Stem cell differentiation, plasticity and regenerative mechanisms in the cholinergic system : implications for Alzheimer’s disease

Abstract

Stem cells are immature cells with self-renewal capacity. They are able to differentiate into multiple lineages that may serve as a source of expandable cells for various applications. To generate cell populations of a specific lineage, it is crucial to understand the regulatory role of local environmental cues, intrinsic factors and signaling pathways that control their cellular phenotype.

The main purposes of the present work were to examine factors that act as induction signals and modulate neuronal differentiation and plasticity, especially as regards the cholinergic phenotype, and also to examine possible interactive mechanisms between neurotrophic factors, cholinergic receptors and the amyloid precursor protein (APP) in stem cell biology.

Human embryonic stem (hES) cells from six different lines were used to evaluate defined conditions for neural differentiation in adherent and suspension culture systems. In serum-free and feeder-free cultures, a dynamic transition into neuroepithelial and radial glial cells was observed, and these neurogenic cells finally gave rise to neurons. For these lines of hES cells, a similar progression of neural differentiation occurred, suggesting that hES cells may serve as an important model system to study early neuronal developmental processes.

Alzheimer’s disease (AD) is associated with amyloid plaques, neurofibrillary tangles, and a marked loss of basal forebrain cholinergic neurons and neuronal nicotinic acetylcholine receptors (nAChRs) expressed on these neurons.

Examination of the subregional lineage of hES cell-derived neurons revealed expression of transcription factors specifying telencephalic neuronal identity. Moreover, these cells also expressed α3, α4 and α7 nAChR subunits and M1, M2 and M3 muscarinic receptor subtypes. Upon stimulation with neurotrophic factors, including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF), the expression of the cholinergic enzyme choline acetyltransferase was increased, which was also observed in the septal SN56 cell line following treatment with retinoic acid (RA). Additionally, RA treatment upregulated the expression of α3 but reduced the level of α4 nAChR subunits in SN56 cells. Stimulation with neurotrophic factors also upregulated the α3 nAChR subunit and M3 mAChR subtype in hES cell-derived neurons, suggesting differential regulation of various AChR subtypes in both septal SN56 and hES cell-derived neurons. The expression of functional cholinergic receptors was demonstrated by a calcium increase evoked by acetylcholine (ACh), and the proportion of responding cells was dependent on the concentration of ACh, which suggests that multiple subtypes of cholinergic receptors were expressed in hES cell-derived neurons.

An understanding of stem cell biology during pathological conditions is crucial for future stem cell-based therapeutical approaches. As disturbances in the microenvironment due to brain injury or pathology could affect the microenvironmental equilibrium, the cells may be exposed to cues that are different from those in their normal conditions that may ultimately affect their phenotype and function. To study this, APP23 transgenic mice were treated with the novel drug (+)-phenserine, which reduced the expression of APP. This treatment also increased the neuronal differentiation of transplanted human neural precursor cells (NPCs), whereas astrocytic differentiation was reduced, indicating a role for APP as cell fate determinant of NPCs in vivo. These findings suggest that a combined treatment and transplantation approach may be necessary in order to further examine the potential of transplantable cells for developing future cell-based treatment strategies of neurodegenerative diseases, including AD.

In conclusion, the findings in the present study demonstrate that hES cells may serve as an important model system to study cellular mechanisms in early human neural development. Exploring the mechanisms of action of RA, BDNF, NT3, CNTF and NGF, which act as inductive signals for differentiation processes and plasticity of hES cells, NPCs and cholinergic neurons, may also lead to the development of novel strategies for future therapeutic interventions in AD.