (20 mW at 470 nm, 5 Hz, 1 ms) were represented. (F) The amounts of norepinephrine release after 15 min illumination with continuous wide-field blue light (26 mW/mm2 at 488 nm) or no light, were analyzed by using commercial ELISA kit (*P < 0.05; unpaired Student’s t-test; n = 3). (G–H) Top, schematic diagrams for the control of beating rate of NMVM; bottom, (G) increased beating rates were observed upon the photoactivation of hChR2-expressing sympathetic neurons with continuous wide-field blue light (see Experimental Procedures; 26 mW/mm2 at 488 nm; **P < 0.01; ***P < 0.001; unpaired Student’s t-test; n = 5 for i, n = 14 for ii, n = 14 for iii, n = 13 for iv). (H) Before and after treatment of β-blocker (1 μM propranolol) for 5 min, neuronal-photostimulation-induced beating rate change on NMVM was measured (*P < 0.05; unpaired Student’s t-test; n = 3). Orange arrows connect the data, before and after treatment of 1 μM propranolol, obtained from the same NMVM syncytium. Red lines indicate each mean value. (I) Photoactivation of hChR2-expressing sympathetic neurons co-cultured with NMVM using continuous wide-field blue light or continuous focal blue light. Different from conventional wide-field illumination, the field diaphragm in the epi-illumination pathway of the microscope was fully closed in order to achieve focal illumination (area, 0.08 mm2). Light intensity was 26 mW/mm2 at 488 nm. (I, left panel) Example images representing focal and wide-field illumination. Blue dotted circle indicates an illuminated region with blue light. White dotted circle indicates a beating region of NMVM. (I, right panel) With focal (n = 3) or wide-field (n = 3) illumination, spontaneous beating rates were measured in the order of three sections (~20 sec each): i) ‘no illumination just before photostimulation’, ii) ‘blue light illumination for photostimulation’, and iii) ‘no illumination just after photostimulation’. All error bars represent mean + S.E.M. Scale bars, 100 μm. See also Figure S5 and Movie S4.
Figure 6
Figure 6. Physical Interaction Between hPSC-Derived Sympathetic Neurons and Ventricular Myocytes Leads to Neuronal Maturation Phenotypes
(A) The amounts of norepinephrine release after 50 mM KCl administration were analyzed by using commercial ELISA kit (*P < 0.05; unpaired Student’s t-test; n = 3). Neurons, hESC-derived PHOX2B::eGFP+ sympathetic neurons. NMVM, neonatal mouse ventricular myocytes. (B–C) We used the ‘neurons only with NMVM-conditioned medium’ control (one day conditioned medium of NMVM was collected and fed to ‘neuron only’ sample daily for 7 days). The RNA from ‘neurons only’ samples and ‘NMVM only’ samples were mixed together and used as a ‘neurons only + NMVM only’ control. qRT-PCR analyses were performed by using indicated human-specific primers (*P < 0.05; **P < 0.01; ***P < 0.001; unpaired Student’s t-test; n = 3). (D–G) The changes in intracellular calcium concentrations [Ca2+]i induced by 0.1 μM nicotine were measured by ratiometric Fura-2 imaging. Calcium responses were calculated as the ratio of Fura-2 light emission on excitation at 340 and 380 nm (340/380) or the normalized ratio (ΔF/F0; ΔF = (F − F0), F = the 340/380 at a given time point, F0 = the mean basal, unstimulated 340/380 of each cell). White boundaries in (D) indicate the cell bodies of neurons which satisfy the criterion for selecting neurons as described in Supplemental Experimental Procedures. (E) Example traces of ΔF/F0 intensity from the neurons co-cultured with (dark orange lines) or without (gray lines) NMVM exposed to 0.1 μM nicotine. Each trace is a response from a unique cell (n = 18). (F) ΔF/F0 intensity plot showing the response of individual cells to 0.1 μM nicotine (***P < 0.001; unpaired Student’s t-test; neurons only, n = 41; neurons with cardiomyocytes, n = 60). Red lines indicate each mean value. (G) The percent of total responder neurons (ΔF/F0 > threshold) in each sample. All error bars represent mean + S.E.M. See also Figure S6 and Movie S5.

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