Preparation of Highly Pure hiPSC-Derived Motor Neurons Through Assembling a Co-culture System

Modeling motor neuron (MN)-related diseases using human induced pluripotent stem cells (hiPSCs) requires culture conditions replicating MN development, differentiation, and maturation in a dish. Current hiPSC-derived MN applications face challenges in survivability and functional properties over time, emphasizing the need for enhanced long-term culture conditions. In this chapter, we introduce and describe an indirect co-culture method involving culture inserts for the extended cultivation of hiPSC-derived MNs. Our findings demonstrate that MNs cultured using the insert-based approach exhibit superior suitability for preparation of highly pure hiPSC-MNs at late maturation stages. Immunocytochemistry and qPCR data from the late-stage MNs prepared with culture inserts reveal key molecular markers and significant shifts in gene expression patterns throughout MN maturation and synaptic connection, validating the effectiveness of this co-culture system in the preparation of mature iPSC-MNs.