TY - JOUR
T1 - Serotonin neurons derived from rhesus monkey embryonic stem cells
T2 - Similarities to CNS serotonin neurons
AU - Salli, Ugur
AU - Reddy, Arubala P.
AU - Salli, Nurgul
AU - Lu, Nick Z.
AU - Kuo, Hung Chih
AU - Pau, Francis K.Y.
AU - Wolf, Don P.
AU - Bethea, Cynthia L.
N1 - Funding Information:
Supported by NIH grants MH62677 to CLB, RR15199 to DPW, NDS40601 to FKYP, U54 contraceptive Center Grant HD 18185 and RR000163 for the operation of ONPRC.
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2004/8
Y1 - 2004/8
N2 - We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERβ), and progestin receptor (PR), but not estrogen receptor-alpha (ERα). Less than 2-3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A- autoreceptor, ERβ, and PR in the differentiated population. A low level of expression of ERα mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.
AB - We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERβ), and progestin receptor (PR), but not estrogen receptor-alpha (ERα). Less than 2-3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A- autoreceptor, ERβ, and PR in the differentiated population. A low level of expression of ERα mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.
KW - Differentiation
KW - Embryonic stem cells
KW - Estrogen receptors
KW - Progestin receptors
KW - Rhesus macaque
KW - Serotonin
KW - Serotonin reuptake transporter
KW - Tryptophan hydroxylase
UR - http://www.scopus.com/inward/record.url?scp=3042708570&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3042708570&partnerID=8YFLogxK
U2 - 10.1016/j.expneurol.2004.04.015
DO - 10.1016/j.expneurol.2004.04.015
M3 - Article
C2 - 15246835
AN - SCOPUS:3042708570
SN - 0014-4886
VL - 188
SP - 351
EP - 364
JO - Neurodegeneration
JF - Neurodegeneration
IS - 2
ER -