Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells

Abstract

BackgroundHuman pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves.ResultsIn this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6+ and SOX1+ neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9+ and ISL-1+ motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9e438::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis.ConclusionsOur motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0172-4) contains supplementary material, which is available to authorized users.

A three-dimensional culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction and disease modeling

“…Consistently, CHRNE transcripts are detected in rodent 2D and 3D skeletal muscle fiber cultures when co-cultured with nerve cells (Bach et al, 2003;Ostrovidov et al, 2017;Smith et al, 2016;Vilmont et al, 2016). However, despite significant progress toward directing human pluripotent stem cells (PSCs) to the motor neuron lineage Lippmann et al, 2014;Maury et al, 2015;Shimojo et al, 2015;Zhang et al, 2001) and establishing electrically and chemically responsive human muscle fibers in vitro (Madden et al, 2015), the first reports of human NMJ models -2D human muscle fiber and motor neuron co-cultures -were unable to demonstrate synapse maturation via the gamma to epsilon AChR subunit switch (Guo et al, 2011;Steinbeck et al, 2016), and there are no reports of epsilon AChR protein expression or function in culture in the absence of enforced gene expression.…”

Personalized Hydrogels for Engineering Diverse Fully Autologous Tissue Implants

“…Recently, the potential of neuronal treatment for regenerating injured spinal cord was reported . In order to engineer patient‐specific motor neuron implants, the cell‐containing hydrogels were exposed to a differentiation protocol that included dual SMAD inhibition, retinoic acid and purmorphamine . The cells assembled into neuronal networks with multiple intertwined motor neurons (Figure i) and synapses (Figure j), as judged by expression of the homebox gene HB9, and colocalization of MAP2, NFM, and SYP.…”

Unveiling synapse pathology in spinal bulbar muscular atrophy by genome-wide transcriptome analysis of purified motor neurons derived from disease specific iPSCs

“…We first established iPSCs from skin fibroblasts isolated from four SBMA patients (SBMA1, 2, 3, and 4), and three healthy age-matched control adults (TIG114, YF, KA) (Table 1), by introducing OCT4, SOX2, KLF4, L-MYC, LIN28, and shTP53 via episomal vectors. Three of the control iPSC clones, TIGE-9, YFE-16, and EKN3, have already been described previously [20,21] (Table 1). All the SBMA and control iPSC clones showed characteristic human embryonic stem cell-like morphology and expressed pluripotent stem cell markers, Oct4 and Nanog, as revealed by immunocytochemical analysis (Fig.…”