RRC ID 35571
Author Shimojo D, Onodera K, Doi-Torii Y, Ishihara Y, Hattori C, Miwa Y, Tanaka S, Okada R, Ohyama M, Shoji M, Nakanishi A, Doyu M, Okano H, Okada Y.
Title Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.
Journal Mol Brain
Abstract BACKGROUND:Human 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.
RESULTS:In 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 (HB9 (e438) ::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.
CONCLUSIONS:Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.
Volume 8(1)
Pages 79
Published 2015-12-1
DOI 10.1186/s13041-015-0172-4
PII 10.1186/s13041-015-0172-4
PMID 26626025
PMC PMC4666063
MeSH Adult Animals Cell Culture Techniques / methods Cell Differentiation* Cells, Cultured Coculture Techniques Genes, Reporter Human Embryonic Stem Cells / cytology Humans Lentivirus / metabolism Male Mice, Inbred NOD Mice, SCID Motor Neurons / cytology* Muscle Fibers, Skeletal / drug effects Muscle Fibers, Skeletal / metabolism Neuromuscular Junction / metabolism Pluripotent Stem Cells / cytology* Small Molecule Libraries / pharmacology Young Adult
IF 4.686
Times Cited 31
DNA material pCAG-HIVgp (RDB04394) pCMV-VSV-G (RDB04392).