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.
Highlights
Human pluripotent stem cells are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders
Neural cells derived from disease-specific Human induced pluripotent stem cell (hiPSC) from patients with neurological disorders have been especially useful as in vitro disease models recapitulating in vivo pathogenesis, as cells in the nervous system cannot be usually obtained from patients themselves
Because the derivation of neural stem/progenitor cells (NS/PCs) from embryoid body (EB) using the previously established method takes 1 month, we first utilized dual SMAD inhibition to facilitate the neural differentiation of EBs [8]
Summary
Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. 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. Neural cells derived from disease-specific hiPSCs from patients with neurological disorders have been especially useful as in vitro disease models recapitulating in vivo pathogenesis, as cells in the nervous system cannot be usually obtained from patients themselves. Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA) are motor neuron diseases. These motor neuron diseases exhibit different molecular pathologies, they share a common phenotype: motor neuron degeneration. The efficiency of these methods tends to be low, and show variability depending on hPSC clones referred to as clonal variations [5]
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