Objective

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell type of the human body, making them an interesting tool for studying disease; particularly given the lack of ethical concerns compared to using embryonic stem cells. The use of iPSCs is especially promising in the fields of neurodevelopment and neurological diseases due to the difficulties associated with obtaining tissue samples from patients. These cells also have potential in the development of more efficacious and physiologically relevant toxicity tests and drug screening models to replace cell line based techniques; their three dimensional culture also offers an opportunity to bridge the gap between two dimensional in vitro culture and in vivo models, possibly leading to a reduction in animal experimentation. The aim of this study was to validate and refine a published protocol for neuronal differentiation (Rigamonti et al. 2016) using three dimensional aggregates, and to make the protocol easily scalable through the use of a benchtop bioreactor.

Human iPSCs obtained from the European Bank for Induced Pluripotent Stem Cells (EBiSC) were cultured in suspension using the BioLevitator^TM (Hamilton, Nevada, USA) system at 37 °C in an atmosphere enriched with 5 % CO₂. Single cell suspensions were cultured in maintenance medium for four days to allow spheroids to self-assemble. Cortical neuronal differentiation was then induced over 40 days largely according to the aforementioned published protocol.

Three initial cell concentrations of 0.75, 4.5, and 9 million cells/tube were investigated, and the uniformity of the resultant spheroids was assessed by light microscopy. At defined time points spheroids were then cultured in 2D for four days to allow assessment of neural rosette formation and neurite growth and maturation, through the use of immunocytochemistry and fluorescence microscopy.

An initial concentration of 4.5 million cells/tube was found to be optimum due to the assembly of spherical spheroids with a relatively homogenous size distribution. This concentration also resulted in the fastest neurite growth and the expression of a marker of neurite maturation. Overall, a number of immunocytochemical markers of neural stem cells and neural progenitors (Nestin and SOX2), early cytoskeletal neurites (TUJ1) and mature neurites (MAP2) were observed during the study.

These findings validate a published protocol for neuronal differentiation and confirm the suitability of the BioLevitator^TM bioreactor system for the production and maintenance of human iPSC spheroids, as well as their subsequent differentiation. The BioLevitator^TM system allows rapid expansion of spheroids neuronal differentiation on a potentially large scale. Further refinement of this system is likely to allow efficient and reproducible production of human iPSC derived neuronal progenitors for applications such as toxicity testing and drug screening. The additional possibility of using diseased iPSC lines in this system further reinforces its potential.

       

Rigamonti, A. et al. (2016) "Large-scale production of mature neurons from human pluripotent stem cells in a three-dimensional suspension culture system" Stem Cell Reports 6(6) p. 993-1008.