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

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.

Generation of Human iPSC‐Derived Neurons on Nanowire Arrays Featuring Varying Lengths, Pitches, and Diameters

“…Such a value is comparable to other studies conducted in regular well plates. [76] The majority of the identified neurons (≈65%) were also immunoreactive for another mature neuronal marker, namely, NeuN, similar to other studies. [77] A small fraction of neurons was also positive for TH (about 2.5%) indicating the development toward a dopaminergic phenotype.…”

“…Also, the yields compare well considering the short differentiation period of only 14–15 days versus >30 days used in other studies. [ 76–78 ] In this context, it is also striking that the neurons, in addition, were functional already. One potential reason for the fast maturation of the neurons might be the early application of DAPT (on day 6) to the maturation medium to enhance the neuronal differentiation.…”

The Efficiency of Direct Maturation: the Comparison of Two hiPSC Differentiation Approaches into Motor Neurons

“…Characteristic proteins essential for the identification of postmitotic and functional MNs are MNX1, ISL1, and ChAT [46]. MNX1 (also known as Hb9) promotes the specification of MNs and is considered to be a marker for lower MNs [47,48].…”

Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells

“…Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to generate various cell types in vitro and hold promise for disease modeling and cell-replacement therapies (Ebert and Svendsen, 2010; Karagiannis et al, 2019; Takahashi et al, 2007; Thomson et al, 1998; Yu et al, 2007). Many motor neuron differentiation protocols have been developed over the past decades (Ben-Shushan et al, 2015; Cutarelli et al, 2021; Du et al, 2015; Hudish et al, 2020; Lee et al, 2007; Li et al, 2005; Li et al, 2008; Patani et al, 2011; Qu et al, 2014). Generally, hPSCs were firstly induced to form neuroepithelial (NE) cells at embryonic body (EB) or monolayer culture, subsequently specified to OLIG2 positive motor neuron progenitors (MNPs) under the activation of retinoic acid (RA) and sonic hedgehog (SHH).…”