Optimization of long-term human iPSC-derived spinal motor neuron culture using a dendritic polyglycerol amine-based substrate

Abstract

Human induced pluripotent stem cells (h-iPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on h-iPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using h-iPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current h-iPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of h-iPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve iPSC-based studies of human MN biology and disease.

Comparing the Characteristics of Microglia Preparations Generated Using Different Human iPSC-Based Differentiation Methods to Model Neurodegenerative Diseases

“…Human iPSC line NCRM-1 was obtained from the National Institutes of Health Stem Cell Resource (Bethesda, MD, USA). Human iPSC lines CS52iALS-C9n6.ISOxx (referred to as “CS52” hereafter for sake of brevity) and CS29iALS-C9n1.ISOxx (“CS29” hereafter), corresponding to corrected, isogenic lines derived from two distinct parental lines generated from ALS patients with C9orf72 gene mutations, were obtained from Cedars-Sinai (Los Angeles, CA, USA) (Thiry et al, 2022). Undifferentiated state of human iPSCs was assessed by testing for expression of the stem cell markers NANOG and OCT4 using rabbit anti-NANOG (1/1,000; Abcam; Cambridge, UK; Cat.…”

Comparing the potential of microglia preparations generated using different human iPSC-based differentiation methods to model microglia-mediated mechanisms of amyotrophic lateral sclerosis pathophysiology

“…Human iPSC line NCRM-1 was obtained from the National Institutes of Health Stem Cell Resource (Bethesda, MD, USA). Human iPSC lines CS52iALS-C9n6.ISOxx and CS29iALS-C9n1.ISOxx, corresponding to corrected, isogenic lines derived from two distinct parental lines generated from ALS patients with C9orf72 gene mutations, were obtained from Cedars-Sinai (Los Angeles, CA, USA) (Thiry et al, 2022). Maintenance and quality control of iPSCs was conducted as described previously (Soubannier et al, 2022).…”

An optimized workflow to generate and characterize iPSC-derived motor neuron (MN) spheroids

“…The cell bodies of the MNs often form clusters with their axons entangled ( Fig 1A ), making them inaccessible for some analyses. For instance, monitoring cell morphology, electrical activity and protein expression through immunocytochemistry becomes a challenging task (25, 26). Moreover, the clustering cells are prone to detachment from the surface, preventing their long-term culture.…”

“…Maintaining cultures over several weeks is a critical aspect in MND modelling considering that disease phenotypes might only appear after a long period of culture. While some groups are optimizing 2D protocols to reduce the aggregation of iPSCderived MNs (25,27), other groups are developing techniques to generate 3D cultures which include spinal organoids (28)(29)(30)(31) and MN spheroids (32)(33)(34)(35)(36) to address some of the technical issues that arise with 2D monolayers and to enhance MN maturation by exposing the cells to a 3D environment.…”