Differentiation of iPSC-derived neural progenitors into motor neurons in 3D-printed bioscaffolds

Abstract

Translational medicine for neurodegenerative diseases can advance through the use of in vitro models incorporating human neural cells derived from patient-specific induced pluripotent stem cells (iPSCs). Previously, we investigated whether motor neuron (MN) progenitors derived from human iPSCs could differentiate from MNs within three-dimensional (3D)-printed scaffolds. While extensive neurite arborization was observed on the scaffold surface, no neurite outgrowth occurred within the scaffold interior. Here we showed the extensive growth of the neurites from iPSC-derived neural progenitors, imbedded into the gelatin scaffolds during 30 days of experimental time. We presented a bioink formulation that softens the scaffold while preserving its 3D structure, thereby facilitating neurite outgrowth throughout the scaffold. MN differentiation, evidenced by extensive neurite arborization and the expression of choline acetyltransferase (ChAT), was verified in 3D images deep within the scaffold structure. Notably, the degree of MN differentiation appeared to depend on two factors as follows: the delivery of MN differentiation factors via mesoporous silica particles (MSPs) embedded in the bioink and the method used to generate MN progenitors prior to 3D printing. In this paper, we provide a detailed protocol for 3D-printing human iPSC-derived MN progenitors, enabling their differentiation and survival within gelatin scaffolds. This protocol could be expanded to incorporate additional cell types, allowing the creation of more complex and standardized 3D neural tissues. Such advancements could facilitate investigations into the pathophysiology of motor neuron diseases and the development of new therapeutic strategies.

Keywords

3D bioscaffold / Differentiation / Gelatin / Induced pluripotent stem cells / Mesoporous silica / Motor neuron