Derivation of Motor Neurons from three Clonal Human Embryonic Stem Cell Lines.
Human embryonic stem cells (hESC) demonstrate a remarkable proliferative and developmental potential and thus have huge therapeutic potential. To direct the differentiation of hESC to a specific lineage of high purity for cell transplantation is highly desirable. Here we describe a modified in vitro procedure to direct differentiation of three clonal hESC lines, hES 3.1, hES 3.2 and hES 3.3 efficiently to spinal motor neurons by using various differentiation factors namely retinoic acid (RA), sonic hedgehog (Shh), bone morphogenetic protein-2 (BMP-2) and Wnt3A. The highest number of motor neurons (58.0 +/- 7.6%) were obtained by an early treatment of embryoid bodies with a combination of RA + Shh from all the clonal hESC lines combined. The hES 3.1 line, however, produced relatively more motor neurons (69.5 +/- 11.8%) compared to other two hES clones, 3.2 (52.4 +/- 13.1%) and 3.3 (52.3 +/- 15.5%). Immunolocalisation studies revealed the expression of neuronal specific marker, beta omega-tubulin and motor neuron specific marker, HB9/HLXB9 in all the three hESC clones after 45 days of differentiation. The RT-PCR analyses showed the presence of the neuron-specific genes. This modified differentiation protocol provides a mean of obtaining an enriched population of motor neurons from hESC for possible use in studies of lineage development, drug discovery and also as a potential cell therapy for motor neuron disease.
Lim
U-Ming
UM
Stem Cell Division, Diabetes Transplant Unit, Prince of Wales Hospital and University of New South Wale, Australia.
Sidhu
Kuldip S
KS
Tuch
Bernard E
BE
D015415:Biomarkers / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology*; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D053823:Hedgehog Proteins / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D009046:Motor Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D051153:Wnt Proteins / Q000494:pharmacology; D060508:Wnt3 Protein; D060509:Wnt3A Protein
C525718:BMP2 protein, human; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; C490003:MNX1 protein, human; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D014404:Tubulin; C555510:WNT3A protein, human; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D014212:Tretinoin