Dopaminergic differentiation of human embryonic stem cells.
In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)-positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K(+). When transplanted into 6-hydroxydopamine-treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.
Zeng
Xianmin
X
Cellular Neurobiology Research Branch, National Institute on Drug Abuse, Baltimore, Maryland 21224, USA. xzeng@intra.nida.nih.gov
Cai
Jingli
J
Chen
Jia
J
Luo
Yongquan
Y
You
Zhi-Bing
ZB
Fotter
Erin
E
Wang
Yun
Y
Harvey
Brandon
B
Miura
Takumi
T
Backman
Cristina
C
Chen
Guann-Juh
GJ
Rao
Mahendra S
MS
Freed
William J
WJ
D015102:3,4-Dihydroxyphenylacetic Acid / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000502:physiology; D001921:Brain / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D004396:Coloring Agents / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D004298:Dopamine / Q000737:chemistry / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004622:Embryo, Mammalian / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D000199:Actins; D000964:Antimetabolites, Antineoplastic; C525809:BMP4 protein, human; C525811:Bmp4 protein, rat; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D004396:Coloring Agents; D017931:DNA Primers; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D015102:3,4-Dihydroxyphenylacetic Acid; D014446:Tyrosine 3-Monooxygenase; D001142:Aromatic-L-Amino-Acid Decarboxylases; D001973:Bromodeoxyuridine; D011188:Potassium; D004298:Dopamine; D009638:Norepinephrine