Common pitfalls of stem cell differentiation: a guide to improving protocols for neurodegenerative disease models and research

Abstract

Induced pluripotent stem cells and embryonic stem cells have revolutionized cellular neuroscience, providing the opportunity to model neurological diseases and test potential therapeutics in a pre-clinical setting. The power of these models has been widely discussed, but the potential pitfalls of stem cell differentiation in this research are less well described. We have analyzed the literature that describes differentiation of human pluripotent stem cells into three neural cell types that are commonly used to study diseases, including forebrain cholinergic neurons for Alzheimer's disease, midbrain dopaminergic neurons for Parkinson's disease and cortical astrocytes for neurodegenerative and psychiatric disorders. Published protocols for differentiation vary widely in the reported efficiency of target cell generation. Additionally, characterization of the cells by expression profile and functionality differs between studies and is often insufficient, leading to highly variable protocol outcomes. We have synthesized this information into a simple methodology that can be followed when performing or assessing differentiation techniques. Finally we propose three considerations for future research, including the use of physiological O2 conditions, three-dimensional co-culture systems and microfluidics to control feeding cycles and growth factor gradients. Following these guidelines will help researchers to ensure that robust and meaningful data is generated, enabling the full potential of stem cell differentiation for disease modeling and regenerative medicine.

Keywords: Alzheimer’s disease; Astrocytes; Cholinergic neurons; Differentiation; Dopaminergic neurons; Neurodegeneration; iPS cells.

Fig. 1

Schematic diagram of basal forebrain cholinergic neuron differentiation. The differentiation of basal forebrain cholinergic neurons from pluripotent stem cell colonies is driven by transitions between two- and three-dimensional culturing stages, as well as timed exposure to essential growth factors, such as NGF. The presence or absence of developmental and maturation markers are essential guides to monitor the differentiation progress at each culturing stage towards mature, functional forebrain cholinergic neurons

Fig. 2

Schematic diagram of midbrain dopaminergic neuron differentiation. The differentiation of midbrain dopaminergic neurons from pluripotent stem cell colonies is driven by the transition from spherical to adherent cultures and a staggered supplementation of growth factors, particularly SHH, FGF8 and later TGF3β. The presence or absence of developmental and maturation markers are essential guides to monitor the differentiation progress at each culturing stage towards mature, functional midbrain dopaminergic neurons

Fig. 3

Schematic diagram of astrocyte differentiation. The differentiation of astrocytes from pluripotent stem cell colonies follows the early neuronal developmental progress through spherical and adherent culture stages. Glial progenitor formation is triggered by the supplementation of EGF and FGF2, with CNTF being required for transition to mature astrocytes and aided by the neurotrophic factor NRG1β. The presence or absence of developmental and maturation markers are essential guides to monitor the differentiation progress at each culturing stage towards mature, functional astrocytes