Geometrical constraints dictate assembly and phenotype of human iPSC-derived motoneuronal spheroids

Abstract

Background Neuronal spheroids represent an easy and versatile solution to model neuronal tissue in vitro. Conventional approaches to generate spheroids lack accurate size control, scalability, and customizability. This is even more exacerbated in case of pluripotent stem cell (PSC) derived spheroids, which remain challenging to standardize. Microwell devices address these limitations, providing an optimal balance between accessibility and scalability. With the aim of optimizing culture conditions, we parametrically investigated the role of microwell geometry on the formation and maturation of iPSC-derived motor neuron precursor (MNP) spheroids. Methods We developed a customizable mold device using Digital Light Processing (DLP) 3D printing to fabricate agarose microwell arrays with distinct aspect ratios for culturing hiPSC-derived MNP spheroids with high reproducibility. We generated nine different pyramidal microwell array geometries for culturing size-controlled spheroids in the 40–140 μm diameter range. We then evaluated the differential expression of genes related to cell proliferation and motor-neuron differentiation as function of microwell geometry and spheroid size. Results Our results indicate that spheroid size is significantly influenced by the microwell geometry, reliably due to cell partitioning at the seeding stage. Expression of proliferation and differentiation markers, such as motor neuron and pancreas homeobox 1 (MNX1) and Islet-1 (ISL1) transcription factors, is also dependent on microwell geometry and spheroid morphological descriptors. Conclusion Our approach enables the scalable production of size-controlled MNP spheroids and underscores the effect of geometrical confinement on regulating motor neuron differentiation.

1. Introduction

The human nervous system (NS) is organized in a hierarchical complex architecture where neurons and glial cells are connected in intricate functional networks. This makes the NS extremely difficult to model in vitro and only slightly reproducible with classic two-dimensional (2D) cell cultures [1]. The NS multicellular heterogeneity and the challenging accessibility and scalability of human primary cell cultures have always represented an obstacle to NS modelling redirecting the efforts on animal models for both physiological and pathological studies [2].

The advent of human induced pluripotent stem cells (hiPSCs) three-dimensional (3D) neuronal platforms, such as organoids [3], organ-on-chip [4], 3D bioprinted models [5], scaffold-based [6] and scaffold-free [7] cell cultures has marked a fundamental improvement in neurobiology [8]. The remarkable neuronal differentiative potential of iPSCs [9] combined with their almost unlimited renewal capability and non-invasive derivability from patients, has offered a new horizon for a more personalized medicine approach [10]. The 3D configuration is more effective in mimicking the brain tissue architecture, promoting more accurate cell-to-cell and cell-to-matrix interactions, and a better fluidic dynamic exchange for nutrients, oxygen, and drugs than standard 2D cultures [11, 12].

Among 3D neuronal models, spheroids appear as an easy and versatile solution [13] to better model neuronal tissue in vitro. Spheroids are self-assembled cell clusters formed by spontaneous or forced aggregation in either scaffold or scaffold-free environment [14]. Spheroids have been used to recapitulate the fundamental features of brain tissues in terms of self-organization, cell differentiation [15] electrophysiology, and neural networking in 3D [16]. Neuronal spheroids have also demonstrated useful models in the field of neurodegenerative disorders such as Alzheimer’s disease [17, 18] or Amyotrophic Lateral Sclerosis [19, 20] making them suitable for high throughput [21, 22] and drug screening purposes [23, 24]. Such translational applications require high reproducibility, scalability, and customizability, which are often lacking in conventional spheroid generation methods.