The controlled generation of functional basal forebrain cholinergic neurons from human embryonic stem cells

Abstract

An early substantial loss of basal forebrain cholinergic neurons (BFCN) is a constant feature of Alzheimer's disease and is associated with deficits in spatial learning and memory. The ability to selectively control the differentiation of human embryonic stem cells (hESCs) into BFCN would be a significant step toward a cell replacement therapy. We demonstrate here a method for the derivation of a predominantly pure population of BFCN from hESC cells using diffusible ligands present in the forebrain at developmentally relevant time periods. Overexpression of two relevant human transcription factors in hESC-derived neural progenitors also generates BFCN. These neurons express only those markers characteristic of BFCN, generate action potentials, and form functional cholinergic synapses in murine hippocampal slice cultures. siRNA-mediated knockdown of the transcription factors blocks BFCN generation by the diffusible ligands, clearly demonstrating the factors both necessary and sufficient for the controlled derivation of this neuronal population. The ability to selectively control the differentiation of hESCs into BFCN is a significant step both for understanding mechanisms regulating BFCN lineage commitment and for the development of both cell transplant-mediated therapeutic interventions for Alzheimer's disease and high-throughput screening for agents that promote BFCN survival.

Figure 1

Generation of basal forebrain cholinergic neurons (BFCN) through sequential growth factor treatments. (A, B): Confocal microscopy demonstrates that cells generated through BMP9 treatment of fibroblast growth factor 8/sonic hedgehog pretreated neural progenitors express ChAT, p75, and Map2 and have a projection neuron morphology, whereas control neurons are only Map2-positive. (C, D): Confocal microscopy of equivalent cells stained only for ChAT and Map2 show the same BMP9 response and long ChAT+, Map2-axons. Scale bar = 20 μM. (E, F): BMP9-mediated ChAT immunopositivity is associated with expression of the VChaT. Lower magnification analysis of fields of neurons stained for VChAT. Scale bar = 100 μM. (G): qRT-PCR analysis shows 12- to 40-fold increases of RNA levels for markers characteristic of the BFCN. Bars are standard error, n = 4. All increases were significant by ANOVA (*, p < .0001; **, p = .0014; ***, p < .0001; ****, p < .0001). Data are from four replicate experiments; error bars show SEM. Data in (A–F) are from five replicate experiments, data in (G) is from four replicate experiments; error bars show SEM. Abbreviations: AChE, acetylcholinesterase; BMP9, bone morphogenetic protein-9; ChAT, choline acetyltransferase; Map2, microtubule-associated protein 2; VChAT, vesicular acetylcholine transporter.

Figure 2

Generation of basal forebrain cholinergic neurons through transcription factor overexpression. (A): Confocal analysis of FACS-purified neurons shows strong expression of ChAT, p75, and Map2. (B): Confocal microscopy shows FACS-purified neurons immunostained only for ChAT and Map2. (C): FACS-purified neurons shown at higher power contain large numbers of vesicles positive for VChAT. Scale bars = 20 μM. (D): qRT-PCR analysis shows large increases in ChAT and p75 RNA transcript levels. Bars show SEM, n = 4. Both increases were shown to be significant by ANOVA (*, p = .0002; **, p = .0031). Abbreviations: ChAT, choline acetyltransferase; Map2, microtubule-associated protein 2; VChAT, vesicular acetylcholine transporter.

Figure 3

Lhx8 siRNA blocks BMP9 effects on basal forebrain cholinergic neurons (BFCN) differentiation. (A): qRT-PCR analysis demonstrates that Lhx8 siRNA nucleofection blocks the BMP9-mediated increase in Lhx8 levels, causing a reduction in Lhx8 transcript to levels below basal expression when compared with scrambled siRNA nucleofection after BMP9 treatment of dissociated and plated neural progenitors. (B): qRT-PCR analysis indicates that Lhx8 siRNA inhibits the BMP9-mediated BFCN differentiation of human neural progenitors, with only a twofold but still significant increase in levels of ChAT mRNA (*, p = .0225) after the siRNA treatment. (C): Neurons generated from Lhx8 siRNA-expressing neural progenitors fail to become ChAT immunopositive. Scale bar = 20 μM. Data in (B, C) are from three replicate experiments; error bars show SEM. Abbreviations: BMP9, bone morphogenetic protein-9; ChAT, choline acetyltransferase; Map2, microtubule-associated protein 2; N.S., not significant.

Figure 4

Quantification of neuronal differentiation into BFCN. (A): A high percentage of neurons from the FACS-purified (94% ± 1.53%) or BMP9-treated (85.59% ± 1.31%) populations are ChAT immunopositive, whereas control (0.89% ± 0.24%) and Lhx8 siRNA-treated (1.26% ± 1.33%) populations fail to express ChAT. All populations are significantly different by Mann-Whitney U test (*, p = .035; **, p < .001; ***, p = .001; ****, p = .006) except BMP9 versus nucleofected (#, p = .066). n = 4,700 control, 2,565 siRNA, 2,582 BMP9, or 1,718 nucleofected cells from four (control, BMP9 and nucleofected) or three (siRNA) replicate cultures. Error bars show SEM. (B): Representative ChAT immunohistochemistry demonstrates the clear distinction of ChAT immunopositivity between positive and negative cells. Scale bar = 20 μM. Abbreviations: BMP9, bone morphogenetic protein-9; ChAT, choline acetyltransferase; FACS, fluorescence-activated cell sorting; Map2, microtubule-associated protein 2.

Figure 5

Immunohistochemical and electrophysiological evidence for and characterization of functional synaptic transmission after engraftment of FACS-purified neurons into murine ex vivo hippocampal slice cultures. (A): FACS-purified neuronal populations stably engraft in mouse hippocampal ex vivo slice cultures and project long networks of axons. All green fluorescence in Figure 5 is the enhanced-green fluorescent protein (eGFP) expression from the adenovirally labeled FACS-purified Lhx8/Gbx1 transiently overexpressing neurons. Scale bar = 50 μM. (B): Murine presynaptic terminals ((synapsin1, red channel) line the axons of the engrafted cells, giving immunohistochemical verification of the electrophysiologically detected synaptic inputs to these cells. Scale bar = 5 μM. (C): Transcription factor-generated basal forebrain cholinergic neurons contain presynaptic terminals (synapsin1, red channel) within their axons, indicating that they are generating synapses with other neurons. Please refer to Supporting Information Figure 8 for orthogonal view confirming inclusion of synapsin1 immunopositivity within the engrafted neuron. Scale bar = 2 μM. (D): α-Bungarotoxin labeling indicates that eGFP+ axons from FACS-purified neurons terminate on regions with postsynaptic α7 nicotinic acetylcholine receptors, strongly indicating the presence of cholinergic neurotransmission. Scale bar = 2 μM. (E): (i) Spontaneous action potentials recorded from an EGFP-expressing neuron. This cell was held at −60 mV in current clamp mode (n = 3). Second trace (ii) illustrates the gradual decrease in action potentials amplitude after addition of TTX (500 nM). Lower traces (iii) illustrate action potentials before and after TTX addition (from i and ii). (F): Voltage-dependent Na currents recorded under voltage clamp conditions from an eGFP-positive cell (i), illustrating block of the inward current by TTX (ii) (n = 4). (G): Spontaneous γ-aminobutyric acid-ergic PSCs were detected in eGFP-expressing cells (i). Representative traces of PSCs recorded under voltage clamp conditions. Using high KCl in the pipette, at −70 mV eGFP cells displayed numerous PSCs. The frequency of PSCs was greatly reduced after application of BIC (100 mM) and CNQX (10 mM) (*, p < .01; n = 7) (ii). PSCs reappeared after 10 minutes washing (iii). We observed that all PSCs were blocked by BIC when this question was specifically examined. Nevertheless, we always also included CNQX as well to block any 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid–mediated currents were they to occur. (H): (i) PSCs recorded from an eGFP-negative cell in close proximity to an eGFP-expressing cell. PSCs were recorded under whole-cell voltage clamp (−70 mV) conditions. (ii) These PSCs were partially blocked by BIC (100 mM) and CNQX (10 mM). (iii, iv) The frequencies of PSCs were further blocked by the nicotinic antagonists MLA (10 nM) and DHβE (1 mM). (v) PSCs reappeared following washout of these drugs. (I): In eGFP-expressing cells the frequency (**, p < .01) and amplitude (*, p < .05) of PSCs were significantly blocked by BIC (100 mM) plus CNQX (10 μM, n = 5). (J): In non-eGFP-expressing cells closely juxtaposed to eGFP-expressing cells MLA and DHβE produced a significant decrease in PSC frequency, (n = 7). Abbreviations: BIC, bicuculline; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DHβE, dihydro-β-erythroidine; MLA, methyllycaconitine; PSC, postsynaptic current; TTX, tetrodotoxin.