Culture of embryonic stem (ES) cells at high density inhibits bothβ-catenin signaling and neural differentiation. ES cell density does not influence β-catenin expression, but a greater proportion ofβ-catenin is targeted for degradation in high-density cultures. Moreover,in high-density cultures, β-catenin is preferentially localized to the membrane further reducing β-catenin signaling. Increasing β-catenin signaling by treatment with Wnt3a-conditioned medium, by overexpression ofβ-catenin, or by overexpression of a dominant-negative form of E-cadherin promotes neurogenesis. Furthermore, β-catenin signaling is sufficient to induce neurogenesis in high-density cultures even in the absence of retinoic acid (RA), although RA potentiates the effects of β-catenin. By contrast,RA does not induce neurogenesis in high-density cultures in the absence ofβ-catenin signaling. Truncation of the armadillo domain ofβ-catenin, but not the C terminus or the N terminus, eliminates its proneural effects. The proneural effects of β-catenin reflect enhanced lineage commitment rather than proliferation of neural progenitor cells. Neurons induced by β-catenin overexpression either alone or in association with RA express the caudal neuronal marker Hoxc4. However, RA treatment inhibits the β-catenin-mediated generation of tyrosine hydroxylase-positive neurons, suggesting that not all of the effects of RA are dependent upon β-catenin signaling. These observations suggest thatβ-catenin signaling promotes neural lineage commitment by ES cells, and that β-catenin signaling may be a necessary co-factor for RA-mediated neuronal differentiation. Further, enhancement of β-catenin signaling with RA treatment significantly increases the numbers of neurons generated from ES cells, thus suggesting a method for obtaining large numbers of neural species for possible use in for ES cell transplantation.

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.

An early substantial loss of basal forebrain cholinergic neurons (BFCNs) is a constant feature of Alzheimer’s disease (AD) and is associated with deficits in spatial learning and memory. Induced pluripotent stem cells (iPSCs) derived from patients with AD as well as from normal controls could be efficiently differentiated into neurons with characteristics of BFCNs. We used BFCNs derived from iPSCs to model sporadic AD with a focus on patients with ApoE3/E4 genotypes (AD-E3/E4). BFCNs derived from AD-E3/E4 patients showed typical AD biochemical features evidenced by increased Aβ42/Aβ40 ratios. AD-E3/E4 neurons also exhibited altered responses to treatment with γ-secretase inhibitors compared to control BFCNs or neurons derived from patients with familial AD. BFCNs from patients with AD-E3/E4 also exhibited increased vulnerability to glutamate-mediated cell death which correlated with increased intracellular free calcium upon glutamate exposure. The ability to generate BFCNs with an AD phenotype is a significant step both for understanding disease mechanisms and for facilitating screening for agents that promote synaptic integrity and neuronal survival.