Abstract
We have developed a four-part protocol to differentiate human embryonic stem cells (hESCs) to oligodendrocyte progenitor cells (OPCs) according to developmental principles. In the first 2 weeks, hESCs are induced to differentiate into neuroepithelial cells, which form neural tube-like rosettes. In the following 10 d, these neuroepithelial cells are specified to OLIG2-expressing progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH). Upon treatment with fibroblast growth factor 2 (FGF2) for another 10 d, these progenitors convert to OLIG2 and NKX2.2-expressing pre-OPCs. Finally, the pre-OPCs take 8-9 weeks to differentiate into OPCs, which express additional markers of oligodendrocytes, such as SOX10, platelet-derived growth factor receptor alpha (PDGFRalpha) and NG2. The unique aspects of the protocol are the use of FGF2 to promote the differentiation of gliogenic pre-OPCs in the third part and the removal of FGF2 during the transition of pre-OPCs to OPCs. This 3-month differentiation protocol consistently yields OPCs of high purity capable of producing myelin sheaths in vivo.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Basic Helix-Loop-Helix Transcription Factors / metabolism
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Cell Culture Techniques / methods*
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Cell Differentiation / drug effects
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Fibroblast Growth Factor 2 / pharmacology
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Hedgehog Proteins / pharmacology
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Homeobox Protein Nkx-2.2
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Homeodomain Proteins / metabolism
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Humans
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Nerve Tissue Proteins / metabolism
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Neuroepithelial Cells / cytology
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Neuroepithelial Cells / drug effects
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Neuroepithelial Cells / metabolism
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Nuclear Proteins
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Oligodendrocyte Transcription Factor 2
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Oligodendroglia / cytology*
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Oligodendroglia / drug effects
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Oligodendroglia / metabolism
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Pluripotent Stem Cells / cytology*
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Pluripotent Stem Cells / drug effects
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Pluripotent Stem Cells / metabolism
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Transcription Factors / metabolism
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Tretinoin / pharmacology
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Zebrafish Proteins
Substances
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Basic Helix-Loop-Helix Transcription Factors
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Hedgehog Proteins
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Homeobox Protein Nkx-2.2
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Homeodomain Proteins
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NKX2-2 protein, human
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Nerve Tissue Proteins
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Nuclear Proteins
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OLIG2 protein, human
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Oligodendrocyte Transcription Factor 2
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SHH protein, human
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Transcription Factors
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Zebrafish Proteins
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nkx2.2b protein, zebrafish
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Fibroblast Growth Factor 2
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Tretinoin