A Monolayer System for the Efficient Generation of Motor Neuron Progenitors and Functional Motor Neurons from Human Pluripotent Stem Cells.

Alessandro Cutarelli,Daniele Arosio,Luciano Conti,Alice Tata,Ingrid Battistella,Carlo Musio,Vladimir A Martínez-Rojas,Daniela Rossi

doi:10.3390/cells10051127

Abstract

Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications.

Highlights

Motor neurons (MNs) are cells located in specific areas of the central nervous system (CNS), including the cerebral cortex, brain stem and spinal cord [1]
To establish a robust and simplified monolayer procedure for rapid and efficient MN progenitor cells (MNPCs) from human induced pluripotent stem cells (hiPSCs), we focused on mimicking the developmental features underlying spinal cord formation by using the minimum required molecules in a xeno-free environment
At DIV6, the cultures were characterized by the absence of residual hiPSCs as shown by the lack of OCT4+ve and NANOG+ve cells, and by the presence of neural cells arranged in neural rosette structures and exhibiting marked immunoreactivity for NESTIN and for the neural rosette markers ZO1 and aPKC (Figure 1B) [29,30]

Summary

Introduction

Motor neurons (MNs) are cells located in specific areas of the central nervous system (CNS), including the cerebral cortex (upper MNs), brain stem and spinal cord (lower MNs) [1]. They are involved in governing voluntary actions and in general body movements through the participation and contribution of large neuronal circuits. MNs are the only cells in the body that can directly stimulate skeletal muscles, modulating their contraction Their loss results in diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (SLA) [2,3]. Once the neural precursors are obtained, the following steps involve the patterning into MN progenitor cells (MNPCs) by mirroring in vitro the spinal cord developmental-relevant timing and physiological events, and the subsequent maturation of this cell population into mature

Methods

Results

Conclusion